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99mtc‘labeled nanocolloid drugs development methodsVladimir Sadkin1 Viktor Sкuridin1 Evgeny Nesterov1 Elena Stasyuk1 Alexander Rogov1 Natalya Varlamova1 Roman Zelchan2The work considers the problem of obtaining nanocolloid radiopharmaceuticals RPs and studying their functional suitability for diagnosing sentinel lymph nodes SLN in cancer patients Two principal approaches to the formation of technetium99mlabeled ps based on inanic and anic matrices were considered when carrying out research to develop methods for the production of nanocolloid RPs The composition of the reagents and the conditions for obtaining nanocolloid radiopharmaceuticals were determined The functional suitability of new RPs for scintigraphic diagnostics of sentinel lymph nodes has been studiedThe identification of sentinel lymph nodes”the first nodes that stand in the way of metastasizing of malignant neoplasms attracts increasing interest in modern oncological practice1“ It is believed that if the SLN is not affected by the metastatic process then all other regional lymph nodes are intact so the results of biopsy of these nodes are an objective diagnostic criterion for the spread of malignant process Fig a0 The optimal method of detecting SLN is the use of colloid nanomaterials labeled with technetium99m for scintigraphic or radiometric determination of node localization6“ Not so much the chemical nature of such ps but their size is the determining factor in the choice of the indicator in this case Thus according to Schauer14 a colloid with a p size of less than a0nm can accumulate not only in the SLN but also at nodes of and orders of magnitude Ps with the sizes of more than a0nm slowly migrate from the injection site The colloid with the p size from to a0nm was recognized as the optimal one for detecting SLNThe simplest method of obtaining colloids with given sizes and properties is immobilization of 99mTc on the surface of nanosized materialsTechnetium99m is by far the most popular radionuclide for conducting diagnostic studies practically in all fields of medicine15“ This is primarily due to its nuclearphysical characteristics a relatively short halflife a0h and Îradiation energy of a0meV providing a low exposure dose and at the same time sufficient penetrating power for radiometric measurementsToday the Tc99m Tilmanocept radiopharmaceutical is widely used which has proven itself well and gives good results But its production is quite timeconsuming and requires expensive components We offer a less laborious method from the simple components1920Materials and methodsMaterials All the reagents were purchased from SigmaAldrich ACS grade and used without further purification Technetium99m was obtained from chromatographic 99Mo99mTc generator œ99mTcGTTOM produced by Tomsk Polytechnic University TPU”Tomsk RussiaThree types of nanops were selected to obtain nanocolloids labeled with 99mTc The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriaminepentaacetic acid DTPA It should be noted that the DTPA molecule itself like its complexes with metals is hydrophilic and does not tend to form colloidal ps The introduction of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified DTPA complexes21 The original substance of the modified DTPA DTPAmod was synthesized in Tomsk Polytechnic University Preparation of colloid solution DTPAmod was produced using the following method A sample of modified DTPA with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of NaHCO3 solution by heating to a0°C After that the volume was adjusted with the same solvent up to the mark In order to reduce the p size the container with suspension was heated in water to a0°C and treated with ultrasound for a0min 1Tomsk Polytechnic University Lenina Avenue Tomsk Russia 2Tomsk National Research Medical Center Russian Academy of Sciences Kooperativny street Tomsk Russia email sadkintpuruScientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorFigure a0 The general scheme for the synthesis of 99mTcDTPAmodwhich reduced the average p radius up to a0nm The general scheme for the synthesis of 99mTcDTPAmod is shown in Fig a0The second type of colloids is iron nanops coated with a carbon shell of FeC Fig a03a These ps were obtained from the Institute of Metal Physics UrB RAS Ekaterinburg Russia In order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates ADT onto the surface of these ps has been developed An effective method for the synthesis of ADT followed by their application onto the carbon surface of ps was developed at the Tomsk Polytechnic University22 The general scheme for the synthesis of FeC ps and their subsequent interaction with 99mTc is shown in Fig a03bIn the third type of colloids technetium99m was adsorbed on aluminum oxide powder A powder of lowtemperature cubic modification of gammaoxide Al2O3 prepared from aluminum hydroxide powder AlOH3 by its calcination in a muffle furnace was used The substance was synthesized in Tomsk Polytechnic UniversityA reducing agent”tin II chloride dihydrate was used in order to obtain complexes of 99mTc with colloidsGelatin powdered Ph Eur USPNF pure pharma grade CAS Number was purchased from AppliChem GmbH Darmstadt GermanyMethods Method for preparation of 99mTc labeled nanocells The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl2ˆ™2H2O “ a0mgml in different ratios and then adding a a0ml of eluate of 99mTc “ a0MBqml to the mixtures The mixtures were incubated for a0min at a temperature of “ a0°C The preparation is ready for use after cooling at room temperature The reducing agent SnCl2ˆ™2H2O was used as a hydrochloric acid solution The amount of a0g of tin chloride II is added to the vial and a0ml of a0M hydrochloric acid HCl Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Carbon encapsulated iron nanop B the general scheme for the synthesis of FeC psis then added for its preparation After dissolution the volume is adjusted with distilled water to a0ml Dissolution was carried out in an inert gas argon mediumDetermination of the size of 99mTc labeled colloidal ps The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox p size analyzer œSympatec GmbH Germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm Three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity Calculations of the yield of products with different p sizes were determined according to the formulas given belowC220 Avc ˆ’ A1Avc C100 A1 ˆ’ A2A1 C50 A2 ˆ’ A3A2where Avc is the activity of the initial suspension prior to filtration A1 is the activity measured after filtration through a a0nm filter A2 is the activity after filtration through a0nm filter A3 is the activity measured after filtration through a0nm filterIn parallel determination of the radiochemical purity RCP of preparations by thin layer chromatography method was carried outThin‘layer chromatography TLC procedure To determine radiochemical purity of 99mTc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip Sorbfil Russia — a0 cm To determine Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cAmount of SnCl2ˆ™2H2O mgA[Sn99mTc]A ATcVIIA Table Change in relative activities of the complex [Sn99mTc] and 99mTc VIIpertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min In this system pertechnetate migrated with the front of the mobile phase Rf To determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min In this system the 99mTc“nanocolloid migrated with the front of the mobile phase Rf Stability The stability of 99mTc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mTc“nanocolloid following by incubation at a0°C for a0h At different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24Determination of the functional suitability of preparations for scintigraphic detection of SLN A study to assess the functional suitability of new nanocolloid RPs was performed in series of experiments involving white Wistar male rats weighing “ a0g Injection of RP in a dose of “ a0MBq was performed between the first and second fingers of the rat™s hind paw The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study Since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix Static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ECAM Signature gamma camera Siemens Germany The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œEuropean Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes Strasbourg The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee Protocol number All invasive manipulations with animals were performed using inhalation or drug anesthesiaStatistical analysis All mean values are expressed as IDg ± SD Data were analyzed statistically using methods of general statistics with a commercially available software package œStatistics for Windows StatSoft Inc Version Results and discussionTo carry out the labeling of colloids 99mTc extracted from a standard generator in the form of pertechnetate ions contained in a NaCl solution was used It has a higher degree of oxidation VII in this chemical form and is not prone to complex formation A reducing agent”tin II chloride dihydrate widely used for the preparation of various compounds labeled with 99mTc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 As a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess SnCl2·2H2O or the additional formation of a complex of reduced 99mTc with tin26 All this required preliminary experimental studies to establish the necessary and sufficient amount of SnCl2·2H2O in the reaction mixtureDuring the experiments it was found that the optimal concentration of Sn II in the composition of the reaction mixture when it interacts with 99mTc should be in the range of “ a0mgml Table a0It was found that when the eluate with the preliminarily reduced 99mTc VII was added to the nanops the Sn II concentration introduced in the RP was CSn a0mgml almost the entire amount of 99mTc has time to enter the composition of the largesize complex with tin even before its mixing with nanops This means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the Sn II solution into the reaction mixture In this connection the reduction of 99mTc with tin II was carried out in the presence of the selected substance In this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex The technique of applying of the 99mTc label to the surface of nanosized ps is given in the previous sectionAs a result of the studies reagent compositions and conditions for obtaining three nanocolloid RPs were determined Table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmProceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mTc VII in the obtained preparations is “ Preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm Scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in Fig a0Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cComposition of the preparation per a0mlDTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Al2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Colloid yield “ a0nm RCP ± ± ± Table Composition of reagents for production of technocium99 a0m nanocolloidsFigure a0 Distribution of the preparation in the rat when the preparation is administered [Al2O3 99mTc Sn II] A immediately after the administration of the drug B a0min after the administration C a0min after the administrationComposition of preparations per a0mlAl2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n DTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n Yield of colloid “ a0nm RCP ± ± ± Table Indicators of RCP and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsScintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mTc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid Along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed Gelatin G was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system Matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mTc The results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm Table a0In addition the size of these ps was determined on a Nanophox p analyzer The obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in Fig a0 A B C The average p size diameter is and a0nm respectivelyStability test showed that complex 99mTc“nanocolloid was stable in normal serum at least for a0h Radiochemical purity of the tracer at the end of the experiment was ± A study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes Fig a0 Table a0 displays the Al2O3 99mTc DTPAmod 99mTc FeC 99mTc biodistribution data at different time points postinjectionThe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionAs a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid RPs were determined An experimental dependence of the change in the content of 99mTc VII impurities on the concentration of tin II was established and its minimum amount a0mgml was determined to reach a RHP greater than In this case the yield of the target colloid with p sizes of ± a0nm is “ Preliminary tests of the developed preparations on experimental animals showed that accumulation of RP in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range Increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml In addition there was an increase in the yield of the colloid Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Change in the density of the distribution of the number of ps from their size in radiopharmaceuticals A œ99mTcAl2O3 B œ99mTcFeC C œ99mTcDTPAmodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ Repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cStomachTime h99mTcAl2O399mTcDTPAmod99mTcFeC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figure a0 Distribution of the preparation in the rat with injection of suspension [Al2O3 99mTc Sn II Gelatin] A immediately after the administration of the preparation B a0min after the administration C a0min after the administration D a0min after the administration The numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BloodmlHeart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table Biodistribution data up to a0h after injection of “ a0MBq of 99mTc in healthy male rats Data represent the average value n accumulation in the SLN Thus the level of accumulation of RP œ99mTcDTPAmod and RP œ99mTcFeCADT in the SLN is and respectively At the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityReceived March Accepted July References Jakobsen J K Sentinel node biopsy in urooncology A history of the development of a promising concept Urol Oncol “ Weixler B et al Sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival A prospective singlecenter trial World J Surg 101007s0026 Beasley G M et al Sentinel Lymph node biopsy for recurrent elanoma A multicenter study Ann Surg Oncol Moser J et al Sentinel node biopsy in melanoma A singlecentre experience with consecutive patients Br J Dermatol 101245s1043 “ Buda A et al Optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer Comparison of realtime fluorescence with indocyanine green and methylene blue Int J Gynecol Cancer “ Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c“ Sahbai S et al Pericervical injection of 99mTcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in SPECTCT Clin Nucl Med “ Hoogendam J P et al 99mTcnanocolloid SPECTMRI fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer J Nucl Med “ Stoffels I Leyh J P¶ppel T Schadendorf D Klode J Evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies Prospective randomized clinical trial to compare ICG99mTcnanocolloid hybrid tracer versus 99mTcnanocolloid Eur J Nucl Med Mol Imaging “ Beisani M et al Initial experience in sentinel lymph node detection in pancreatic cancer Rev Esp Med Nucl Imagen Mol Schubert T Uphoff J Henke R P Wawroschek F Winter A Reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer Verification in consideration of the European guidelines BMC Urol “ Jaukovic L et al Lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions Hell J Nucl Med “ Subramanian S Pandey U Shah S Rangarajan V Samuel G An indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection Nucl Med Commun “ RuizDom­nguez J M IbarzServio L Garc­ade Manuel G Calaf Peris© O Intraoperative injection of 99mTcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery Actas Urol “ Schauer A J Specific developments in sentinel node labling using 99mTccolloids In The Sentinel Lymph Node Concept Springer Berlin Wang Y et al Gasphase chemistry of technetium carbonyl complexes Chem Phys “ O™Connor M K et al Comparison of Tc99m maraciclatide and Tc99m sestamibi molecular breast imaging in patients with Wang J Zhang R Evaluation of 99mTcMIBI in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer EJNMMI Res Br J Radiol Costa P et al Scintigraphic imaging with technetium99Mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats Acta Cir Bras “ Vera D R Wallace A M Hoh C K Mattrey R F A synthetic macromolecule for sentinel node detection 99mTcDTPAmannosyldextran J Nucl Med “ Hoh C K Wallace A M Vera D R Preclinical studies of [99mTc]DTPAmannosyldextran Nucl Med Biol “ Skuridin V et al Modified DTPA moleculebased nanocolloid radiopharmaceuticals J Radioanal Nucl Chem “ Filimonov V D et al Unusually stable versatile and pure arenediazonium tosylates Their preparation structures and synthetic applicability Lett “ Lukasz K Thin Layer Chromatography in Drug Analysis “ CRC Press London Skuridin V et al Radiopharmaceutical drug based on aluminum oxide Indian J Sci Technol 1017485 ijst2015v8i36 Sazonova S I et al Synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging Iran J Nucl Med “ Skuridin V S et al Synthesis and biological characterization of 99mTclabeled ciprofloxacin Pharm Chem J “ AcknowledgementsThis work was financially supported by Ministry of Education and Science of the Russian Federation RFMEFI57514X0034Author contributionsVS Conducting experimental research analysis and interpretation of the data Final approval for manuscript publication VS development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication EN development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication ES development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication AR conducting experimental research analysis and interpretation of the data Final approval for manuscript publication NV conducting experimental research analysis and interpretation of the data Final approval for manuscript publication RZ conducting tests of the functional suitability of drugs Preparation of the section Evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and Figures „– Final approval of the manuscript for publication of the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to VSReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0c Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c'

"A decrease in Hsp70 by RNAi promoted cisplatin-dependent activation of Bax. A549 cells treated with Hsp70 or control siRNA were incubated in presence or absence of cisplatin; each cell extract was immunoprecipitated with an anti-active Bax antibody followed by immunoblotting with anti-Bax antibody. The data are representative of three separate experiments Synergistic effect of Hsp70 suppression on the cisplatin-mediated activation of caspase-9. (a) A549 cells were treated with cisplatin and/or ibuprofen and cell extracts were immunoblotted with active caspase-9 antibody. The lower panel shows the measurement of each caspase-9. (b) A549 cells exposed to siRNA targeting Hsp70 or control siRNA were incubated with or without cisplatin and the active caspase-9 was detected by western blot using an anti-caspase-9 antibody. The quantity of each protein was estimated by densitometric analysis (lower panels). (c and d). Assay for enzymatic activity of caspase-9 using a fluorogenic substrate. (c) After the incubation of the A549 cells with cisplatin (10??M) and/or ibuprofen (400??M) the caspase-9 activity of each cell extract was measured as described in Materials and methods section. (d) A549 cells transfected with Hsp70 siRNA or control siRNA were exposed to cisplatin for 48?h. The caspase-9 activity was then assessed using an enzymatic assay as described earlier. The value of caspase-9 activity was presented relative to the activity in untreated cells set at 1.0. The data represent mean values of three separate experiments. Significances were determined by Student's t-test (*P<0.05) Effects of nonsteroidal anti-inflammatory drugs on the expression of Hsp70 in A549 cells NSAIDs Hsp70 expression (%) Ibuprofen (400??M) 22.7±2.8 Aspirin (2500??M) 95.1±7.8 Diclofenac (200??M) 97.2±5.6 Sulindac (15??M) 98.9±2.9 Piroxicam (60??M) 96.6±6.2 Indometacin (10??M) 95.0±15.1 Mefenamic acid (25??M) 100.5±6.0 Values are shown as means±S.D. The expression of Hsp70 was measured by immunoblotting with an anti-Hsp70 antibody. The quantity of Hsp70 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each NSAID on the A549 cells for 48?h Effects of nonsteroidal anti-inflammatory drugs on the expression of HSF-1 in A549 cells NSAIDs HSF-1 expression (%) Ibuprofen (400??M) 16.2±3.9 Aspirin (2500??M) 93.5±2.9 Diclofenac (200??M) 96.7±6.6 Sulindac (15??M) 99.8±3.6 Piroxicam (60??M) 96.3±4.7 Indometacin (10??M) 98.1±1.6 Mefenamic acid (25??M) 98.5±1.1 Values are shown as means±S.D. The expression of HSF-1 was measured by immunoblotting with anti-HSF-1 antibody. The quantity of HSF-1 protein was estimated by densitometric analysis using Scion Image. The values in parentheses are the highest non-toxic concentrations (approximately 90% viability) used for each of the NSAID on the A549 cells for 48?h PLoS One one 1932-6203 Public Library of Science San Francisco USA 24505298 3914822 PONE-D-13-39952 .0087629 Research Biology Population Biology Epidemiology Medicine Clinical Research Design Epidemiology Drugs and Devices Drug Research and Development Epidemiology Cancer Epidemiology Clinical Epidemiology Oncology Cancers and Neoplasms Lung and Intrathoracic Tumors Non-Small Cell Lung Cancer Cancer Treatment Radiology Nuclear Medicine PET imaging [18F]FDG Positron Emission Tomography within Two Weeks of Starting Erlotinib Therapy Can Predict Response in Non-Small Cell Lung Cancer Patients Theranostic Use of FDG-PET in NSCLC Patients Hachemi Mammar 1 2 Couturier Olivier 1 2 3 Vervueren Laurent 1 2 Fosse Pac´me 1 2 Lacœuille Franck 1 2 3 Urban Thierry 1 3 4 5 Hureaux Jos 1 3 4 5 * 1 LUNAM Universit 49 000 Angers France 2 Universit d'Angers CHU Angers P´le de Radiologie Service de Mdecine Nuclaire Angers France 3 INSERM UMR_S 1066 Micro et Nanomdecines Biomimtiques Angers France 4 Universit d'Angers CHU Angers P´le des Spcialits Mdicales et Chirurgicales Intgres Dpartement de Pneumologie Angers France 5 Universit d'Angers Equipe Pyver Angers France Singh Pankaj K. Editor University of Nebraska Medical Center United States of America * E-mail: JoHureauxchu-angers.fr Competing Interests: The authors have declared that no competing interests exist. Conceived and designed the experiments: OC TU JH. Performed the experiments: MH OC LV PF FL JH. Analyzed the data: MH OC FL TU JH. Contributed reagents/materials/analysis tools: MH OC TU JH. Wrote the paper: MH OC JH. 2014 5 2 2014 9 2 e87629 29 9 2013 26 12 2013 2014 Hachemi et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. Purpose The aim of this prospective study was to evaluate whether [18F]FDG-PET/CT performed within two weeks of starting erlotinib therapy can predict tumor response defined by RECIST 1.1 criteria after 8 weeks of treatment in patients with inoperable (stage IIIA to IV) non-small cell lung cancer patients. Patients and Methods Three [18F]FDG-PET/CT scans were acquired in 12 patients before (5±4 days) and after 9±3 days (early PET) and 60±6 days (late PET) of erlotinib therapy. Conventional evaluation including at least chest CT (baseline versus after 8 weeks of treatment) was performed according to RECIST 1.1 criteria. Change in [18F]FDG uptake was compared with conventional response progression-free survival (PFS) and overall survival (OS). Results By using ROC analysis the Area Under the Curve for prediction of metabolic non-progressive disease (mNP) by early PET was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) at a cut-off of 21.6% reduction in maximum Standardized Uptake Value (SUVmax). This correctly classified 11/12 patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). Non-progressive disease after 8 weeks of treatment according to RECIST 1.1 criteria was significantly more frequent in patients classified mNP (P?=?0.01 Fisher's exact test). mNP patients showed prolonged PFS (HR?=?0.27; 95% CI 0.04 to 0.59; P<0.01) and OS (HR?=?0.34; 95% CI 0.06 to 0.84; P?=?0.03). Late PET analysis provided concordant results. Conclusion Morphologic response PFS and OS survival in non-small cell lung cancer patients can be predicted by [18F]FDG-PET/CT scan within 2 weeks after starting erlotinib therapy. The authors have no support or funding to report. Introduction Lung cancer is the leading cause of cancer-related death in both Europe[1] and the United States of America.[2] The most common forms of lung cancer are non-small cell lung cancer (NSCLC) histological subtypes. Systemic chemotherapy has contributed to a significant improvement in NSCLC therapy but progress appears to be stagnating.[3] [4] Over the last decade a better knowledge of cellular pathways has allowed the development of new therapies based on NSCLC-driving genetic abnormalities. Targeted therapies have been developed to block pathological cellular pathways involved in cancer cell survival proliferation and metastasis. Epidermal Growth Factor Receptor (EGFR) is overexpressed in NSCLC[5] and has been extensively studied as a potential therapeutic target. Two EGF Receptor blockers gefitinib and erlotinib have been demonstrated to be effective in front-line therapy in patients with inoperable NSCLC harboring EGFR-activating mutations.[6] [7] Erlotinib is also authorized after failure of previous chemotherapy and as maintenance therapy.[8] [9] In clinical practice evaluation of tumor response is based on changes in tumor size according to criteria proposed by the World Health anization[10] or RECIST criteria.[11] [12] This morphological evaluation may lead to underestimation of the efficacy of cytostatic therapeutic agents such as erlotinib that stabilize the disease in non-mutated patients whereas conventional cytotoxic drugs induce shrinkage of tumor dimensions in the case of tumor response. NSCLC tumor size evaluation can also be difficult due to atelectasis of normal lung. The major limitations to morphological imaging methods are their inability to assess response to therapy at an early stage and their inability to identify cancer in residual masses after treatment. In patients with NSCLC [18F]FDG-PET has been recognized as an adequate staging tool[13] [14] and several studies also suggest that the standardized uptake value (SUV) has a prognostic value in NSCLC.[15] [16] The value of SUV for evaluation of tumor response to targeted therapy is currently being investigated. We designed a preliminary study to evaluate tumor response in NSCLC patients eligible for erlotinib therapy. The aim of this prospective study was to determine whether [18F]FDG-PET/CT performed several days after starting erlotinib therapy could predict tumor response defined by RECIST 1.1 criteria and [18F]FDG-PET/CT after 8 weeks of treatment. Materials and Methods Patients Twelve consecutive eligible patients with stage IIIA to IV NSCLC (7 adenocarcinomas 3 large cell carcinomas 2 squamous cell carcinomas) in whom erlotinib therapy was indicated were studied at the Angers University Hospital France. Screening for EGF receptor mutations was carried out (patient characteristics are shown in ). Eligibility criteria were: histologically or cytologically proven NSCLC; unresectable stage III/IV disease or recurrent disease after surgery; age over 18 years; measurable disease according to RECIST 1.1 criteria; Eastern Cooperative Oncology Group (ECOG) performance status between 0 to 2; adequate bone marrow function liver function and renal function. Patients were not included if they had previous lung diseases such as interstitial pneumonitis or lung fibrosis identified by chest Computed Tomography (CT) scan or diabetes mellitus that could artefact PET imaging. Life expectancy was predicted to be longer than 12 weeks. Erlotinib was administered orally in a dosage of 150 mg/day on an empty stomach until clinical disease progression unacceptable toxicity or patient refusal. The medical ethics committee of the CHU of Angers approved the study protocol. All patients gave informed written consent before inclusion according to local medical ethical committee regulations and in accordance with the guidelines established by the World Medical Association Declaration of Helsinki. .0087629.t001 Clinical characteristics of the study population. Patients Male 6 (50) Female 6 (50) Total 12 (100) Histology Adenocarcinoma 7 (58) Large cell carcinoma 3 (25) Squamous cell carcinoma 2 (17) Clinical stage IIIA or IIIB 2 (17) IV 10 (83) Smoking status Current 5 (42) Former 2 (17) Never 5 (42) EGFR mutation status Presence 2 (17) Absence 10 (83) Previous chemotherapy Yes 10 (83) No 2 (17) Size of primary tumor (cm) 1.0€“2.0 4 (33) 2.1€“3.0 3 (25) 3.1€“5.0 5 (42) >5.1 1 (8) Metastasis Lymph nodes 12 (100) Lung 4 (33) Liver 2 (17) Bone 4 (33) Adrenal glands 0 Work Plan (study design) [18F]FDG PET/CT imaging Three [18F]FDG PET/CT scans were planned: PET1 before starting therapy PET2 within 2 weeks after starting therapy and a third [18F]FDG PET/CT scan (PET3) 8 weeks after starting erlotinib therapy. PET/CT examinations were obtained in 2D mode from the vertex to mid-thighs (5 minutes of emission scan per bed position with an average of 7 bed positions at 15 cm intervals) (Discovery-ST GE Healthcare France). Patients were instructed to fast for at least 6 hours prior to scanning. Unenhanced CT scan was performed from the skull base to the upper thighs. CT parameters were 120 kVp 100 mAs 0.8 second rotation 3.27 mm slice collimation and Pitch 1.5. CT data were used for attenuation correction and PET images were reconstructed by clinical standard 2D-iterative algorithm (ordered subset expectation maximization using 4 iterations and 16 subsets; zoom 100%; image matrix size: 128—128; and Gaussian post-smoothing of 5 mm in full width at half maximum). No corrections for partial volume effect lean body mass or blood glucose levels were applied. Conventional evaluation Conventional staging and follow-up were performed according to standards of care.[11] [12] Conventional evaluation included at least clinical examination plus CT scan performed before (CT1; 7±6 days) and 8 weeks after (CT2; 58±8 days) starting erlotinib therapy. None of the patients underwent additional CT scanning during the 2 weeks after starting erlotinib therapy. Chest abdomen and pelvis CT scans (Brillance 64 PHILIPS Medical System® France) were acquired from the lung apex to the symphysis pubis after an intravenous embolus of 130 mL of iodinated contrast agent (Xenetix350®). Helical scanning parameters were 130 kVp 120 mAs 1 second rotation 4 mm slice collimation 8 mm/s bed speed and 3 mm section width. Image analysis and response evaluation CT data were interpreted by two experienced physicians specialists in thoracic oncology blinded to PET/CT results according to the Response Evaluation Criteria in Solid Tumors (RECIST 1.1 criteria[12]) by comparison of baseline CT scan (CT1) and final CT scan (CT2). Therapeutic response evaluation was defined as: 1) complete response (CR: disappearance of all target lesions); 2) partial response (PR: at least 30% decrease in the sum of the longest diameter of five target lesions); 3) progressive disease (PD: at least a 20% increase in the sum of the longest diameter of five target lesions); and 4) stable disease (SD: neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD). Patients were then classified in the progressive disease (P) group or the non-progressive disease (NP) group including CR PR and SD therapeutic response. [18F]FDG PET interpretation was performed on an Imagys® workstation (Keosys Saint-Herblain France) qualitatively and semi-quantitatively by two experienced nuclear medicine physicians blinded to clinical and conventional evaluation results. Any focus of increased [18F]FDG uptake over background not located in areas of normal [18F]FDG uptake and/or [18F]FDG excretion was considered to be positive for tumor. For semi-quantitative analyses of [18F]FDG uptake 3D regions of interest (VOIs) were placed over all lesions considered to be positive for tumor by using Imagys® software (Keosys France). The maximum standardized uptake value (SUVmax) was calculated using the single hottest pixel inside the tumor VOI. SUV peak was also calculated using a 1.2 cm diameter spherical VOI containing the SUVmax. Bone lesions were not taken into account as they were considered to be non-measurable lesions. For patients with more than one tumor lesion the sum of SUVmax and SUVpeak were calculated and used for evaluation of changes between PET1 and PET2. PET measurements were performed in up to a maximum of five measurable target lesions. All SUVs were normalized to the injected dose and patient body weight. The percentage changes in SUV between PET1 and PET2 were finally calculated as follows: ?SUV?=?(SUV1?SUV2)/SUV1. The same protocol was used for PET1 and PET3. Statistical analysis Data are expressed as mean±SD excepted for survival data that were expressed as the median. The primary endpoint of the study was comparison of changes in tumor [18F]FDG uptake on PET2 versus PET1 PET3 versus PET1 and subsequent CT scan evaluation at 8 weeks after initiation of erlotinib therapy. Friedman test was used for non-parametric comparison of repeated measures. The secondary endpoints were to determine the Receiver Operating Characteristic (ROC) analysis for [18F]FDG changes with regard to predicting response to erlotinib therapy. The relationship between metabolic response (patients stratified according to the median value of SUV variations) and clinical response was analyzed by Fisher's exact test. Progression-free survival (PFS) and overall survival (OS) were determined by standard Kaplan-Meier survival analysis and between-group comparison was performed by log-rank test. PFS was defined as the time interval from the date of enrolment in the study until the first signs of progression. OS was calculated from the date of enrolment until death from any cause. All analyses were performed using Graphpad prism version 4.0 b 2004 (Graphpad Software San Diego CA). The limit of significance was set at 0.05. Results Population Twelve eligible patients with NSCLC 6 women (50%) and 6 men (50%) with a mean age of 60±13 years were included. Two patients presented tumors harboring an activating Epidermal Growth Factor Receptor mutation (2573T>G substitution (p.Leu858Arg) in exon 21 in one patient; deletion (L747_E749del) in exon 19 in the other patient). Patient characteristics are described in . The median duration of erlotinib therapy was 75 days. Due to rapid progression and death PET3 and CT3 could not be performed in 2 patients. Tumor 18F-FDG uptake The three [18F]FDG PET/CT scans were acquired as follows: PET1 5±4 days before starting therapy PET2 9±3 days after starting therapy and PET3 60±6 days after starting erlotinib therapy. Scanning started 68±17 min (PET1) 71±16 min (PET2) and 64±13 min (PET3) after [18F]FDG injection of 271±53 MBq (PET1) 270±61 MBq (PET2) and 263±54 MBq (PET3). Blood glucose level was less than 1.5 g/L for all PET examinations i.e. 1.1±0.1 g/L for PET1 1.1±0.2 g/L for PET 2 and 1.1±0.2 g/L for PET3. Non-parametric Friedman tests did not show any significant difference between PET1 PET2 and PET3 for FDG uptake time injected FDG dose or blood glucose. Fifty-five lesions were described on PET1 before treatment and 45 lesions were defined as target lesions for PET evaluation of response to treatment (up to five most hypermetabolic lesions per patient; mean 3.8 lesions/patient). The mean tumor SUVmax of the most [18F]FDG€“avid lesion (SUVmax) was 10.0±4.7 for PET1 and did not vary significantly over time with a mean of 10.1±6.6 for PET2 and a mean of 9.1±5.6 for PET3 (P?=?0.97). The SUVpeak was 8.6±4.3 for PET1 8.1±5.4 for PET2 and 7.1±4.6 for PET3 and did not vary over time (P?=?0.60). No variation over time was observed for the sums of SUV. The mean sum of tumor SUVmax of all target lesions was 30.1±19.5 for PET1 27.5±17.7 for PET2 and 28.3±22.4 for PET3 (P?=?0.83). Sums of SUVpeak of all target lesions were 22.7±14.3 for PET1 20.6±13.4 for PET2 and 22.2±18.6 for PET3 (P?=?0.44). [18F]FDG-PET response versus conventional evaluation CT scan data were interpreted by chest physicians blinded to PET/CT scan results (). Evaluation of response to treatment according to RECIST 1.1 criteria demonstrated 7 patients with progressive disease (group P) and 5 patients with non-progressive disease (group NP) including 4 cases of stable disease (SD) and 1 partial response (PR). .0087629.t002 CT and PET assessments of response rates OS and PFS. Patient PET2 versus PET1 PET3 versus PET1 RECIST 1.1 Evaluation PFS OS New lesion ? SUVmax * ? SUVpeak * ? SUVmax * ? SUVpeak * Response to Treatment Progressive (P) or not (NP) days days on PET3 #1 ?21.6 ?17.6 18.6 ?1.5 SD NP 267 915 ? #2 25.9 26.9 70.3 77.4 PD P 57 316 + #3 9.0 7.6 23.4 23.3 PD P 216 447 + #4 ?18.6 ?15.0 ?3.2 ?2.6 PD P 67 414 + #5 ?20.3 ?11.1 42.1 51.1 PD P 53 152 + #6 ?56.7 ?59.9 ?72.1 ?70.6 PR NP 190 296 ? #7 ?22.0 ?26.0 ?31.3 ?24.3 SD NP 727 1249 + #8 ?32.0 ?25.1 3.9 ?3.9 SD NP 317 1146 ? #9 16.4 7.8 ?5.4 ?10.8 SD NP 77 359 ? #10 2.1 4.4 MD MD PD P 37 92 MD #11 36.1 20.0 30.3 25.7 PD P 104 734 ? #12 ?7.2 ?10.5 MD MD PD P 61 71 MD * For patient with more than one tumor lesion the sum of SUVmax and of SUVpeak were calculated and used for the evaluation of changes between PET1 and PET2 (or between PET1 and PET3). Missing data are indicated as MD. On ROC analysis the AUC for prediction of non-progressive disease by PET2 was 0.86 (95% CI 0.62 to 1.1; P?=?0.04) corresponding to a maximum specificity of 0.80 and sensitivity of 0.86 for non-progressive disease at a cut-off of 21.6% reduction in SUVmax (Figure 1) and a positive predictive value (PPV) of 0.86 a negative predictive value (NPV) of 0.80 an accuracy of 0.83 and a maximum Youden index of 0.65. The use of this SUVmax cut-off value correctly classified 11/12 patients (7 with true progressive disease (Figures 2 and 3); 4 with true non-progressive disease (Figures 4 and 5); 1 with false progressive disease (). Non-progression after 2 months of treatment was significantly more frequent in patients with an early decrease in SUVmax of 21.6% or more (P?=?0.01 Fisher's exact test). The only misclassified patient (patient #9 false progressive disease on PET2 versus PET1) displayed a 16.4% increase of SUVmax but metabolic progression was not confirmed on PET3 with a 5.4% decrease of SUVmax compared to PET1. Similar results were observed for SUVpeak as non-progressive disease after 2 months of treatment was significantly more frequent in patients with a decrease in SUVpeak of at least 17.6% on PET2 (P?=?0.01 Fisher's exact test). Similar results were also obtained in terms of AUC sensitivity specificity PPV NPV and accuracy and with the same classification of patients (7 with true progressive disease; 4 with true non-progressive disease; 1 with false progressive disease). .0087629.g001 Figure 1 Percentage change in SUVmax on 18F-FDG PET/CT (cut-off: ?21.6%) within 2 weeks of starting erlotinib therapy in relation to conventional imaging response. Each red or green bar represents a patient NP or P respectively. .0087629.g002 Figure 2 Example of a progressive patient on PET (mP) and conventional imaging. Progressive patient with right upper lobe NSCLC associated with mdiastinal lymphadenopathy lung and bone metastases (patient #2). Sum of the SUVmax of the 5 most hypermetabolic lesions (2 lung lesions 2 mediastinal lymph nodes one hilar lesion) were 35.2 44.3 (+26%) and 59.9 (+70%) for PET1 PET2 (% versus PET1) and PET3 (% versus PET1) respectively. Based on a SUVmax cut-off value of ?21.6 the patient was classified as mP on PET2 in accordance with RECIST evaluation on CT scan (performed 57 days after starting erlotinib). mP was confirmed on PET3 with the appearance of a new lesion (subcarinal adenopathy) and a 70% increase of SUVmax. .0087629.g003 Figure 3 New subcarinal adenopathy on PET3 (same patient as Figure 2). .0087629.g004 Figure 4"

recently the current pandemic of coronavirus disease covid characterized by a pulmonary infection in humans is caused by a novel virus strain from family coronaviridae known as severe acute respiratory syndrome coronavirus sarscov2 the previous outbreak of severe acute respiratory syndrome sars in “ and middle east respiratory syndrome mers in has demonstrated the lethality of coronaviruses when they cross the species barrier and infect humans so far six coronaviruses infecting humans have been identified and the novel coronavirus is the seventh one described to date as being responsible for a respiratory infection sarscov and merscov and the new sarscov2 belong to the betacoronavirus family [“] the coronaviruses have the largest genome around k among the rna viruses sarscov2 was closely related from “ identity to two batderived severe acute respiratory syndrome sarslike coronaviruses batslcovzc45 and batslcovzxc21 but it was more distant from sarscov from “ and merscov about furthermore the performed bioinformatic analysis showed that the nucleotide sequence of sarscov2 is similar to those of other betacoronaviruses with nucleotide identities of ‰¥ there are currently no effective licensed therapies for human coronaviruses hcov infections and existing treatment strategies are generally limited to symptomatic treatment and supportive care email addresses kuzunovahqtchaikapharmacom k uzunova efilipovahqtchaikapharmacom e filipova vpavlovahqtchaikapharmacom corresponding author v pavlova tvekovmuplevenabvbg t vekov 101016jbiopha2020110668 received may received in revised form august accepted august biomedicinepharmacotherapy1312020110668availableonline24august2020075333222020theauthorspublishedbyelseviermassonsasthisisanopenaccessundertheccbyncndlicensehttpcreativecommonslicensesbyncnd40 0csuch as solidarity who recovery k uzunova in the absence of a specific treatment for this novel virus the effort of researchers is focused on understanding and controlling the disease and on preventing and controlling the replication and spread of the virus to devise therapeutic strategies to counteract the sarscov2 infection numerous potential treatment options are being evaluated in ongoing clinical trials many antiviral and immunological treatments being investigated against coronaviruses are summarized by who in landscape analysis of therapeutics as of march the realtime dashboard of completed ongoing and planned clinical trials for covid includes drugs and promising therapies such as remdesevir lopinavirritonavir hydroxychloroquine il6 inhibitors tocilizumab and sarilumab convalescent plasma therapy stemcell transfusion vaccine candidates traditional chinese medicines which are of top interventions of the presented network among them remdesivir an analogue of adenosine seems to have a more promising future due to proven in vitro and in vivo antiviral efficacy till the beginning of june promising therapies involving lopinavirritonavir and chloroquine or hydroxychloroquine were part of treatment guidelines in many countries but currently they are excluded from covid19 treatment protocols because of uncertainty regarding their risks and benefits and it is recommended that they should be used only in the context of clinical trials [“] in spite of its known in vitroin vivo efficacy and safety profiles some trials evaluating these drugs for covid19 infection treatment uk ntc04381936 and discovery inserm ntc04315948 discontinued hydroxychloroquine and lopinavirritonavir arms the interim trial results showed that hydroxychloroquine and lopinavirritonavir produced little or no reduction in the mortality of hospitalized covid19 patients when compared to standard of care nevertheless some countries worldwide continue to recommend chloroquinehydroxychloroquine as a treatment option [“] the existing drugs that target viral proteins associated with enzymatic activities or blocking viral replication machinery or host proteins involved in viral life cycle regulating the function of the immune system or other cellular processes in host cells have great potential and are available on the market our review aims to highlight the potential molecular mechanisms of the therapeutic options available for the cure of other health conditions and their repurposing for the treatment of this novel coronavirus sars cov2 selected treatments of sarscov2 remdesivir gs5734 “ polymerase inhibitor deltacoronavirus genus pdcov which have the most divergent rdrp of known cov as compared to sars and merscov an in silico test of the covid19 rdrp built model suggested the effectiveness of remdesivir as a potent drug sarscov and sarscov2 both belong to the betacoronaviruses of the b lineage and the rdrp amino acid sequences of the two viruses are identical whereas merscov belongs to the betacoronaviruses of the c lineage and is only identical with sarscov2 another in vitro and in vivo proof came from sheahan who examined if gs5734 could inhibit replication of sarscov and mers cov in primary human airway epithelial hae cell cultures they found out a dosedependent reduction in replication with average ic50 values of μm sarscov and μm merscov moreover the compound inhibits a broad range of diverse cov including circulating human zoonotic bat cov and prepandemic zoonotic cov with both prophylactic and therapeutic 1dpi dosing of gs5734 a reduction in replication below a diseasecausing threshold in mouse model of sars cov pathogenesis was demonstrated therapeutic gs5734 substantially reduced the sarscov induced weight loss in infected animals and significantly suppressed virus lung titers p thus demonstrating that therapeutic administration of gs5734 can reduce disease and suppress replication during an ongoing infection furthermore remdesivir has the potential to block sarscov2 infection in vitro at lowmicromolar concentration and in treatment of merscov and sarscov infections in vivo it demonstrated a significant improvement of pulmonary pathology in mice the rnadependent rna polymerase rdrpmediated mechanism of cov inhibition by gs5734 is proven even in the setting of intact exoribonuclease exonmediated proofreading using the model coronavirus murine hepatitis virus mhv it was demonstrated that gs5734 dramatically inhibited viral replication and viral rna synthesis in wildtype wt virus while an nsp14 exon mutant lacking proofreading demonstrated increased susceptibility to gs5734 45fold more active this suggests that gs5734 is recognized at least partially by a functional exon but that the exon activity is not sufficient to prevent potent inhibition of cov replication the results provide strong evidence that rdrp is the target for remdesivir and support the hypothesis that gs5734 directly inhibits viral rna synthesis the mechanism of inhibition of rdrp of merscov by remdesivir was studied by gordon et al they coexpressed the merscov nonstructural proteins nsp5 nsp7 nsp8 and nsp12 rdrp in insect cells as a part of a polyprotein coexpression of the mers nsp5 protease with nsp7 nsp8 and nsp12 in insect cells yielded a stable complex composed of nsp8 and nsp12 the triphosphate form of the inhibitor rdvtp is utilized as a substrate and competes with its natural counterpart atp and they observed that incorporation of the nucleotide analogue was significantly more efficient once added into the growing rna chain the inhibitor does not cause immediate chain termination the presence of the ²hydroxyl group allows the addition of three more nucleotides until rna synthesis is arrested at position i3 therefore the main possible mechanism of action is delayed rna chain termination recently the same authors obtained almost identical results with sarscov merscov and sarscov2 rdrps they provided evidence that all three coronavirus rdrp complexes terminated rna synthesis at position i3 almost all viruses encode polymerases in the central steps of replication and transcription therefore polymerases are becoming the most attractive and suitable targets for antiviral development there are two major types of polymerase inhibitors i nucleoside and nucleotide substrate analogs and ii allosteric inhibitors nucleoside analogs are first triphosphated by the host cell to produce the active inhibitor and then act as an inhibitor by competing with the natural nucleoside triphosphates and terminating the growing viral nucleic acids to date most of the approved antiviral drugs for antihiv therapy utilize this mechanism remdesivir is a nucleotide analogue with a proved mechanism of action as an inhibitor of rnadependent rna remdesivir rdv is an investigational compound with a broad spectrum of antiviral activities against rna viruses including sarscov and merscov gs5734 was originally developed for the treatment of the ebola virus disease gs5734 the single sp isomer of the 2ethylbutyl lalaninate phosphoramidate prodrug effectively bypasses the rate limiting first phosphorylation step of the nuc nucleoside ribose analogue the mechanism of action of nuc requires intracellular anabolism to the active triphosphate metabolite ntp which is expected to interfere with the activity of viral rnadependent rna polymerases rdrp gs5734 selectively inhibits ebola virus replication by targeting its rdrp and inhibiting viral rna synthesis following efficient intracellular conversion to ntp in nonhuman primates this compound shows a broad spectrum of antiviral activities against several rna viruses including respiratory syncytial virus rsv junín virus lassa fever virus and middle east respiratory syndrome virus but was inactive against alphaviruses or retroviruses furthermore remdesivir dosedependently inhibits endemic human cov229e and covoc43 replications which typically cause upper respiratory infection in children but can cause more severe lower respiratory infection in adults with underlying respiratory conditions ie asthma copd and the elderly as well as a member of the biomedicinepharmacotherapy13120201106682 0c lopinavirritonavir “ protease inhibitor the proteases encoded by most viruses play a crucial role in the viral life cycle the protease inhibitors pis bind competitively to the substrate site of the viral protease this enzyme is responsible for the post translational proteolysis of a polyprotein precursor and the release of functional viral proteins allowing them to function correctly and individually in replicationtranscription and maturation inhibition results in the production of immature virus ps coronavirus proteases of which there are two in sarscov a papainlike cysteine proteinase plpro nsp3 and a 3clike proteinase 3clpro or mpro nsp5 and three in several other coronaviruses cleave the orf1 polypeptide as it is translated enabling the formation of the viral replication complex the substratebinding pockets are highly conserved among cov 3clpro suggesting the possibility for a widespectrum inhibitor design targeting this region in the 3clpro of all covs it is postulated that the 3clproinhibiting activity of lopinavirritonavir contributes at least partially to its anticov effects in silico binding studies of the drugs using the identified crystal structure of mpro and employing the hex program to conduct the docking of the ligands to the sarscov main proteinase revealed that lopinavir and ritonavir could basically bind to the active site of sars main proteinase but the efficacy of lopinavirritonavir was predicted to be poor according to the latest report of the structure of 3clpro from sarscov2 pdb code 6lu7 and the available structure of 3clpro from sarscov pdb code 1uk4 the two main proteases differ by only amino acids comparing ligand binding free energies for the main proteases has confirmed that good binders for sarscov are in general and sarscov k uzunova polymerases this mechanism is probably involved in an antiviral activity against sarscov2 biochemical data provided by gordon suggested a unifying mechanism of inhibition of sarscov merscov and sarscov2 fig and future emerging covs may be similarly susceptible to the inhibition by remdesivir comparable replication with also good binders for sarscov2 3clpro protease inhibitors a class of drugs best known for success against hiv block the final step of virion assembly in the treatment of human immunodeficiency virus infection with proven efficacy the combination of lopinavir with ritonavir is widely used as a boosted protease inhibitor in the treatment of hiv infection because of low oral bioavailability of lopinavir and its extensive metabolism by the cyp3a4 isoenzyme lopinavir needs to be coadministered with ritonavir to achieve drug concentrations high enough to inhibit viral replication [ “] so far the reported results from studies in different cell lines animal models and patients for lopinavirritonavir are not so convincing in their inhibition action in human coronaviruses screening the library of fdaapproved drugs for antimerscov activity in cell culture has identified four compounds chloroquine chlorpromazine loperamide and lopinavir which inhibit merscov replication effective concentration ec50 3cid0 μmoll in vitro lopinavir inhibited mers cov efficacy ec50 μm and a maximal protective effect were observed at a dose of μm it was previously shown that lopinavir but not ritonavir inhibit sarscov chymotrypsinlike 3cl protease at the concentration of μm moreover it was suggested that lopinavir blocks a postentry step in the merscov replicative cycle in vitro the detectable antiviral activities of ribavirin rimantadine lopinavir and baicalin were shown by using the frhk4 cell line and in vero e6 cells infected with sarscov2 lopinavir inhibit replication with ec50 at μm during the sars outbreak treatment with lopinavir in combination with ritonavir was explored with some success in nonrandomized clinical trials patients with sarscov treated with lopinavirritonavir showed a progressive decrease of viral load and reduction of the composite adverse outcome at day recently the antiviral activity of remdesivir and ifn was found to be superior to that of lopinavirritonavirifn against merscov in vitro and in vivo the efficacy of lopinavirritonavir with or without ribavirin is evaluated in sarscov2 patients under randomized control trials currently it was demonstrated that this combination has no benefits in adult patients with severe covid19 although protease inhibitors are a common class of medication used in the treatment of hiv1 infection their efficacy in human coronavirus infections is not convincing moreover several antihiv pis are also known to influence other intracellular pathways it was demonstrated that hiv protease inhibitors indinavir saquinavir and lopinavir independently from any viral infection can hinder lymphocyte apoptosis by influencing mitochondrial homeostasis in view of the weak antiviral activity of protease inhibitors further studies should be done to ascertain whether the clinical benefit could be attributed to their antiapoptotic rather than their antiviral activity hence even if the molecular target of lopinavirritonavir is the main protease 3clpro in sarscov2 infected cells fig there are no biochemical and molecular studies confirming the interaction and associating this with clinical efficacy of the protease inhibitor chloroquinehydroxychloroquine chloroquine chq was introduced into clinical practice in as a prophylactic treatment for malaria hydroxychloroquine hcq differs from chloroquine by the presence of a hydroxyl group at the end of the side chain the nethyl substituent is hydroxylated currently chq and its hydroxyl form hcq are used as antiinflammatory agents for the treatment of rheumatoid arthritis lupus erythematosus and amoebic hepatitis in addition chq has been studied as a potent antiviral agent against hiv1aids [“] hcov229e sarscov [ ] influenza a h5n1virus influenza a and b and many other rna and dna viruses many recent reports and published studies suggested that chq and hcq were associated with reduced fig inhibition of viral infection by lopinavirritonavir and remdesivir biomedicinepharmacotherapy13120201106683 0ck uzunova progression of the covid19 and decreased duration of the symptoms [“] there are in fact overall more than trials currently underway around the world on its impact either as a prophylactic or treatment for covid19 it is noteworthy that the usefulness of hydroxychloroquine and chloroquine is intensively investigated chloroquine was found to exert an antiviral effect during pre and postinfection conditions suggesting to have both prophylactic and therapeutic advantages timeofaddition assay demonstrated that chq functioned at both entry and postentry stages of the sarscov2 infection in vero e6 cells however it did not reduce viral replication in sarscov infected mice hydroxychloroquine is significantly more potent than chq in vitro ec50 values and μm respectively and has a lower potential for drugdrug interactions than chloroquine pharmacokinetic models demonstrate that hydroxychloroquine sulfate is significantly superior days in advance to chloroquine phosphate in inhibiting sarscov2 in vitro and was demonstrated to be much less toxic than chq in animals on the other hand data presented by liu demonstrated that the antiviral effect of hcq against sarscov2 infection was comparable with chq in vitro cc50 μm and μm for chq and hcq respectively moreover they suggested that both chq and hcq blocked the transport of sarscov2 from early endosomes ees to endolysosomes els and caused noticeable sizemorphological changes in ees and els they surmised that endosome maturation might be blocked at intermediate stages of endocytosis resulting in failure of further transport of virions to the ultimate releasing site hydroxychloroquine shares the same mechanism of action as chloroquine apart from the probable role of chq and hcq as antiviral agents their mechanisms of action are not fully understood and it was demonstrated that they have multiple effects on mammalian cells ace2 is known to be a cell receptor for sarscov the high similarities of the amino acid sequences and predicted protein structures of the receptorbinding domain of sarscov2 and sarscov suggest that sarscov2 may efficiently use human ace2 as a receptor for cellular entry and employ the cellular serine protease tmprss2 for s protein priming zhou confirmed that sarscov2 used the ace2 receptor to enter cells and did not use other coronavirus receptors such as aminopeptidase n apn and dipeptidyl peptidase dpp4 so the primary mechanism by which cell infection is prevented by these drugs may be at the stage of binding with the surface receptor and endosomemediated viral entry two independent in vitro studies confirmed that chq inhibits the replication of the sarscov chloroquine inhibits the early stage of sarscov replication in vero e6 cells with a effective concentration of ± μml the antiviral activity of chq was indicative at the time point at virus attachment or penetration vincent established that the drug might interfere with terminal glycosylation of the cellular receptor ace2 when chq was added prior to infection the impairment of terminal glycosylation of ace2 may result in reduced binding affinities between ace2 and sarscov spike protein and negatively influence the initiation of sarscov infection when chq or nh4cl were added after infection these agents could rapidly raise the ph and interrupt ongoing fusion events between the virus and endosomes thus inhibiting the infection on the basis of in vitro experiments they suggested that the primary mechanism by which infection was prevented was the poor affinity of sarscov spike protein to underglycosylated ace2 in vitro studies with hiv infected cells also identified that inhibition of glycosylation might be a possible mechanism of action of chq chq inhibits hiv replication at a postintegration stage resulting in the production of immature virions it was demonstrated that the sole mechanism explaining the antihiv activity of chq was a decrease in the infectivity of the newly produced virus associated with defective production of the heavily glycosylated 2g12 epitope of gp120 according to in vitro results the antiretroviral effects of chq are attributable to the inhibition of viral p glycosylation these effects appeared to be specific since the chq concentrations effective in vitro neither affected any other step in hiv1 replication nor were cytotoxic thus there is direct evidence that chq is an inhibitor of glycosylation of gp120 and these alterations may be responsible for the decreased infectivity of hiv grown in the presence of chloroquine when added after the initiation of infection these drugs might affect the endosomemediated fusion and subsequent virus replication sarscov pseudoviruses may enter cells via receptordependent clathrin and caveolaeindependent phsensitive endocytosis likely through a process involving lipid rafts a later study however suggests that the entry of coronaviruses into the host cells occurs through clathrinmediated endocytosis murine hepatitis virus mhv a prototypic member of the cov family requires trafficking to lysosomes for proteolytic cleavage at the fp proximal position of its spike s protein membrane fusion to occur many authors indicated that s protein cleavage is an important step for fusion activity and subsequent internalization of the sarscov virus genome into cells [“] adding chq prior to infection results to inhibition of endosome maturation and strongly decreased mhv infection and fusion which was not observed when the drug was added at hpi indicating that the compound mainly affects mhv entry chloroquine is a weak base that is known to increase the ph of lysosomal and transgolgi network tgn vesicles leading to the dysfunction of enzymes necessary for proteolytic processing and posttranslational modifications of newly synthesized viral proteins chloroquine is able to prevent the processing of prm protein to m protein in flavivirusinfected mammalian and mosquito cells by raising the ph of the postgolgi vesicles in which this cleavage occurs as a result virions from infected cells which had been treated with acidotropic amines late in the virus replication cycle contained prm protein rather than m protein and this reduced the infectivity of the virus the chloroquinemediated rise in endosomal ph modulates iron metabolism in a variety of cell types decreasing in intracellular concentration of iron affects the function of several cellular enzymes involved in pathways leading to the replication of cellular dna and to the expression of different genes [“] autophagy is a lysosomedependent degradative pathway chq and its analogue hcq are known clinically relevant autophagy inhibitors chq is a weak base that inhibits lysosomal acidification which prevents the fusion of autophagosomes with lysosomes and subsequent autophagic degradation inhibition of autophagy with chq stimulates superoxide generation ubiquitinconjugated protein accumulation and apoptosis in a colon cancer xenograft model chq treatment clearly inhibited autophagy in mouse lung and efficiently ameliorated acute lung injury and dramatically improved the survival rate in mice infected with live avian influenza a h5n1 virus h5n1 virusinduced autophagic cell death in alveolar epithelial cells through a pathway involving the kinase akt the tumor suppressor protein tsc2 and the mammalian target of rapamycin and autophagyblocking agents might be useful as prophylactics and therapeutics against infection of humans by the h5n1 virus furthermore prentice suggested that authophagy was induced by the coronavirus mouse hepatitis virus mhv and was required for formation of double membranebound mhv replication complexes which significantly enhanced the efficiency of replication replication of the virus was impaired in atg5 knockout embryonic stem cells the same authors also examined the sarscovs and found out similar colocalization of the key viral replication proteins with endogenous lc3 a protein marker for autophagosome it could be assumed that autophagy inhibitors like chq could inhibit virus replication at present the exact role of autophagy in cov infection remains debatable and there is much evidence suggesting that the endocytic pathway plays a key role in mediating viral entry for many covs including sarscovs merscovs and possibly sarscov2 the antiinflammatory properties of chqhcq should also be considered several studies have suggested that multiple an failure biomedicinepharmacotherapy13120201106684 0chas not yet been identified in sarscov2 infected patients and probably multiple pathways could be involved fig conclusion the sarscov2 is the cause of the coronavirus disease covid19 that has been declared a global pandemic by the world health anization who in despite some clinical characteristics that differentiate covid19 from sarscov merscov and seasonal influenza the pathogen sarscov2 has the same phylogenetic similarity to sarscov and mers cov most of the encoded proteins exhibited high sequence identity between sarscov2 and the related batderived coronaviruses batslcovzc45 and batslcovzxc21 a notable difference was a longer spike protein encoded by sarscov2 compared with the bat sarslike coronaviruses sarscov and merscov in addition sarscov2 was distinct from sarscov in a phylogeny of the complete rnadependent rna polymerase rdrp gene moreover the receptorbinding domain of sarscov2 which directly engages the ace2 receptor for cell entry was more closely related to those of sarscovs “ amino acid identity since the outbreak researchers have released many agents that could have potential efficacy against covid19 there is currently no clinically proven specific antiviral agent available for sarscov2 infection like sarscov and merscov certain agents like chloroquine hydroxychloroquine lopinavirritonavir and remdesivir are being used in ongoing clinical trials all over the world with hopes to further delineate their role in the treatment and prophylaxis of covid19 furthermore due to their availability and using for decades and proven safety records it is reasonable to suggest that they may be appropriate for treatment of covid19 remdesivir an adenosine analogue with wellstudied mechanism of action in cov infections can target the rnadependent rna polymerase and block viral rna synthesis and has been a promising antiviral drug antiviral studies in cell culture and animal models the available human safety data as well as the clear mechanism of action characterize rdv as a directacting antiviral since some authors found that lopinavir“ritonavir treatment did not significantly accelerate clinical improvement hence antiviral effects as an inhibitor of the sarscov main 3cl protease should be further investigated although chq and hcq are wellknown drugs for the treatment of k uzunova observed in fatal cases are most likely associated with not only the direct viral infection and destruction of susceptible cells eg endothelial cells but also the effects of proinflammatory cytokines chemokines and other mediators released from infected and activated cells such as monocytes and macrophages the clinical worsening of individuals with sars in week is apparently unrelated to uncontrolled sars coronavirus replication but may be related to immunopathological damage another study reveals that the presence of viral elements within endothelial cells and the accumulation of inflammatory cells led to endotheliitis in several ans as a direct consequence of viral involvement and to host inflammatory response moreover chq has immunomodulatory effects suppressing the productionrelease of tumour necrosis factorα and interleukin6 which mediate the inflammatory complications of several viral diseases chloroquinehcq was reported to inhibit the production of soluble mature tnf in macrophage cell line inhibit tnfα receptor in human histocytic u937 cells inhibit tnfα ifnγ and il6 in peripheral blood mononuclear cells pbmc reduce tnfα production and lipopolysaccharide lpsinduced il1 release in human monocytic cells it is suggested that chq exerts antiinflammatory and immunomodulatory effects predominantly by pretranslational and nonlysosomotropic mechanisms chloroquineinduced inhibition of tnf and il6 production is not mediated through a lysosomotropic mechanism and chloroquine probably acts on tnf secretion by disrupting iron homeostasis besides its antiviral activity and due to its suppressive effects on the productionrelease of tnfα and interleukin chqhcq may be effectively used in the treatment of viral infections characterized by symptoms associated with inflammatory processes andor immunehyperactivation antiinflammatory effects of chq remain poorly understood regulation of proinflammatory cytokines chq can also act on the immune system through cell signaling chq inhibits the activation of p38 mapk in hcov229einfected cells and evokes the activation of erk independently of infection these results suggested that chq may inhibit cov replication by suppressing the p38 activation additionally chq strongly inhibited phosphorylation of mitogenactivated protein kinase mapk p38 and to a lesser extent cjun nterminal kinase and extracellular signalregulated kinase ½ chloroquine could also inhibit innate immune responses trough downregulation of tlr9 signaling pathways requiring endocytosis and acidification of endosomes within plasmacytoid dendritic cells pdcs and act as novel antagonists to chemokine receptor cxcr4 that suppress pancreatic cancer cell proliferation on the other hand another hypothesized mechanism of chq is via the inhibition of antigen degradation and improving the crosspresentation efficiency of dcs in vitro in vivo evidence suggested that a short course of treatment with chq followed by a booster dose of a soluble antigen immunization can efficiently enhance human cd8 t cell responses and single vaccination with inactivated influenza virus combined with chloroquine treatment elicits a higher t cell immunity in mice regulation of nlrp3 inflammasome activation may offer a promising therapeutic approach by inhibiting or slowing down the process of acute respiratory distress syndrome hcq is a known nlrp3 inhibitor and its potential clinical effectiveness is certainly based on the downregulation of il1 expression the major proinflammatory cytokine interleukin1beta il1 is elevated in plasma from hospitalized covid19 patients and its associated signaling pathway seems to drive sarscov2 pathogenicity il1 secretion is primarily initiated by inflamm

LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathwaysXue Wu123 XiaoFeng Li123 Qian Wu4 RuiQi Ma123 Jiang Qian123 Rui Zhang123·Basic Research·1Department of Ophthalmology Eye ENT Hospital of Fudan University Shanghai China2NHC Key Laboratory of Myopia Fudan University Shanghai China 3Laboratory of Myopia Chinese Academy of Medical Sciences Shanghai China4Department of Pathology West China Hospital Sichuan University Chengdu Sichuan Province ChinaCofirst authors Xue Wu and XiaoFeng LiCorrespondence to Rui Zhang Department of Ophthalmology Eye ENT Hospital of Fudan University Fen Yang Road Shanghai China zhangrui936163comReceived Accepted Our research suggests that SNHG15 may play a vital role as a potential marker in UM that predicts poor prognosis Besides GSEA indicates the underlying signaling pathways enriched differentially in SNHG15 high expression phenotype KEYWORDS SNHG15 uveal melanoma the Cancer Genome Atlas pathology prognosis Gene Set Enrichment Analysis1018240ijo20200804Citation Wu X Li XF Wu Q Ma RQ Qian J Zhang R LncRNA SNHG15 predicts poor prognosis in uveal melanoma and its potential pathways Int J Ophthalmol Abstract— AIM To evaluate the role of long noncoding RNA lncRNA SNHG15 and its potential pathways in uveal melanoma UM METHODS The SNHG15 mRNA expression level and corresponding clinicopathological characteristics of patients with UM were obtained from the Cancer Genome Atlas TCGA database and further analyzed The SPSS statistical software package was used for statistical analyses To investigate the potential function of SNHG15 in UM we conducted indepth research on Gene Set Enrichment Analysis GSEA— RESULTS The univariate analysis revealed that the age tumor diameter pathological type extrascleral extension cancer status and high expression of SNHG15 were statistical risk factors for death from all causes The multivariate analysis suggested that the mRNA expression level of SNHG15 was an independent risk factor for death from all causes as was age and pathological type KaplanMeier survival analysis confirmed that UM patients with high SNHG15 expression might have a poor prognosis In addition SNHG15 was significantly differentially expressed in the different groups of tumor pathologic stage metastasis and living status Besides the logistic regression analysis indicated that high SNHG15 expression group in UM was significantly associated with cancer status pathologic stage metastasis and living status Moreover the GSEA indicated the potential pathways regulated by SNHG15 in UM INTRODUCTIONU veal melanoma UM the most common intraocular cancer in adult worldwide[] is a malignant tumor that originates in melanocytes of the choroid plexus ciliary body and iris of the eye At present despite definitive radiotherapy or removal of the primary lesion numerous patients eventually develop metastases and subsequently prognosis is significantly poor[] In addition UM tends to metastasize to liver through hematogenous pathway a distant site relative to their origins in the eye[] There is an incubation period between the enucleation of the primary tumor and the emergence of metastasis which can range from a few months to several decades[] Despite the advancement of UM management there are currently no effective therapy once the metastases occurred[] Therefore close followup and further research on the pathogenesis and novel makers exploration of UM are of great significance for accurate diagnosis appropriate therapy and prognosis prediction Long noncoding RNA lncRNA is a class of noncoding transcripts with a length of larger than nucleotides[] which has been involved widely in biological processes of different cancers including cell cycle apoptosis cell differentiation[] In the development of UM lncRNA is also reported to play a vital role in cell cycle cell proliferation apoptosis invasion and autophagy[] For example silencing of lncRNA PVT1 prevents the development of UM by impairing microRNA173pdependent MDM2 upregulation[] ZNNT1 can suppress Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomathe progression of UM by inducing the expression of crucial autophagy gene[] The lncRNA RHPN1AS1 facilitates the tumorigenesis of UM by influencing cell proliferation and migration[] However the study of vital lncRNAs in UM still remains to be exploredSNHG15 a novel lncRNA located on chromosome 7p13[] is identified to play a key role in many types of human tumors such as osteosarcoma[] papillary thyroid carcinoma[] pancreatic ductal adenocarcinoma[] colorectal carcinoma[] hepatocellular carcinoma[] prostate cancer[] and breast cancer[] To our knowledge the potential impact of SNHG15 on the tumorigenesis of UM seems unclear recently Thus the purpose of this study was to evaluate the pivotal role of SNHG15 in the progression of UM In addition the relationship between SNHG15 expression and clinicopathologic characteristics in UM was preliminarily demonstrated To explore the underlying mechanisms of the biological pathways involved in UM we conducted a research on Gene Set Enrichment Analysis GSEA MATERIALS AND METHODSEthical Approval The study protocol was approved by the Ethics Committee of the Eye ENT Hospital of Fudan University and all procedures were complied with the principles of the Declaration of Helsinki All datasets of our present study were downloaded from an database TCGA so there was no written informed consent from participantsRNASequencing Patient Data and Bioinformatics Analysis The RNASeq gene expression level and clinicopathological characteristics including cases were obtained from the official website of the Cancer Genome Atlas TCGA UM project portalgdccancergov Patients with UM were classified as two groups based on the median SNHG15 expression level cutoff value794 FPKM Finally patients with UM were retained and their clinicopathological characteristics were further analyzed including the detailed information of age gender tumor diameter thickness pathological type extrascleral extension cancer status pathological stage metastasis living status SNHG15 expressionGene Set Enrichment Analysis GSEA is a common bioanalysis used to interpret and analyze microarray and other similar data and to speculate related pathways that can significantly enrich regulatory genes[] Through TCGA UM project we obtained the RNASeq gene expression level of UM patients And the analysis was conducted using GSEA v30 software In this study according to the association with SNHG15 expression the ordered gene list was generated firstly by GSEA Subsequently GSEA was conducted to clarify statistically significant differences between the two groups with high and low SNHG15 expression A total of permutations were performed The SNHG15 expression level was identified as a phenotype label The related pathways statistically enriched in each phenotype were selected with the nominal P005 and an false discovery rate FDR Statistical Analysis The SPSS statistical software package SPSS Inc USA was used for statistical analyses Both the univariate and multivariate analyses using Cox analysis were performed to demonstrate independent prognostic biomarkers for UM patients The survival curve was generated by conducting KaplanMeier method To compare the significant differences in overall survival OS the logrank test was conducted The plot chart was performed to visualize the difference of SNHG15 expression level for diverse variables through Graphpad The relationship between the SNHG15 expression and clinicopathological characteristics were analyzed using logistic regression The median value of SNHG15 expression was selected as the cutoff value P005 was considered statistically significantRESULTSPatient Characteristics The records of primary UM with both RNASeq gene expression level and clinicopathological characteristics were obtained from TCGA database The mean age of UM patients was years old including males and females The mean value of tumor diameter and thickness were and mm respectively In our study cohort the pathological type of UM included epithelioid cell dominant type and spindle cell dominant type of tumors were epithelioid cell dominant and were spindle cell dominant There were cases without extrascleral extension and cases with extrascleral extension The cancer status included tumorfree cases and cases with tumor Pathologic stage II was found in cases and stage IIIIV in cases And of cases had metastases of cases had no metastases Of cases cases died of all causes Survival Outcomes and Multivariate Analysis Prognostic factors of UM were analyzed using univariate and multivariate Cox regression The univariate analysis suggested that high SNHG15 expression was a risk factor for death from all causes Other clinicopathologic variables related to poor prognosis included age tumor diameter pathological type extrascleral extension cancer status Table In a multivariate analysis SNHG15 was an independent risk factor for death from all causes as was age and pathological typeSNHG15 Expression Associated with Clinical Pathological Characteristics A total of UM cases with SNHG15 expression data and clinicopathologic characteristics were analyzed from TCGA KaplanMeier survival analysis 0cTable Prognostic parameters in UM were analyzed using univariate and multivariate Cox regressionDeath from all causesParametersnmeanUnivariate analysisPHR95CIAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelioid cell dominantSpindle cell dominantExtrascleral extensionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVSNHG15HighLowUM Uveal melanomaMultivariate analysisPHR95CIdemonstrated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group Figure 1A P005 As shown in Figure 1B1D SNHG15 was statistically differentially expressed in diverse groups of the tumor pathologic stage stage II vs IIIIV P00257 metastasis P00071 living status P00017 To clarify the clinicopathologic impact of SNHG15 we also used logistic regression and concluded that the SNHG15 expression based on median value of FPKM as a categorical variable was statistically related to clinicopathologic features Table High SNHG15 expression was significantly related to cancer status pathologic stage metastasis living status in UM all P005 Table These results demonstrated that UM with high SNHG15 expression were prone to progress to cancer status of survival with tumor a more advanced stage metastasis and poor living status when compared to the low SNHG15 expression group However there was no statistically significant difference in age gender tumor diameter thickness pathological type extrascleral invasionMain Enriched Pathways in UM Tissues with High SNHG15 Expression To explore the SNHG15related potential signaling pathways activated in UM GSEA was performed In the current study based on the association with SNHG15 expression the gene list was generated firstly Figure The SNHG15 expression was associated with clinical pathological characteristics A Patients with high SNHG15 expression had a shorter OS when compared with the low SNHG15 expression group P002 BD The expression of SNHG15 was statistically different in diverse groups of the tumor pathologic stage P00257 metastasis P00071 living status P00017 aP005 bP001by GSEA To clarify the statistically significant differences between high and low SNHG15 expression groups GSEA was Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaTable Association between SNHG15 expression and clinicopathologic variables using logistic regressionParametersAge yGenderFemaleMaleTumor diameter mmThickness mmPathological typeEpithelialNonepithelialExtrascleral invasionNoYesCancer statusTumor freeWith tumorPathological stageIIIIIIVMetastasesNoYesLiving statusAliveDeadnmeanSNHG15 expressionLowHighPOR95CINESNominal PvalESTable Enriched pathways for differential SNHG15 expression in UMNameSpliceosomeCell cyclePyrimidine metabolismDNA replicationNucleotide excision repairRNA degradationHomologous recombinationMismatch repairUM Uveal melanoma ES Enrichment score NES Normal enrichment score FDR False discovery rateFDR Qvalconducted subsequently The results indicated that there were significant differences in spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair among patients with high SNHG15 expression phenotype Figure Table DISCUSSIONAccumulating evidences indicate that SNHG15 plays a dual role in the tumorigenesis and development of different tumors[] Previously SNHG15 has been demonstrated as a carcinogenic lncRNA which is usually upregulated in tumor tissues compared with normal tissues[] It exerts 0cFigure Enrichment plots from GSEA Spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair are enriched significantly in SNHG15 high expression phenotypean oncogenic effect via various epigenetic mechanisms[] For example it can suppress the expression of miR3383p and facilitate the proliferation of colorectal cancer cells[] It plays a carcinogenic role by affecting miR3383pFKBP1A axis in prostate cancer[] It can also enhance hepatocellular carcinoma progression by negative regulation of miR1413p[] However there are reports that SNHG15 has a tumor suppressive effect suggesting that low SNHG15 expression is related to poor prognosis in thyroid cancer and upregulating expression of SNHG15 can significantly suppress cell proliferation[] At present the impact of SNHG15 on UM is still unclear Therefore vital roles and potential biological mechanism of SNHG15 in UM needs to be elucidated In this study we revealed that high SNHG15 expression was related to clinicopathologic features in UM Through RNASeq gene expression level and clinicopathological characteristics obtained from the TCGA UM project we analyzed the relationship among SNHG15 expression clinicopathological features and prognosis of UM The univariate analysis demonstrated that SNHG15 expression level age tumor diameter pathological type extrascleral extension and cancer status were risk factors for death from all causes The multivariate analysis suggested that high SNHG15 expression along with age and pathological type was an independent risk factor for death from all causes Therefore the results demonstrated that high SNHG15 expression was an independent predictor of poor prognosis in UM through univariate and multivariate analysis KaplanMeier survival analysis also indicated that high SNHG15 expression group had a worse prognosis when compared to low SNHG15 expression group in UM In addition an analysis was conducted to further explore the relationship between SNHG15 and clinicopathological features The SNHG15 expression was statistically different in diverse groups of the tumor pathologic stage metastasis and living status Besides high SNHG15 expression based on median expression value of FPKM in UM was associated with cancer status of survival with tumor advanced pathologic stage metastasis and living status It demonstrated that high SNHG15 expression in UM was strongly related to poor prognosis The mechanisms of SNHG15 dysregulation in malignant tumors are quite complex and are far from being completely understood Previous studies have suggested that SNHG15 is involved in diverse pathological and physiological processes of many tumors through their abnormal expressions including cell proliferation invasion migration and autophagy[] To explore the biological mechanism of SNHG15 in UM GSEA was conducted It indicated that spliceosome cell cycle pyrimidine metabolism DNA replication nucleotide excision repair RNA degradation homologous recombination and mismatch repair were all enriched differentially in SNHG15 high expression phenotype Alternative splicing is essential for gene regulation and abnormal splicing plays a vital role in inactivating tumor suppressor genes or activating oncogenes[] SNHG15 may have an impact on the invasion Int J Ophthalmol Vol No Aug18 wwwijocnTel Email ijopress163com 0cLncRNA SNHG15 predicts prognosis in uveal melanomaand migration of UM cells by affecting spliceosomal related factors The abnormal cell proliferation of tumor is related to the lack of checkpoint control over the cell cycle which is the basis of genetic instability[] Evidence shows that the lack of homologous recombination may facilitate the disturbance of cell cycle the instability and accumulated mutations of genome during the progression and development[] Mismatch repair proteins have an significant role in DNA hypermethylation alteration and tumorigenesis[] SNHG15 is closely related to DNA replication and mismatch repair demonstrating that SNHG15 may promote the occurrence of UM by affecting DNA replication and DNA mismatch repair It indicated that SNHG15 may be identified as a novel marker of diagnosis therapeutic and prognosis prediction in UM However the related mechanism needs to be further elucidated This research also has some limitations The most important one is the limited number of patients and time of followup In addition some patient characteristics such as ciliary body involvement were not completely recorded in the database In fact ciliary body involvement plays a critical role in UM[]In conclusion this study aims to demonstrate the vital role of SNHG15 in UM and the potential relationship between SNHG15 expression and clinical parameters SNHG15 expression may be a valuable biomarker for poor survival in UM Moreover we have preliminarily explored the crucial pathway associated with SNHG15 in UM However further experimental validation is needed to be performed for clarifying the significant impact of SNHG15 And it is of great significance to further identify its independent prognostic value in a largescale standardized researches on UMACKNOWLEDGEMENTSFoundations Supported by the National Natural Science Foundation of China No81970835 No81800867 Conflicts of Interest Wu X None Li XF None Wu Q None Ma RQ None Qian J None Zhang R NoneREFERENCES van Raamsdonk CD Griewank KG Crosby MB Garrido MC Vemula S Wiesner T Obenauf AC Wackernagel W Green G Bouvier N Sozen MM Baimukanova G Roy R Heguy A Dolgalev I Khanin R Busam K Speicher MR O€™Brien J Bastian BC Mutations in GNA11 in uveal melanoma N Engl J Med Carvajal RD Sosman JA Quevedo JF Milhem MM Joshua AM Kudchadkar RR Linette GP Gajewski TF Lutzky J Lawson DH Lao CD Flynn PJ Albertini MR Sato T Lewis K Doyle A Ancell K Panageas KS Bluth M Hedvat C Erinjeri J Ambrosini G Marr B Abramson DH Dickson MA Wolchok JD Chapman PB Schwartz GK Effect of selumetinib vs chemotherapy on 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Intervertebral disc degeneration IDD is the most common degenerative disease all over the word Our previous studyconfirmed that the downregulated circGRB10 directly interacts with miR3285p which modulate ERBB2 and leads tothe degeneration of intervertebral disc however the underpinning mechanism of circGRB10 dysregulation remainsunclear We identified that FUS and demonstrated that circGBR10 biosynthesis in nucleus pulposus NP cells waspromoted by FUS whose expression was controlled by miR1413p In addition ERBB2 downregulation led todecreased Erk12 phosphorylation which enhanced miR1413p production in NP cells In vivo data indicated that circGRB10 inhibited IDD in rat model The present study revealed that miR1413p and FUS are key factors that regulatecircGRB10 synthesis in NP cells In addition circGBR10 participates in the molecular circuitry that controls human IDDdevelopment These findings provide a basis for further functional diagnostic and therapeutic studies of circGRB10in IDDIntroductionThe Global Burden of Disease Study stated that lowback pain LBP represents an important cause of disability worldwide1 LBP is tightly associated with intervertebral disc degeneration IDD which involves ofall LBP cases causing significant economic and socialburdens worldwide23 According to previous reports of the world population have low back pain during theirlifetime with being chronically disabled4Currently IDD pathogenesis is largely unclear howeverit could be due to microenvironmental alterations in theintervertebral discs caused by various factors such asgenetic features aging sex a predisposing injury and theCorrespondence Wei Guo guow0319163com1Department of Orthopaedics Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R China2Department of Breast Surgery Hebei Province Cangzhou Hospital ofIntegrated Traditional and Western Medicine Cangzhou No2 Hospital Huanghe Road Cangzhou Hebei Province P R ChinaFull list of author information is available at the end of the Edited by G Calinenvironment56 The main feature of IDD pathology iselevated biosynthesis of catabolic enzymes combined withreduced extracellular matrix ECM accumulation causedby imbalanced anabolism and catabolism7 Intervertebraldiscs comprise a central nucleus pulposus NP a peripheral annulus fibrosus AF and cartilaginous endplates which connect overlying capillary beds craniallyand caudally The NP maintains homeostasis by producing an ECM mostly comprising type II collagen andproteoglycans the main functional components of intervertebral discs which are indispensable to maintain thedisc height and absorb various mechanical loads8 It iswell known that loss of collagenII and aggrecan is anearly critical event in the degenerative cascade in Intervertebral disc tissue9 MMP13 is the most importantenzymes that hydrolyze collagens10 ADAMTS5 is classified as the major aggrecanases due to their high efficiency in cleaving aggrecan11 A large body of evidencesupporting the involvement of MMP13 and ADAMTS5in IDD pathogenesis12 During IDD the main histologicalalteration involves the centrally located NP cells which The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of after a phenotypic transformation are substituted bysmaller fibrochondrocytelike cells with reduced proteoglycan production and a global shift towards synthesizing fibrotic materials and compromising the structuralintegrity of discs1314 Therefore unveiling the mechanisms underpinning such imbalance is urgently needed forthe development of new therapeutic targets in IDDMounting evidence supports roles for circular RNAscircRNAs in IDD15“ Previous research demonstratedthat circRNAs are closed RNAs produced by backsplicingof single premRNAs18 It is not completely known howcircRNAs are biosynthesized although complementaritybetween inverted sequences in flanking introns and theactivity of RNAbinding proteins RBPs increase thecontiguity of splice sites contributing to backsplicing inmammalian cells19“The RBP FUS affects splicing regulation23 with manysplicing factors termed FUS interactors24“ FUS mutations could lead to protein mislocalization to the cytosolwith decreased nuclear FUS amounts and occurrence ofabnormal cytosolic aggregates2728 The FUS protein isinvolved in regulating intracellular RNA transport mRNAsynthesis alternative splicing and polyadenylation siteselection29 He demonstrated that FUS combinedwith circ_002136 and promoted the generation ofcirc_002136 in Glioma30 It was recently shown that FUScontrols the expression of circRNAs by binding tointrons that flank the splicing junction31 Moreover FUSwas reported to be regulated by many miRNAs includingmiR1413p3233 Studies revealed miR1413p is upregulated in NP tissue specimens from IDD cases anddemonstrated that miR1413p is associated with discdegeneration34 However the function and mechanism ofFUS as well as the interaction between FUS and miR1413p in IDD have not been reportedOur previous research confirmed that circGRB10amounts are markedly reduced in NP cells from IDDpatients which accelerates IDD development by enhancing miR3285p mediated ERBB2 suppression in NPcells15 However the mechanism of circGRB10 downregulation in degenerative NP cells remains unclear Inthis study we demonstrated that the miR1413p whichis significantly increased in degenerative NP cells34 regulate expression of the FUS which is responsible for thegeneration of circGRB10 in NP cells Furthermore weshowed that ERBB2 downregulation led to decreasedErk12 phosphorylation and the decreased levels of Erk1 phosphorylation enhanced miR1413p biogenesis indegenerative NP cells promoting IDD developmentTaken togetherthese findings suggested circGBR10contributes to the molecular circuitry controlling IDDdevelopment in humansOfficial journal of the Cell Death Differentiation AssociationResultsCircGRB10 regulates NP cell functions through the ERBB2Erk signaling pathwayOur previous study revealed circGRB10 promotes NPcell survival by increasing ERBB2 amounts via suppression of miR3285p However the effect of circGRB10expression on NP cell anabolism or catabolism remainsobscure To further assess circGBR10™s functions in IDDpathogenesis circGRB10 or circGRB10 small interferingRNA siRNA was transiently transfected into culturedprimary human NP cells As shown in Supplementary FigS1 overexpression and knockdown of circGRB10 haveno effect on linear GRB10 but only affect circular GRB10The immunofluorescence results demonstrated that afteroverexpressing circGRB10 in NP cellssignificantlyupregulation of collagen II and aggrecan and decreasedamounts of MMP13 and ADAMTS5 were found Conversely circGRB10 knockdown resulted in oppositeeffects Fig 1a b These findings were confirmed by qRTPCR Fig 1csignificantlyincreased while MMP13Our previous research demonstrated circGRB10 inhibits IDD development by regulating ERBB2 expression inNP cells Increasing evidence supports an important rolefor the ERBB2 gene and Erk signaling pathways in theprogression of many human diseases35“ Meanwhile theErk pathway is altered during IDD38 and plays a significant role in extracellular metabolism39 These resultsprompted us to assess the plausible association of circGRB10 with ERBB2 Erk signaling In this study primaryhuman NP cells underwent transfection with circGBR10circGRB10 siRNA and respective negative controlsrespectively As shown in Fig 1d e western blot assayshowed that pErk12 collagen II and aggrecan amountswereandAMADT5 levels were reduced in NP cells overexpressingandcircGRB10 Conversely pErk12aggrecan were downregulatedandAMADT5 amounts were increased in NP cells transfected with circGRB10 siRNA Fig 1d e FurthermoreERBB2 affected pErk12 in a similar way as circGRB10Fig 1d e suggesting cricGRB10 modulates IDD progression via ERBB2Erk signaling Therefore in order tofurther validated whether ERBB2 was the downstreammediator of circGRB10 in the NP cells We cotransfectedcircGRB10 and ERBB2 siRNA into NP cells andobserved that the positive effects of circGRB10 on NPcells functions were attenuated in the absence of ERBB2Fig 1f g Moreover upregulation of ERBB2 counteracted the inhibitory effect of circGRB10 knockdown onNP cells function Fig 1hi Collectively the abovefindings indicated that circGRB10 associated protectionin IDD may involve ERBB2Erk signalingcollagen IIand MMP13 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig circGRB10 regulates NP cell functions through ERBB2Erk signal pathway a CollagenII aggrecan MMP13 ADAMT5 expression wereanalyzed in circGRB10 or circGRB10 siRNA transfected cultured primary human NP cells using Immunofluorescence staining analysis b Thecorresponding bar graphs show quantitative analysis of the relative fluorescent value of each group n replicates per group p p Scale bar μm c qRTPCR showing the expression levels of collagen II aggrecan MMP13 ADAMT in human NP cells after circGRB10overexpression or knockdown Three independent experiments are presented as mean ± SEM error bars P P d The expressionlevels of CollagenII aggrecan MMP13 ADAMT5 and pErk12 were detected by western blot Quantitative analysis was shown in e and threeindependent repeats were performed in each experiment p f NP cell were cotransfected with circGRB10 and ERBB2 siRNA Western blotassay showed that ERBB2 siRNA blocked the effect of circGRB10 on CollagenII aggrecan MMP13 and ADAMT5 expression Quantitative analysis wasshown in g and three independent repeats were performed in each experiment p h NP cell were cotransfected with circGRB10 siRNAand ERBB2 Western blot assay showed that ERBB2 attenuated the effect of circGRB10 siRNA on CollagenII aggrecan MMP13 and ADAMT5expression Quantitative analysis was shown in i and three independent repeats were performed in each experiment p Key factors regulating circGRB10 formationIn this study we found a highly reverse complementarysequence nt upstream the ² splice site in intron andone nt downstream the ² splice site in intron which were named 2RC reversecomplementarysequence in intron and 6RC reverse complementarysequence in intron respectively Then wildtypesequence spanning from intron to intron of theGRB10 gene and multiple deletion constructs “for circGRB10 were introduced into pcDNA31respectively Fig 2a Upon transfection the wildtypevector unlike the 2RC andor 6RC deletion constructs “ overexpressed circGRB10 indicating 2RCand 6RC may contain the binding sites that regulate circGRB10 biogenesis Fig 2bcould beregulated by RBPs postAs circRNAs are derived from premRNAs and circRNAstranscriptionally18212240 we hypothesized that circGBR10 ismodulated by RBPs posttranscriptionally in IDD development To identify the RBPs which might regulateGRB10 premRNA splicing to generate circGRB10 weincubated biotin labeled sequences cloned from circGRB10 back splicing site nt upstream P1 or ntdownstream P2 with nuclear protein extracts fromnormal human NP cells Fig 2c Nuclear proteins boundto RNA underwent separation by SDSPAGE and silverstaining Fig 2dfollowed by mass spectrometry foridentification A total of proteins SupplementaryTable S1 were retrieved and mapped to the STRINGdatabase screening significant interactions with scoresabove Fig 2e Enrichment analysis demonstratedthat these proteins were mainly involved in thepathways of gene expression processing of capped introncontaining premRNA mRNA splicing mRNA splicingmajor pathway mRNA processing and formation andmaturation of mRNA transcript related signaling pathways Fig 2f Among these proteins were involved inthe mRNA splicing and mRNA splicingmajor pathwaySupplementary Table S2 In addition the web tool CircInteractome predicted RBPs which can potentiallybind circGRB10 premRNA Fig 2g Notably FUS wasOfficial journal of the Cell Death Differentiation Associationthe only RBP that was involved in mRNA splicing andcould potentially bind to circGRB10 premRNA suggesting circGRB10 generation may be associated withFUS expression in NP cellsFUS promotes the generation of circGRB10 in NP cellsRecently FUS was reported to have a role in regulatingcircRNA biosynthesis via binding of introns surroundingthe backsplicing junctions31 As shown in Fig 3A FUSamounts in IDD NP tissues were remarkably lower thanthose of controls In addition Western blot further confirmed this result Figs 3b and S2 To assess whetherFUS contributes to circGRB10 production in NP cells weoverexpressed or suppressed FUS and determined circGRB10 amounts qRTPCR demonstrated that FUSoverexpression led to significantly increased circGRB10amounts in NP cells while FUS knockdown reduced theexpression of circGRB10 Fig 3c Moreover FUS had noeffects on linear GRB10 expression Fig 3c Overexpression of FUS resulted in increased collagenII andaggrecan amounts and decreased MMP13 and ADAMT levels in NP cells while the circGRB10 siRNA attenuated these changes Fig 3d FUS knockdown resultedin downregulated collagenII and aggrecan and upregulated MMP13 and ADAMT5 in NP cells while circGRB10 markedly counteracted the effects of FUS knockdown indicating that FUS exerted its functions throughcircGRB10 Fig 3eTo assess whether FUSbinding sequences are important in circGRB10 biosynthesis FUSbinding sequenceswere searched in circGRB10 and surrounding intronsand two putative FUSbinding sites were detected Fig3f Next two short circGRB10 minigenes were engineeredincluding circGRB10s and circGRB10sEmPrecisely circGRB10s comprises presumed FUSbingingsites on both flanking introns preserved with the inverselyinserted ²intron in circGRB10 removed to preventcomplementary sequences from reacting Fig 3f circGRB10sEm resembles circGRB10s but with FUS sitesdeleted from the surrounding introns Fig 3f RIPrevealed an overt interaction of FUS with circGRB10s 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig RBPs interact with GRB10 premRNA a A schematic drawing of four types of circGRB10 overexpressing vectors to The genomicregion for circGBR10 green bars with its wildtype flanking introns black lines was inserted into the pcDNA31 expression vector 2RC and 6RCare indicated by red bars A series of deletions are indicated by black crosses to b qRTPCR showed the expression of circGBR10 aftertransfection with the four types of circGRB10 overexpressing vectors to Three independent repeats were performed in each experimentp c Schematic diagram of RNAs corresponding to different fragments of GRB10 premRNA P1 P2 produced by in vitro transcription inthe presence of biotin for RNA pulldown experiments d Silver stain acrylamide gel of total nuclear proteins before Input and after pulldown withbiotinlabeled RNA probe P1 P2 M molecular weight marker kDa e Proteins identified from mass spectrometry were integrated to STRINGdatabase and constructed Proteinprotein interaction PPI network A densely connected module which contains proteins including FUS wasscreened from the PPI network and these proteins were participate in biological process of mRNA splicing via spliceosome f Pathways enrichmentanalysis of proteins in PPI network g RBPs which can potentially bind circGRB10 premRNAunlike circBsEm Fig 3g indicating FUS required theputative sites in surrounding introns for binding We nextexpressed circGBR10sin FUS overexpressing orknocked down NP cells and circGRB10s yielded elevated amounts of circGRB10 transcript after FUS overexpression and reduced amounts upon FUS knockdownconfirming FUS is important in circGRB10 biosynthesisin NP cellsFig 3h Next circGRB10sEm wasexpressed in NP cells and it yielded markedly reducedcircGRB10 amounts in comparison with circGRB10sFig 3h This indicated that the putative FUSbindingsequences in the surrounding introns were important incircGRB10 biosynthesis Taken togetherthe abovefindings demonstrated that FUS had a critical regulatoryfunction in circGRB10 biosynthesis in NP cells viabinding to recognition sites in the introns surrounding thecircGRB10forming exonsFUS in NP cells is regulated by miR1413pThe mechanism of FUS downregulation in NP cells ofIDD patients remains unclear Previous studies havedemonstrated that FUS is regulated by miRNAs in manydeseases3233 Therefore we hypothesized that FUS maybe regulated by miRNAs in NP cells Using the Targetscan Microt4 miRanda PITA and RNAhybird databasesall predicted miRNAs were retrieved and submitted toVenn analysis Fig 4a The results showed that FUS waspredicted to be regulated by miRNAs SupplementaryTable S3 including miR1413p Svetoni confirmedthat miR1413p regulates FUS expression during neuraldifferentiation and Ji revealed miR1413p is associated with disc degeneration3334 Furthermore qRTPCRshowed that miR1413p was markedly upregulated in NPtissue samples from IDD cases in comparison with controlvalues Fig 4b Therefore we supposed that FUSexpression was regulated by miR1413p in NP cells Tofurther assess miR1413p interaction with FUS luciferasereporter assays were carried out Cotransfection of FUSWT wild type and miR1413p mimic in primary humanNP cells resulted in markedly decreased luciferase activityin comparison with the FUSmut mutantmiR1413pmimic cotransfection group Fig 4c d These findingsOfficial journal of the Cell Death Differentiation Associationwere further confirmed at the gene and protein levels inhuman NP cells in vitro Fig 4e f pointing to FUS as amiR1413p target Then primary human NP cellsunderwent transfection with miR1413p mimic and miR1413p inhibitor and the corresponding negative controls respectively The results showed that circGBR10was significantly downregulated in cells overexpressingmiR1413p Fig 4g Conversely circGRB10 was upregulated in the miR1413p inhibitor group Fig 4gMoreover upregulation of FUS alleviated the suppressiveeffects of miR1413p on circGRB10 expression Fig 4hwhile FUS knockdown attenuated the effects of miR1413p inhibitor on circGRB10 upregulation Fig 4i Theabove results indicated that miR1413p regulates circGRB10 expression in NP cells primarily through targetingof FUSERBB2 regulates miR1413p in NP cells byphosphorylating Erk12Induced Erk12 signaling causes widespread miRNArepression via suppression of the main steps of miRNAbiogenesis4142 In this study we found decreased levels ofErk12 phosphorylation in circGRB10 or ERBB2 knockeddown NP cells Fig 1d e Previous studies demonstratedthat phosphorylated Erk12 can cause widespreadmiRNA repression through suppressing the major stepsof miRNA biogenesis41“ As ERBB2 amounts werereduced in degenerative NP cells we hypothesized thatmiR1413p may be regulated by Erk12 phosphorylationin NP cells To explore this possibility we overexpressedor knocked down ERBB2 in NP cells qRTPCR resultsdemonstrated that overexpression of ERBB2 significantlydownregulated miR1413p while ERBB2 knockdownincreased miR1413p amounts Fig 5a In addition pretreatment of NP cells with the Erk12 phosphorylationinhibitor U0126 downregulated ERBB2 and attenuatedERBB2 induced phosphorylation of Erk12 decreasing theexpression of FUS Fig 5b Moreover blocking Erk12phosphorylation in NP cellssignificantly attenuatedERBB2™s effects on miR1413p biogenesis Fig 5c anddecrease the expression of circGRB10 Fig 5d Collectivelythe above findings demonstrated that ERBB2 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig FUS regulates the generation of circGRB10 in NP cells a qRTPCR showing FUS mRNA levels in normal and IDD NP tissues Threeindependent repeats were performed in each experiment p b Western blot showing FUS protein amounts were decreased in IDD NPtissues c qRTPCR analysis of circGRB10 expression level after FUS overexpression or knockdown in NP cells FUS overexpression led to significantlyincreased circGRB10 amounts in NP cells while its knockdown reduced circGRB10 levels Moreover FUS had no linear effects on GRB10 expressionThree independent repeats were performed in each experiment p d e qRTPCR analysis of the expression of CollagenII aggrecan MMP and ADAMT5 in NP cells f Schematic illustrating the putative FUSbinding sites on the flanking introns in the circGRB10s minigene The ²terminus of the circular exons of circGRB10 was defined as position Putative FUSbinding sites A and B are located in the intron at the ² terminusof the circGRB10 exon position ˆ’ to ˆ’ and on the intron at the ² terminus of the circGRB10 exon position “ g RIP analysis ofFUSbinding to circGRB10s and circGRB10sEm minigenes in NP cells Bound complexes were pulleddown using an antibody against FUS qRTPCRwas then used to measure circGRB10s binding to FUS Values were normalized to the level of background RIP as detected by an IgG isotype controlh qRTPCR analysis of the expression of circGRB10 relative to GAPDH in NP cells Cells were cotransfected with FUS or FUS siRNA and a circGRB10minigene circGRB10s or circGRB10 minigene containing deleted FUSbinding sites circGRB10Em Quantitative data from three independentexperiments is presented as mean ± SEM error bars P P regulated miR1413p expression in NP cells by phosphorylating Erk12Next we found that decreased ERBB2 amounts indegenerative NP cells could promote miR1413p generation which suppressed the expression of FUS resultingin circGRB10 downregulation our previousstudydemonstrated that circGRB10 downregulation leads toERBB2 reduction by enhancing miR3285p mediatedsuppression of ERBB2 in NP cells15 These findings suggested circGBR10 contributed to the molecular circuitrycontrolling IDD development in humans Fig 5ephosphorylation collagenII and aggrecan upregulationand inhibited the expression of MMP13 ADAMT5 inthe rat model of IDD Fig 6g Moreover Immunofluorescence staining also confirmed that the increasedexpression of collagenII aggrecan and the downregulation of MMP13 ADAMT5 expression in the circGRB10 group compared with noninjection group at weeks Fig 6h Jointly the above findings suggested atherapeutic role for circGRB10 in protecting the discsrevealing circGRB10 as a candidate therapeutic target inIDDIntradiscal injection of circGRB10 alleviates IDD in a ratmodelNeedle puncture has been one of the most commonmethods to establish animal models of IDD4445 We havesuccessfully established a rat model of IDD by needlepuncture according to the above methods in this studySupplementary Fig S3 At and days upon modelingadenoviral human circGRB10 was injected into thepunctured intervertebral discs with 33G needles In vivoRNA FISH indicated circGRB10 in the NP region at weeks after surgery Fig 6b CT scan at and weeks revealed progressive disc space narrowing in allpunctured animals and a significant increase in DHI wasnoted at and weeks post surgery in rats treated bycircGRB10 Fig 6c CT scan revealed that overexpression of circGRB10 markedly preserved the progressive disc space narrowing in rat IDD modelFig 6dAnd safranin O staining results demonstrated that overexpression of circGRB10 can inhibit the degradation ofextracellular matrix of NP cells Fig 6e These resultssuggesting circGRB10 exerted protective effects in surgically induced IDD After injection of adenoviral circGBR10 FUS and ERBB2 amounts in degenerative NPtissues were remarkably elevated Fig 6f while miR1413p amounts were decreased Fig 6f In addition injectionof adenoviral circGBR10 alleviated the degenerativealterations ofincluding enhanced Erk12the NPDiscussionNumerous circRNAs are found in the human transcriptome playing critical roles in the regulation of cellfunctions174647 Our previous study showed that circGRB10 downregulation is associated with human NP cellapoptosis15 Howeverthe mechanism of circGRB10dysregulation in IDD has not been previously describedHere we found that FUS regulated and promoted circGRB10 biosynthesis by interacting with its flankingintrons In addition FUS expression in NP cell wasregulated by miR1413p Our findings suggest a regulatory network in NP cells FUS bound to GRB10 premRNA to regulate circGRB10 synthesis while circGRB10 acted as a molecular sponge for miR3285p withsuppressive effects on ERBB2 production and modulatedIDD development downregulation of ERBB2 decreasedErk12 phosphorylation and promoted the generation ofmiR1413p which bound to the ²UTR region of FUS toinhibit its expression constituting a positive feedbackloop promoting intervertebral disc degenerationThe differential expression of circGBR10 betweennormal and degenerative NP tissues indicates that circGRB10 biosynthesis is controlled differently in variouscells with NP cells possessing specific factors required forcircRNA biogenesis Indeed introns and of the GRB10premRNA had binding sites to regulate circGRB10biogenesis Furthermore multiple RBPs were highlyOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig See legend on next pageOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of see figure on previous pageFig miR1413p inhibits FUS expression in NP cells a The Venn diagram indicates the predicted miRNAs regulate FUS expression miR1413p was intersected predicted by different databases b Expression of miR1413p in IDD NP tissues showing that miR1413p expression wassignificantly higher than that of controls Quantitative data from three independent experiments is presented as mean ± SEM error bars P c Sequence alignment of a putative miR1413pbinding site within the ²UTR of FUS mRNA Bottom mutations in the ²UTR of FUS mRNAsequence to create the mutant luciferase reporter constructs d Luciferase reporter assay in NP cells after transfected with scramble oligo or miR1413p mimics Renilla luciferase vector and the reporter constructs Both firefly and Renilla luciferase activities are measured in the same sample Fireflyluciferase signals were normalized with Renilla luciferase signals Quantitative data from three independent experiments is presented as mean ± SEMerror bars P e f FUS expression level was detected by qRTPCR western blot in primary human NP cells Three independent experimentsis presented as mean ± SEM error bars P g NP cells from control tissues were transfected with miR1413p mimic or miR1413p inhibitorqRTPCR was used to detect the relative expression level of circGRB10 compared with controls Three independent experiments is presented asmean ± SEM error bars P h NP cells from control tissues were transfected with miR1413p with or without FUS overexpress plasmidqRTPCR was used to detect the relative expression level of circGRB10 compared with controls i miR1413p inhibitor with or without FUS siRNA wastransfected into NP cells from control tissues and the expression level of FUS Three independent experiments is presented as mean ± SEM errorbars P Fig ERBB2 regulate miR1413p expression in NP cells a miR1413p expression level in NP cells with ERBB2 overexpression or ERBB2knockdown Three independent experiments is presented as mean ± SEM error bars P b NP cells overexpressing ERBB2 were treated withU0126 or not for one hour Western blot was used to detect the phosphorylated level of Erk12 c d NP cells overexpressing ERBB2 were treated withU0126 or not qRTPCR was used to detect the expression level of miR1413p and circGRB10 Three independent experiments are presented asmean ± SEM error bars P e Schematic representation of mechanisms by which circGRB10 mediates IDD development On the basis offindings described in the manuscript miR1413p downregulates FUS level in NP cells leading to circGRB10 decreased This downregulated circGRB10 in turn enhanced miR3285p mediated suppression of ERBB2 in NP cells leads to decreased Erk12 phosphorylation level And the decreasedErk12 phosphorylation level enhances the generation of miR1413p in NP cellsOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of Fig cricGRB10 attenuates IDD development in vivo a Overview of the experimental setup with injections of circGRB10 or their negativecontrol at and days after surgery b Six weeks after surgery in vivo RNA FISH found circGRB10 located in the NP region Blue fluorescence diamidino2phenylindole DAPI indicating cell nucleus Red fluorescence indicating circGRB10 Scale bar μm c Changes in disc height indexDHI of the indicated groups after needle puncture The DHI was measured at and weeks A significant decrease of the DHI was observedin all puncture groups at week after surgery P A significant increase in DHI was noted at and weeks post surgery in rat treated withcircGBR10 P d CT scan of the indicated groups were obtained weeks after needle puncture e The intervertebral disc degenerationevaluated by Safranin O staining Scale bar µm f qRTPCR showed that the increased levels of FUS ERBB2 and the decreased level of miR1413p in the punctured discs treated with circGRB10 Three independent experiments are presented as mean ± SEM error bars P g Westernblot showing the expression levels of collagen II aggrecan pErk12 MMP13 ADAMT5 in rat NP tissues h Immunostaining for collagenII andaggrecan in IDD model treated by circGRB10 at and weeks Scale bar µmOfficial journal of the Cell Death Differentiation Association 0cGuo Cell Death and Disease Page of enriched in circGRB10™s flanking introns and FUS contributed to circGRB10 biogenesis as shown aboveAlthough FUS induced circGRB10 biosynthesisitssilencing only decreased circGRB10 levels by asshown above It is known that two or more RBPs couldthe synthesis of circRNAs synergistically4048controlwhich might explain the above incomplete suppressionTherefore circGRB10 modulation in NP cells deservesfurther examinationAs shown above altered FUS expression might profoundly affect circGRB10 biogenesis Further deletion ofFUSbinding sequences in the introns flanking of circGBR10 dramatically reduced circGRB10 amounts Takentogether the above findings indicate FUS may directlycontrol backsplicing to upregulate circGRB10 in NPcells by interacting with putative binding sequences onboth flanking introns of circGRB10Recently miR141 has been detected in NP tissuesamples from IDD cases and its levels correlate with discdegeneration Therefore miR141 NPs have been used inIDD treatment with commendable efficacy34 As shownabove miR1413p which is a key regulator of IDDdirectly regulated FUS further revealing the FUScircGRB10 axis as an IDDrelated regulatory pathwayAccumulating evidence indicates that Erk signaling hasan important role in IDD394950 In the present study wefound that circGRB10 significantly upregulated collagenII and aggrecan in NP cells and mediated the protectiveeffects in IDD likely via ERBB2Erk signaling There mightalso be cellular pathways that compensate for ERBB2expression after its knockdown For example the longintergenic noncoding RNA lincRNA BCLIN25 upregulates ERBB2 by inducing promoter CpG methylation ofmiR125b resulting in miR125b repression44 A previousstudy indicated the Erk pathway regulates the miRNAgenerating complex43 In additi

" bridge to surgery bts using a selfexpandable metallic stent sems for the treatment of obstructivecolorectal cancer improves the patient™s quality of life this study aimed to examine prognostic factors ofobstructive colorectal cancermethods we analyzed stage iiiii resectable colon cancer cases cur a retrospectively registered between january and december overall patients with cur a obstructive colorectal cancer were evaluated ofthem underwent emergency surgery es group and of them after bts with sems placement bts group wecompared surgical results and prognoses between the two groupsresults a total of patients underwent endoscopic sems placement which technical success of andmorbidity rate of primary anastomosis rates were in es and in bts p postoperativecomplication in es and in bts p pathological findings of lymphatic invasion in es and in bts p venous invasion were in es and in bts p and recurrence of in esand in bts the 3year overall survival was significantly different between two groups es 868bts bts is worse than es logrank test p venous invasion independently predicted worsened recurrencefreeand overall survivals the vascular invasiveness was correlated with tumor progression after sems placement and thesurvival rate was lower in bts sems potentially worsens prognostic outcomes in stage ii“iii obstructive colorectalcancerkeywords bowel obstruction colorectal cancer selfexpandable metallic stent colorectal cancer crc remains the leading cause ofcancerrelated deaths worldwide because several patientsare initially diagnosed during advanced stages correspondence ohtakmedkindaiacjp1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japanfull list of author information is available at the end of the approximately “ of patients with crc were diagnosed with acute colonic obstruction [“] severe malignancy with bowel obstruction needs urgent surgicalintervention which includes primary lesion resectionand stoma creation leading to increased morbidity andmortality and a potential failure to achieve completeoncological resection [ ]an endoscopic procedure with selfexpandable metallic stent sems is an acceptable bridge to surgery bts the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cohta bmc surgery page of treatment for acute colonic obstruction [“] preoperative sems placement provides an opportunity to perform medical resuscitation comorbidity optimizationbowel preparation tumor staging and observation ofproximal lesions the procedure prevents highriskemergency surgeries and increase oncological resectionand primary anastomosis rates [ ] after the inclusion of colonic sems placement as bts in the coverageof the national health insurance in japan several physicians joined the colonic stent safety procedure researchgroup and developed skills to provide safe treatmentthe largest multicenter prospective study demonstratedthe feasibility and safety of sems placement as bts inpatients with malignant colorectal obstruction the oncological safety and minimalinvasiveness ofthis procedure have confirmed that sems placement asa bridge to elective surgery is not recommended as astandard treatment for symptomatic leftsided malignantcolonic obstruction [ ] several studies reportedthat prognostic factors of malignant colonic obstructionin sems placement had oncological disadvantages compared with those in emergency surgery es [ ] incontrast several trials showed that sems placement as abridge to elective surgery did not improve the survivalrates [“] how sems placement worsens prognosticoutcomes remains unclear [ ]this study aimed to evaluate the induction of curativesurgery in patients with malignant colorectal obstructionafter a sems placement and its longterm results andprognostic factors postoperatively compared to patientswithout sems placement we demonstrated prognosticfactors and overall survival os and recurrencefreesurvival rfs rates for curative surgery after a semsplacementmethodspatientsmedical records of patients who underwent primarycolorectal resection at higashiosaka city medical centerbetween january and december werereviewed all participants provided written informedconsent oralintake and symptoms before and aftersems placement were assessed in table using thecolorectal obstruction scoring system cross from to we recruited patients with all class oftable the colorectal obstruction scoring system crosspatient™s symptom and their condition of an oral intakesolid meal low residue and full diet without symptomcrosscross as stent insertion candidate from we excluded patients with cross and based on updatedstent insertion guideline malignant colorectal obstruction was diagnosed through clinical examinationcross radiography and computed tomography surgery was performed using three approaches es comprised laparotomylymph node dissection as possibleand primary anastomosis on the same day between and bts after sems placement comprised standbylaparoscopy d3 lymph node dissection and primaryanastomosis since january overall patientswith stage iiiii cur a obstructive colorectal cancerwere evaluated of them underwent emergency surgery as es group and of them after bts with semsplacement as bts group we compared surgical resultsand prognoses between the two groupssems devices and the procedurepatients were endoscopically treated with placement ofan uncovered wallflex enteral colonic stent boston scientific corporation natick ma usa or nitis enteralcolonic uncovered stent taewoong inc gimpo southkorea placements were performed as presented in thepreintroduction publicity announcement placement details were mentioned on the website as a brief guideline obstruction structures were determined using aguide wire and a contrast tube was inserted into theproximal colorectal lumen obstructions were measuredusing contrast agents and then the endoscopist determined the number size and type of stent pathologicalbiopsies were recommended after sems locations andintraluminal or extraluminal marking using an endoscopic clip were recommended via visual recognition ofthe endoscopist dilatation of the colonic obstructionbefore sems placement was generally not allowedhistological findingsparaffinembedded specimens were obtained from a cohort of patients diagnosed by the union for international cancer control stage ii“iiisurvival definitionsos was defined as the duration from surgery to anydeath or last followup diagnosis of recurrence was calculated based on recist according to the chemotherapy criteria rfs was defined as the durationfrom surgery to any recurrence includes local recurrenceor distant metastasissolid meal low residue and full diet with symptomliquid or enteral nutrient intakeno oral intakerequiring continuous decompressionstatistical analysisstudent™s ttest and wilcoxon test for continuous variables and the χ2 and fisher™s exact tests for categoricalvariables were conducted survival curves were generated using the kaplan“meier method and compared 0cohta bmc surgery page of table baseline characteristics and outcomes of endoscopicsems placementtable comparison of baseline characteristics in patientsundergoing emergency surgery and bridge to surgerybts n es n pmalefemalemedian range “ “genderagelocationmalefemalemedian rangececumascendingtransversedescendingsigmoidrectumlength of obstructionmedian range cmtechnical successprocedurestentingmorbiditymortalityclinical successthrough the scopethrough the wirewall flex cmnitis cmoverall cda iiicda vcrossbts n “ “ a clavien“dindo classificationusing a logrank test univariate and multivariate survival analyses were performed using the cox proportional hazards regression model all statistical analysesused jmp version sas institute cary nc or statistical scripting language r httpwwwrprojectpvalues of ‰¤ twosided were considered statistically significant this prognostic study complied withthe reporting recommendationsfor tumor markerprognostic studies resultsa total of patients underwent endoscopic semsplacement which was technically safe for malignantcolorectal obstruction with the technical success rate of the clinical success rate was and the patient™ssymptoms and oral intake dramatically improved afterthe sems placement shown in table a total of patients were reviewed and patients underwentes and bts respectively as shown in table baselineclinical characteristics were balanced between the twogroups moreover cases of patients underwent es on the same day as in open surgery the median waiting period for surgery was days for bts thegenderagelocationtype of operationcecumascendingtransversedescendingsigmoidrectumstandbyemergency “ “duration tooperationmedian range dayssurgical procedurelaparotomy laparoscopy timemedian range minblood lossmedian range ml“ ““ “ before afterstoma creation “morbidity30day complication cda iii anastomosticleakagehospital stayaclavien“dindo classificationmedian range days “ “primary anastomosis ratios were in es and in bts p postoperative complication rateswere in es and in bts p postoperative hospital stay was shorter in bts days comp patients withpared to esobstructive crc showed significantinpostoperative complication rate and hospital stay withsems placement operative procedures were dramatically changed and the primary anastomosis rate improved after the sems placementimprovement daysthe pathological tissue type accounted for of differentiated types shown in table tumordepth was similar between the two groups lymphatic vesselinvasion ratios were in es and in bts p and venous invasion ratioswere in es and in bts p recurrence rates were cases in es and cases in bts nodenegative patients stage iimorewhereas nodepositive patients stage iii more frequently had liver metastasis in the kaplan“meier survival analysis in fig 1a the 3year rfs waslung metastasisfrequentlyhad 0cohta bmc surgery page of table comparison of pathological characteristics of emergency surgery and bridge to surgerypt factortotal lymph nodespn factorhistologicallymphatic invasionvenous invasionsurgical clearancet4b t4a t3median rangen0 n1 n2 n3tub1 tub2 othersly v cur a b csignificantly different between the two groups es bts which was significantly low inbts than that in es logrank test p the3year os rate was also significantly different between thetwo groups es bts p shown in fig 1b the relationship between lymph node metastasis and sems placementwas also evaluated the pathological nodenegativestage ii 3year rfs rate was not different betweenthe two groups es bts as shown infig 2athe pathological nodepositivestage iii 3year rfs rate was different between thetwo groups es bts as shown in fig 2bthe stage ii 3year os rate was not different between thetwo groups es bts as shown in fig 2cwhereas stage iii 3year os rate was different between thetwo groups es bts as shown in fig 2dthese results suggestthat vascular invasiveness andpathological nodepositive status were correlated withtumor progression after sems placementthein contrastthuses n “ bts n “ p “survival rate was affected by poor prognosis in the btsgroupresults of adjusted multiple cox proportional hazard regression for rfs and os in all stages and stage iii diseaseare presented in table after adjusting for possible confounders venous invasion and bts independently predicted poor rfs in all stages and venous invasionindependently predicted poor rfs in stage iii disease venous invasion and bts were also significantly associatedwith os in stage iii diseasediscussionacute colonic obstruction requires emergent surgicalintervention a mandatory conventional treatment skillemergent surgicalis associated with highmorbidity mortality and stoma creation rates affectingthe quality of life of patients malignant colorectal obstruction is not only an intestinal obstruction but alsoan advanced stage crc their prognosis was poorerthan that in patients with nonocclusive disease becausetreatmentfig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a recurrencefree survival b overall survival 0cohta bmc surgery page of fig kaplan“meier survival curves in patients undergoing emergency surgery vs bridge to surgery a rfs nodenegative patients b rfs nodepositive patients c os nodenegative patients d os nodepositive patientsof highly invasiveness and distant metastasis [ ]chen revealed that the prognosis in patients withperforation associated with obstruction was poor early intervention in the clinical setting before the colonic perforation has been established endoscopic placement of colonic stents improves the high decompressioneffect and reduces clinical symptoms high postoperative complication rates were correlatedwith poor prognosis in patients with cancer in severalans [“] reducing complication rates can improve the prognosis our results showed high clinicalsuccess rate after sems placement and high primarysurgicalinterventions howeveranastomosis rate stentrelated complications requiredemergentthe stentplacement is safe and feasible in this study moreoverthe laparoscopic rate was high and postoperative complication rate was clinical results including shortterm outcomesin bts after sems were verifiedthrough a metaanalysis [ ]the prognosis was poor in patients with stent perforation and increased local recurrence rate after the colonic stent placementthe longtermprognosis in patients with colorectal obstruction afterbts was not different compared with that in patients however 0cohta bmc surgery page of table multivariate analysis of recurrencefree survival at all stages and stage iiipvaluevariablesrecurrence free suvivalall stages hazard ratio ± sd cistage iii hazard ratio ± sd cipvaluees vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ ˆ’hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ overall suvivales vs semspt factor t3t4pn factor n0n1n2“verous invasion v0“1v2“lymphatic invasion ly0“1ly2“hr ± hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ without obstruction [“] according to the europeansociety of gastrointestinal endoscopy clinical guidelinethat considers the risk of perforation due to colorectalstents only limited uses are allowed therefore colorectalstent placement is not a standard treatment [“]the prognostic outcomes of bts in this study were significantly worse than those of es particularly in lymphnodepositive patientslymphatic and venous invasionseemed to be a significant prognostic factor althoughreduced postoperative complication rate would improve the prognosis our results were contradictoryafter the stent replacement these results suggestedthat stent placement leads to poor prognosis a concern that colonic stents may be associated with adverse effects of mechanical expansion also exists mechanical expansion may be associated with thegrowth of solid tumors particularly lymphatic andvenous invasion [ ]we found that recurrence and os were associatedwith high vascular invasion after a colonic stent placement venous invasion was an independent factor for recurrence and prognosis the ck20 mrna level anepithelial marker is significantly increased in peripheralblood serum suggesting stent deployment into the vasculature alliteratively ki67 level associated withcellular proliferation and p27 gene assisting cell cycleprogression were measured using specimens obtainedbefore and after sems insertion next the ki67 leveldecreased in the specimen after an sems placementcompared with that before and cell proliferation wassuppressed the prognostic nutritionalindex andserum albumin levels were significantly decreased afterstenting suggesting its disadvantage as bts theduration from stent placement to surgery was daysoncological and nutritional factors might change in theblood and contribute to poor prognosis during the waiting period mechanical expansion of the replacement hr ± ˆ’ hr ˆ’ ± ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ hr ± hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ˆ’ ± ˆ’ ˆ’ hr ± ˆ’ should be minimized to prevent perforation and molecular cytological factors to improve the materials expansion and establishment of new mechanism are necessaryin colorectal obstruction [ ]thisisafirstretrospectivethese findings should be considered in light of severallimitationsnonrandomized small sample sized study from a single institution thereby the heterogeneity of the surgical strategy may have affected the prognostic factors secondalthough validated endoscopic procedures were validated stent devices used in this study had differentlengths types and thickness and obtained from differentvendors lastly we performed stent placement in the patients with cross and who are not indicated forstent insertion until to investigate the oncological longterm prognosis ofcolonic sems placement as a bridge to elective surgerylarge sample size and prospective randomized controlledstudies are warranted to develop a treatment strategy forcrc with obstructionvascular invasiveness was correlated with tumor progression after a sems placement and os and rfs rateswere lower in bts sems placement potentially worsensprognostic outcomes in stage ii“iii malignant colorectalobstructionabbreviationsbts bridge to surgery crc colorectal cancer cross colorectal obstructionscoring system es emergency surgery esge european society ofgastrointestinal endoscopy jsccr japanese society for cancer of the colonand rectum recist response evaluation criteria in solid tumors version os overall survival rfs recurrencefree survival sems selfexpandablemetallic stent wses world society of emergency surgeryacknowledgementsthe authors thank for contribution as endoscopic technical adviser kenkonishi md phd from department of surgery hyogo prefecturenishinomiya hospital 0cohta bmc surgery page of authors™ contributionsall authors have read and approved the manuscript ko protocolproject development data collection and management andmanuscript writingediting mi protocolproject developmentmanagement and manuscript writingediting mu protocolprojectdevelopment and data collection and management jm se jm and ikdata collection and management yt and sn data collection ki andtn data analysis and manuscript writingediting mt and ty dataanalysis and managementfundingauthors have no grant support and no financial relationship for this studyavailability of data and materialsthe datasets used and analyzed during this study are available from thecorresponding author upon reasonable requestethics approval and consent to participateall procedures performed in studies involving human participants were inaccordance with the ethical standards of the institutional researchcommittee and with the helsinki declaration and its later amendmentsor with comparable ethical standards all participants or their guardians haveprovided their written informed consent and that the study protocol wasapproved by higashiosaka city medical center ethical committee on humanresearch assignment number “consent for publicationno applicablecompeting intereststhe authors declare that they have no conflict of interest to discloseauthor details1gastroenterological surgery higashiosaka city medical center osaka japan2department of gastroenterological surgery kindai university nara hospital otodacho ikomacity nara japan 3thoracic surgeryhigashiosaka city medical center osaka japan 4digestive surgery kawasakimedical school okayama japan 5gastroenterology higashiosaka citymedical center osaka japanreceived june accepted august referencesjemal a bray f center mm ferlay j ward e forman d global cancerstatistics ca cancer j clin “ pubmed pmid epub eng winner m mooney sj hershman dl feingold dl allendorf jd wright jd incidence and predictors of bowel obstruction in elderly patients withstage iv colon cancer a populationbased cohort study jama surg “ pubmed pmid pubmed central pmcid pmc45 epub engjullumstro e wibe a lydersen s edna th colon cancer incidencepresentation treatment and outcomes over years color dis “ pubmed pmid epub engcheynel n cortet m lepage c benoit l faivre j bouvier am trends infrequency and management of obstructing colorectal cancers in a welldefined population dis colon rectum “ pubmed pmid epub engcuffy m abir f audisio ra longo we colorectal cancer presenting assurgical emergencies surg oncol ““ pubmed pmid epub eng mcardle cs hole dj emergency presentation of colorectal cancer isassociated with poor 5year survival br j surg “ pubmedpmid epub eng mainar a tejero e maynar m ferral h castanedazuniga w colorectalobstruction treatment with metallic stents radiology “pubmed pmid epub engzhang y shi j shi b song cy xie wf chen yx comparison of efficacybetween uncovered and covered selfexpanding metallic stents inmalignant large bowel obstruction a systematic review and metaanalysiscolor dis 2012147e367“ pubmed pmid epub engtilney hs lovegrove re purkayastha s sains ps westonpetrides gk darziaw comparison of 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pmid pubmed central pmcid pmc5737828 epub eng mcshane lm altman dg sauerbrei w taube se gion m clark gm reportingrecommendations for tumour marker prognostic studies remark eur jcancer “ pubmed pmid epub eng hashiguchi y muro k saito y ito y ajioka y hamaguchi t japanesesociety for cancer of the colon and rectum jsccr guidelines for thetreatment of colorectal cancer int j clin oncol pubmed pmid epub eng cortet m grimault a cheynel n lepage c bouvier am faivre j patterns ofrecurrence of obstructing colon cancers after surgery for cure a population 0cohta bmc surgery page of based study color dis “ pubmed pmid epub eng nojiri t maeda h takeuchi y funakoshi y kimura t maekura r predictive value of btype natriuretic peptide for postoperative atrialfibrillation following pulmonary resection for lung cancer eur jcardiothorac surg “ pubmed pmid epub engpubmed pmid pubmed central pmcid pmc4128744 epub engkaragiannis gs poutahidis t erdman se kirsch r riddell rh diamandis epcancerassociated fibroblasts drive the progression of metastasis throughboth paracrine and mechanical pressure on cancer tissue mol cancer res“ pubmed pmid pubmed central pmcidpmc4399759 epub eng nojiri t inoue m yamamoto k maeda h takeuchi y funakoshi y b maruthachalam k lash ge shenton bk han af tumour celldissemination following endoscopic stent insertion br j surg “ pubmed pmid epub eng matsuda a miyashita m matsumoto s sakurazawa n kawano y yamahatsuk colonic stentinduced mechanical compression may suppresscancer cell proliferation in malignant large bowel obstruction surg endosc“ pubmed pmid epub eng haraguchi n ikeda m miyake m yamada t sakakibara y mita e colonic stenting as a bridge to surgery for obstructive colorectal canceradvantages and disadvantages surg today “ pubmedpmid epub eng zhu z li b liao w lv n chen y shu x novel predictive nomogram foridentifying difficult guidewire insertion in patients with malignant colorectalobstruction and sphincterotomeassisted guidewire insertion for improvingthe success rate of selfexpandable metal stent insertion front oncol pubmed pmid pubmed central pmcid pmc7237730epub eng miyasako y kuwai t ishaq s tao k konishi h miura r newlydeveloped selfexpandable nitis md colonic metal stent for malignantcolonic obstruction world j gastrointest surg “ pubmedpmid pubmed central pmcid pmc7215972 epub engpublisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationstype natriuretic peptide as a predictor of postoperative cardiopulmonarycomplications in elderly patients undergoing pulmonary resection for lungcancer ann thorac surg “ pubmed pmid epub eng cowie mr struthers ad wood da coats aj thompson sg poolewilsonpa value of natriuretic peptides in assessment of patients withpossible new heart failure in primary care lancet “pubmed pmid epub eng cuthbertson bh card g croal bl mcneilly j hillis gs the utility of btypenatriuretic peptide in predicting postoperative cardiac events and mortalityin patients undergoing major emergency noncardiac surgery anaesthesia“ pubmed pmid epub engsabbagh c browet f diouf m cosse c brehant o bartoli e isstenting as a bridge to surgery an oncologically safe strategy for themanagement of acute leftsided malignant colonic obstruction acomparative study with a propensity score analysis ann surg “ pubmed pmid epub engtung kl cheung hy ng lw chung cc li mk endolaparoscopic approachversus conventional open surgery in the treatment of obstructing leftsidedcolon cancer longterm followup of a randomized trial asian j endoscsurg “ pubmed pmid epub eng gianotti l tamini n nespoli l rota m bolzonaro e frego r aprospective evaluation of shortterm and longterm results from colonicstenting for palliation or as a bridge to elective operation versus immediatesurgery for largebowel obstruction surg endosc “pubmed pmid epub eng yang sy park yy han yd cho ms hur h min bs oncologicoutcomes of selfexpandable metallic stent as a bridge to surgery andsafety and feasibility of minimally invasive surgery for acute malignantcolonic obstruction ann surg oncol “ pubmed pmid epub engvan hooft je van halsema ee vanbiervliet g beetstan rg dewitt jmdonnellan f selfexpandable metal stents for obstructing colonic andextracolonic cancer european society of gastrointestinal endoscopy esgeclinical guideline endoscopy “ pubmed pmid epub eng ansaloni l andersson re bazzoli f catena f cennamo v di saverio s guidelenines in the management of obstructing cancer of the leftcolon consensus conference of the world society of emergency surgerywses and peritoneum and surgery pns society world j emerg surg pubmed pmid pubmed central pmcid pmc3022691epub eng arezzo a balague c targarona e bhi f giraudo g ghezzo l colonic stenting as a bridge to surgery versus emergency surgery formalignant colonic obstruction results of a multicentre randomisedcontrolled trial esco trial surg endosc “ pubmedpmid epub eng amelung fj burghgraef ta tanis pj van hooft je ter b f siersema pd critical appraisal of oncological safety of stent as bridge to surgery inleftsided obstructing colon cancer a systematic review and metaanalysiscrit rev oncol hematol “ pubmed pmid epub eng domingo s puertolas s graciavilla l puertolas ja mechanical comparativeanalysis of stents for colorectal obstruction minim invasive ther alliedtechnol “ pubmed pmid epub engsuzuki n saunders bp thomasgibson s akle c marshall m halligan scolorectal stenting for malignant and benign disease outcomes incolorectal stenting dis colon rectum “ pubmed pmid epub eng voutouri c mpekris f papageis p odysseos ad stylianopoulos t roleof constitutive behavior and tumorhost mechanical interactions in thestate of stress and growth of solid tumors plos one 201498e104717 0c"

"Type and diabetes confer an increased risk of pancreatic cancer PaC of similar magnitudesuggesting a common mechanism The recent finding that PaC incidence increases linearly with increasing fastingglucose levels supports a central role for hyperglycaemia which is known to cause carbonyl stress and advancedglycation endproduct AGE accumulation through increased glycolytic activity and nonenzymatic reactions Thisstudy investigated the impact of hyperglycaemia on invasive tumour development and the underlying mechanismsinvolvedMethods Pdx1CreLSLKrasG12D mice were interbred with mitosis luciferase reporter mice rendered diabetic withstreptozotocin and treated or not with carnosinol FL92616 a selective scavenger of reactive carbonyl speciesRCS and as such an inhibitor of AGE formation Mice were monitored for tumour development by in vivobioluminescence imaging At the end of the study pancreatic tissue was collected for histologyimmunohistochemistry and molecular analyses Mechanistic studies were performed in pancreatic ductaladenocarcinoma cell lines challenged with high glucose glycolysis and glycoxidationderived RCS their proteinadducts AGEs and sera from diabetic patientsResults Cumulative incidence of invasive PaC at weeks of age was in untreated diabetic vs in FL92616gtreated diabetic and in nondiabetic mice FL92616 treatment suppressed systemic and pancreaticcarbonyl stress extracellular signalregulated kinases ERK activation and nuclear translocation of Yesassociatedprotein YAP in pancreas In vitro RCS scavenging and AGE elimination completely inhibited cell proliferationstimulated by high glucose and YAP proved essential in mediating the effects of both glucosederived RCS andtheir protein adducts AGEs However RCS and AGEs induced YAP activity through distinct pathways causingreduction of Large Tumour Suppressor Kinase and activation of the Epidermal Growth Factor ReceptorERKsignalling pathway respectivelyContinued on next page Correspondence giuseppepuglieseuniroma1it Stefano Menini and Carla Iacobini contributed equally to this work1Department of Clinical and Molecular Medicine œLa Sapienza University Viadi Grottarossa Rome ItalyFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cMenini Journal of Experimental Clinical Cancer Research Page of Continued from previous pageConclusions An RCS scavenger and AGE inhibitor prevented the accelerating effect of diabetes on PainINsprogression to invasive PaC showing that hyperglycaemia promotes PaC mainly through increased carbonyl stressIn vitro experiments demonstrated that both circulating RCSAGEs and tumour cellderived carbonyl stressgenerated by excess glucose metabolism induce proliferation by YAP activation hence providing a molecularmechanism underlying the link between diabetes and PaC and cancer in generalKeywords Pancreatic ductal adenocarcinoma Hyperglycaemia Reactive carbonyl species Methylglyoxal Advancedglycation endproducts Carnosine derivatives Yesassociated protein Large tumour suppressor kinase Epidermalgrowth factor receptor Extracellular signalregulated kinases BackgroundPancreatic cancer PaC is the tenth most common incident cancer but the seventh leading cause of cancerrelated death worldwide [] because of the poor 5yearsurvival outcomes [] Due to the rising prevalence ofrisk factors such as obesity and type diabetes PaC isexpected to become the second leading cause of cancerrelated death in the US by [] Type diabetes wasfound to be associated with a “7fold higher risk ofPaC in the first year after diabetes diagnosis and nearlytwofold thereafter [ ] Though type diabetes is themain contributor to this problem the entity and temporal trajectory of PaC risk were recently reported to besimilar in type diabetes [] suggesting a commonmechanism related to hyperglycaemia This concept issupported by the recent finding that PaC incidence increases linearly with increasing fasting glucose levelseven within the normal range []thus hindering the understanding ofPrevious studies have shown that type diabetes induced by a highfat diet promotes PaC [ ] Howeverthis experimental model of the metabolic syndrome doesnot allow assessing the role of hyperglycaemia independent of confounding factors such as obesity and hyperinsulinemiathemechanisms underlying the risk conferred by hyperglycaemia We have recently demonstrated that advancedglycation endproducts AGEs promote proliferation ofhuman pancreatic ductal adenocarcinoma PDA cell linesand that exogenous AGE administration markedly accelerates invasive tumour development in a mouse model ofKrasdriven PaC [] Accumulation of AGEs in diabetesis mainly due to increased formation of reactive carbonylspecies RCS derived from glucose autooxidation egglyoxal GO but also from cell metabolism of excess glucose through glycolysis eg methylglyoxal MGO []In turn RCS react with amino groups of proteins causingstructural and functional modifications The resulting irreversible adducts ie AGEs accumulate in tissues wherethey can exert further biological effects through interaction with specific receptors [ ]Carnosine betaalanylLhistidine is an endogenousinhibits AGEhistidinecontainingdipeptidethat[]carnosine derivativesformation by scavenging RCS [] Though LCarnosinewas proven to be effective in several carbonyl stressrelated disease conditionsincluding metabolicdisorders [“]its therapeutic use in humans ishampered by the presence of high levels of serum carnothus prompting the search for carnosinasesinase[“] The novelresistantbioavailable compound carnosinol ie 2S23aminopropanoylamino31Himidazol5ylFL [“] was shown to be highly effective in attenuating diabetesassociated vascular complications [] and obesityrelated metabolic dysfunctions [ ]Moreover it was recently shown that Lcarnosine is effective in counteracting glycolysisdependenttumourgrowth by quenching RCS []propanollesionsthe progression of preneoplasticThis study aimed at investigating whether hyperglycaemia associated with experimental type diabetes favourstomalignancy in a wellvalidated mouse model of PaC byincreasing carbonyl stress To this end mice weretreated with the RCS scavenger and inhibitor of AGEformation FL92616 An additional objective was toanalyse the effect of the diabetic milieu and of FL926 on the activity of Yesassociated protein YAP a keydownstream target of KRAS signalling required for progression of pancreatic intraepithelial neoplasias PanINsto invasive PaC [ ] and for MGOinduced tumourgrowth []MethodsIn vivo studyThe experimental protocols comply with the principles ofwwwnc3rsukarriveguidelines and were approved by the National Ethics Committee for Animal Experimentation ofof HealthAuthorization no 14702015PR The mice were housedin single cages with woodderived bedding material in aspecific pathogenfree facility with a 12h lightdark cycleunder controlled temperatures “ °C Mice werecared for in accordance with the Principles of LaboratoryAnimal Care National Institutes of Health publ no “ revised and with national laws and receivedItalian Ministrythe 0cMenini Journal of Experimental Clinical Cancer Research Page of water and food ad libitum The primary and secondaryendpoint were the development of invasive PaC and thedevelopmentprogression of PanINs respectivelyDesignThe effect of diabetes on PaC progression was investigatedin Pdx1CreLSLKrasG12D KC mice which develop autochthonous PaC in a pattern recapitulating human pathology with high fidelity by developing the full spectrum ofPaC progression from preneoplastic lesions PanINs toadenocarcinoma and metastasis [ ] KC mice wereinterbred with mitosis luciferase MITO“Luc reporter miceto obtain KCMito KCM mice [ ] The LSLKrasG12D lineage was maintained in the heterozygousstate Mice were screened by polymerase chain reactionPCR using tail DNA amplified by specific primers to theLoxP cassette flanking mutated KrasG12D wild type KrasCre recombinase and MITO genes as previously reported[ ] In the MITOLuc mouse an artificial minimal promoter derived from the cyclin B2 gene and induced by NFY drives the expression of the luciferase reporter specificallyin replicating cells Therefore both normal eg bone marrow and tumour actively proliferating cells may be localized by a bioluminescence imaging BLIbased screen [ ] We have previously shown that KCM mice developpreinvasive PanINs and invasive ductal PaC with thesame penetrance latency and histological features as thosedescribed for KC mice [] According to the Ethics Committee recommendations to limit the number of animalsthe experiments were stopped when it was sufficient toconfirm or reject the working hypothesis in a statisticallyand clinically meaningful mannerFigure shows the flowchart and timeline of study design Thirtythree KCM mice were rendered diabeticFig Flowchart and timeline of study design Please refer to the text for detailed description In dashed boxes groups of nondiabetic KCMmice Ctr that served as control for the effect of STZ STZnonDiab or FL92616 FL treatment Ctr FL on PaC development and progressionTo avoid unnecessary suffering three diabetic mice Diab and one Diab mouse treated with FL DiabFL were killed and weeks respectivelybefore the end of the study STZ streptozotrocin BLI bioluminescence imaging 0cMenini Journal of Experimental Clinical Cancer Research Page of with streptozotocin STZ and followed for weeksie up to weeks of age After an overnight fast weekold mice were intraperitoneally injected with mg·kgˆ’ STZ SigmaAldrich St Louis MO USA Success rate defined as the percentage of STZinjected micewith glucose levels mgdL for the entire studyperiod was Three days after injection diabetic mice were randomized to receive no treatmentDiab n or FL92616 gift of Flamma SpAChignolo d™Isola Italy [] at a dose of mg·kgˆ’ ˆ™dayˆ’ in the drinking water DiabFL n andinjected weekly with IU of insulin glargine to preventexcessive weight loss and ketoacidosis FL92616 wasshown to have a suitable absorption distribution metabolism excretion and toxicity ADMET profile and thegreatest potency and selectivity toward RCS among allother carnosine derivatives [] The FL92616 dosewas chosen based on previous results from our group[]showing high efficacy in preventing diabetesinduced renal injury and from other investigators indicating a good safety profile at the dose of “ g·kgˆ’ ·dayˆ’ [ ] Neither histological abnormalities of theliver kidney lung and heart nor functional abnormalities attributable to toxicity on these tissues wereobserved in this study or in a previous one [] STZtreated mice not fulfilling the criteria for diabetes diagnosis STZnonDiab n served as control for STZeffect on PaC seven of these mice failed to develophyperglycaemia whereas two had spontaneous recoveryfrom diabetes within weeks Vehicle salineinjectedKCM mice were used as nondiabetic controls and either left untreated Ctr n or treated with FL926 Ctr FL n to check for any drug effectMice were subjected to in vivo BLI every otherweek [ ] and daily manual palpation of the abdomen to check for tumour growth and avoid the lossof animals along with the need to cope with the related ethicalissues ie compliance with the 3Rsprinciples Briefly min after administration of Dluciferin mgkg body weightintraperitoneal Perkin Elmer Hopkinton MA USA photon emissionfrom the different body areas was acquired for minand analysed with a CCD camera Xenogen IVIS Lumina System Perkin Elmer A specific region ofinterest ROI corresponding to the abdominal areaoccupied by the pancreas was manually selected andthe total photon flux ps from this ROI was evaluated with Living Image software Caliper Life Sciences Perkin Elmer [ ]At the end of the study mice were anaesthetizedwith ketamine mg·kgˆ’ Imalgene ip and xylazine mg·kgˆ’ Rompum ip and killed by cervical dislocation According to the Ethics Committeerecommendations to avoid suffering three Diab andone DiabFL mice presenting with both positive BLIand a palpable abdominal mass or poor general condition were killed and weeks respectively beforethe end of the study The lungs and the middle partof the gastrointestinal tractincluding the pancreasand the liver were dissected and exposed to theCCD camera for min for photon emission assessment The pancreas was dissected photographed andthen one part was stored at ˆ’ °C forweightedmolecular analysis whereas the other part was processed for histologicalimmunohistochemical analysis[] At time of collection a technician CC seeAcknowledgementstoallow blinded analysisrecoded biologicalsamplesMetabolic parametersBody weight and blood glucose were monitored weeklyAt the end of the study the levels of haemoglobin HbA1c an indicator of longterm glycaemic control wereassessed by using the Mouse HbA1c Assay Kit Crystal Chem Zaandam Netherlands and serum AGEsand total protein carbonyls PCOs two carbonyl stressmarkers were measured by ELISA OxiSelect„¢ Advanced Glycation EndProduct Competitive ELISA Kitno STA817 and OxiSelect„¢ Protein Carbonyl ELISAKit no STA310 respectively Cell Biolabs Inc SanDiego CA USAPancreas histologySix 4μmthick nonserial pancreatic sections stainedwith haematoxylin and eosin were examined to confirm the presence of invasive PaC Pancreas withoutinvasive PaC were analysed to grade dysplastic ductsie PanINs according to previously established criteria [] The numbers oflowgrade PanIN1ABand highgrade PanIN23 dysplastic ducts werecounted and expressed as a percentage of total ductsin the specimen []Pancreatic AGEsERK phosphrylation status nuclear YAP and itstarget gene connective tissue growth factor CTGFLevels of AGEs pERK and CTGF protein in homogenates and of active nonphosphorylated YAP1 innuclear extracts of pancreas of mice were assessed byWestern blot Human PDA tissues n were obtained from the Pathology Unit of Sant™Andrea HospitalRome Italyin agreement with the ethical guidelinesestablished by the locally appointed Ethics CommitteePancreatic tissue distribution of AGEs and activatedYAP1 in mouse and human specimens were evaluatedby dual label immunofluorescence and immunoperoxidase respectively [ ] For immunofluorescence agoat polyclonalrabbitantiAGE antibodyand a 0cMenini Journal of Experimental Clinical Cancer Research Page of sections weremonoclonal antibody to active nonphosphorylatedYAP1 were used as primary antibodies followed by appropriate secondary fluorescent antibodies see Supplementary Table S1 for antibodies in Additional file Sections were analysed at a fluorescence microscopeZeiss AXIO A1 equipped with an Axiocam colorcamera Carl Zeiss Italy Milan Italy For immunoperoxidase formalinfixed paraffin embedded sections μm thick were rehydrated and treated with H2O2in PBS for min to block endogenous peroxidase activity Heat mediated antigen retrieval was performed withœAntigen Unmasking Solution Citric Acid Based H Vector Laboratories Burlingame CA USA forAGE staining or TrisEDTA buffer pH for YAPstaining both for min Nonspecific binding wasblocked by incubation in Protein block serum free AgilentDako Santa Clara CA USA for min at roomtemperature Thenincubated withAvidinBiotin blocking Kit SP2002 Vector Laboratories for min an antiAGE antibody Abcam Cambridge UK ab23722 or an antibody directed to theactive nonphosphorylated YAP1 Abcam ab205270at °C overnight and the appropriate biotinylated secondary antibody at room temperature for min seeSupplementary Table S1 for antibodies in Additional file Finally sections were stained with UltraTek Horseradish Peroxidase ABL015 ScyTek Laboratories UTUSAfor min followed by 33diaminobenzidineDABH2O2 ChromogenSubstrate Kit High ContrastACV500 ScyTek Laboratories until the reaction product was visualized min and counterstained withhematoxylin AGE positive staining and nuclear expression of YAP were measured in random fields of eachsection at a final magnification of 250X and 400X respectively by means of the interactive image analyzerImagePro Premier ImmaginiComputer MilanItaly AGE positivity was expressed as the mean percentage of field™s area occupied by the specific stain Expression status of active YAP in tumor specimens wasassessed by counting the number of nuclei positive forYAP and expressed as the mean ratio of YAPpositive nuclei to total nucleiand AGEstheIn vitro studyThe in vitro study investigated the putative role ofRCStumourpromoting effect of high glucose HG and the protective effect of the carbonylsequestering agent andAGE inhibitor FL92616as mediators ofDesignHuman MIA PaCa2 Catalogue No Lot No14A02 and Panc1 Catalogue No Lot No10G011 cells SigmaAldrich were used for assessing theeffects of HG and FL92616 on cell proliferation Experiments aimed at investigating the molecular mechanismsunderlying the glucosemediated effects and the protection by FL92616 were conducted on MIA PaCa2 cellsMycoplasma contamination in cell cultures was regularlytested by PCR MycoSPY Kit Biontex Laboratories GmbHMunchen Germany Human PDA cells were maintainedin DMEM supplemented with FBS and incubated indifferent conditions for three daysie normoglycaemia normal glucose mM hyperglycaemia HG mM treated with MGO or GO μM SigmaAldrich two RCS and AGE precursors or the preformed AGE Nεcarboxymethyllysine CML μgmLprepared as previously reported [ ] with or withoutFL92616 mM and exposed to DMEM lowglucose medium containing of pooled sera from nondiabetic or diabetic individuals before and after AGE removalfrom diabetic serum by an immunoadsorptionmethod see below with or without FL92616 mMInformed consent was obtained from nondiabetic anddiabetic individuals Moreover both YAP and EpidermalGrowth Factor Receptor EGFR were silenced to assessthe role of YAP and EGFR pathway in RCS and AGEinduced cell proliferation see belowRemoval of AGEs from diabetic serumAGEs were removed from diabetic serum using animmunoadsorption method To immunoprecipitateAGEmodified proteins μl of diabetic serum wasincubated for h with μl of Pierce NHSactivatedmagnetic beads Thermofisher Scientific covalentlyconjugated with μg of antiAGE antibody Abcamsee Supplementary Table S1 for antibodies in Additional file according to the manufacturer instruction To confirm the efficiency of AGE depletionAGE concentration in both treated unbound serumfraction and untreated diabetic serum was evaluatedin triplicate by ELISA OxiSelect„¢ Advanced GlycationEndProduct Competitive ELISA Kit no STA817Cell Biolabs Inc San Diego CA USA Followingthis procedure the concentration of AGEs in diabeticserum was reduced by about reaching a concentration similar to that of the nondiabetic serum seethe œResults sectionYAP and EGFR silencingYAP and EGFR were silenced using smallinterferingRNAs siRNAs and irrelevant scrambled siRNAs as control Thermo Fisher Scientific Waltham MA USAValidated predesigned siRNA oligonucleotides and related TaqMan assays are detailed in SupplementaryTable S2 see Additional file Lipofectamine RNAiMAX Thermo Fisher Scientific transfections were performed using nM of each siRNA 0cMenini Journal of Experimental Clinical Cancer Research Page of Cell survival and proliferationCell viability and proliferation were evaluated by Cytoselect WST1 Cell Proliferation Assay Cell Biolabs following the manufacturer instructionsYAP1 its upstream regulators large tumour suppressor Kinase 1LATS1 and EGFRERK pathway and itsmolecular targets CTGF WTN5A and EMP2 in inhuman PDA cells Cells were extracted in SDS buffer containing protease and phosphatase inhibitorsSigma Aldrich Nuclear protein extracts were obtainedfrom cell monolayers with the Nuclear Extract Kit Active Motif Corp Carlsbad CA USA Protein concentrations were determined using the Bradford Assay KitBioRad Hercules CA USA Nuclear protein levels ofYAP1 and cellular protein levels of total and EGFRphosphorylated at Tyr1068 pEGFR total and pERK and LATS1 a key kinase of the Hippo pathway []were assessed by Western blotting see SupplementaryTable S1 for antibodies in Additional file KRAS activity was evaluated by the KRAS activation Assay Kit noSTA400K Cell Biolabs Inc according to the manufacturer™s protocol Briefly mg of lysate was subjected topulldown and μg of lysate was used to measure totalKRAS Pulldown and totallysates were subjected toWestern blotting procedure using the primary antibodyagainst KRAS provided by the kit The mRNA levels ofCTGFCCN2 WTN5A and EMP2 three recognized molecular targets of YAP [ ] were assessed by realtime PCR RTPCR using a StepOne RealTime PCRSystem and TaqMan Gene Expression assays ThermoFisher Scientific [] listed in Supplementary Table S3see Additional file Statistical analysisResults are expressed as mean ± SD mean ± SEM orpercentage Differences between cell typestreatmentsor animal groups were assessed by oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisons or twoway ANOVA followed bythe Bonferroni posttest as appropriate Betweengroup differencesin PaC incidence were assessedusing the Chisquared test and Fisher™s exact test tocompute a Pvalue from a contingency table A Pvalue of was considered to be significant Allstatisticalincluding linear regression analysiswere performed on raw data using GraphPad Prismversion for Windows GraphPad Software SanDiego CA USAtestsResultsIn vivo studyMetabolic parametersSTZtreated KCM mice developed hyperglycaemiastartingandabout h postinjection Fig 2ashowed a slight decline in the growth curve vs Ctrmice which reached statistical significance only at and weeks of age Fig 2b Despite no differencein body weight Fig 2c blood glucose Fig 2d andHbA1c levels Fig 2e FL92616 treatment preventedthe diabetesassociated increase in circulating AGEsFig 2f and total PCOs Fig 2g as assessed at theend of the studyInvasive PaC development Representative BLI imagesat the end of the study period and total photon fluxinduction from pancreas at and weeks of ageare shown in Fig 3a At sacrifice pancreas weightwas significantly P increased in Diab ± g vs Ctr ± g and vs DiabFL ± g KCM mice Pancreasbody weight percent ratiowas almost tripled in Diab vs Ctr mice whereas nostatistical difference was observed between DiabFLand Ctr mice Fig 3b and Table As assessed byhistology Fig 3c cumulative incidence ofinvasivePaC at weeks of age was in Diab mice vs in DiabFL and in Ctr mice Fig 3d and Table Representative BLI images and pancreas histologyfrom Ctr Diab and DiabFL are shown in Fig 3cdNeither the Ctr FL nor the STZnonDiab groupshowed significant differences in the incidence invasive PaC and pancreasbody weight percent ratio vsthe Ctr group Table Furthermore no betweengroup differences were observed in tumour invasiveness except for an apparent reduction in DiabFL vsDiab group Table However the few cases of PaCin DiabFL n and Ctr n mice prevent toperform statistical comparisons among groupsformetastatic disease Representative ex vivo BLI andhistology images of liver and lung metastases are presented in Supplementary Fig S1 in Additional file Grading of dysplastic ducts in mice free of invasivePaC Table showed significant differences betweenDiabFL and Diab mice for the percentage of normalducts which was higher and of highgrade PanINswhich was lowerin the FL92616 treated arm Inaddition Ctr FL mice presented with higher normalducts and lowerlowgrade PanINs vs Ctr micewhereas no difference was observed between STZnonDiab and Ctr micePancreatic AGEs ERK phosphrylation status nuclear YAP and connectivegrowth factorCTGF Pancreatic accumulation of AGEs Fig 4aand levels pERK Fig 4b CTGF Fig 4c awellestablished transcriptional target of YAP [ ] and nuclear YAP1 Fig 4d were increased inDiab vs Ctr mice and increments were prevented bytissue 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig Glucose and HbA1c levels body weight and hyperglycaemiaassociated carbonyl stress Blood glucose levels and body weight during thestudy period a and b and at the end of the study period weeks of age1 c and d and HbA1c levels e and serum levels of AGEs f andtotal PCOs g at the end of the study period weeks of age1 in control Ctr Ctr treated with FL92616 Ctr FL diabetic Diab and Diabtreated with FL92616 DiabFL KCM mice Statistical significance between groups for time course of blood glucose a and body weight c wascalculated using twoway ANOVA followed by the Bonferroni posttest Each time point represents mean ± SD of animals until the 17th weekof age and “ animals from the 18th to the 22nd week of age Statistical significance for blood glucose c body weight d serum levels ofAGEs e and PCOs f at weeks of age1 was assessed using oneway ANOVA followed by the StudentNewmanKeuls test for multiplecomparisons Each dot represents one case and bars represent mean ± SEM P or P vs Ctr    P vs Diab 1Except for threeDiab and one Diab FL mice which were killed and weeks respectively before the end of the study see œResults section for further detailslabelFL92616 treatment Dualimmunofluorescenceanalysis confirmed the association between AGEs andnuclear YAP1 in PaC lesions from Diab mice Fig4e A significant positive relationship between AGEaccumulation and nuclear YAP1 levels was also observed in human PDA Fig 4fgIn vitro studyProliferation of human PDA cellsHG concentration mimicking diabetic hyperglycaemiapromoted PDA cell growth and this effect was prevented by FL92616 Fig 5ab The AGE precursors RCS MGO and GO and the preformed AGE 0cMenini Journal of Experimental Clinical Cancer Research Page of Fig In vivo BLI and gross and microscopic examination of pancreas Representative BLI at the end of the study period and total photon fluxps induction from pancreas at and weeks of age1 a pancreasbody weight percent ratio b representative pancreas histology coriginal magnification 100X scale bar μm and cumulative incidence of PaC d in control Ctr diabetic Diab and Diab treated with FL DiabFL KCM mice at the time of sacrifice Statistical significance between groups for pancreasbody weight percent ratio a wascalculated using oneway ANOVA followed by the StudentNewmanKeuls test for multiple comparisons Each dot represents one case and barsrepresent mean ± SEM Statistical significance for PaC incidence b was assessed using the Chisquared test and Fisher™s exact test P vsCtr  P vs Diab Is islet invasive PaC arrows PanINs 1Except for three Diab and one Diab FL mice which were killed and weeksrespectively before the end of the study see œResults section for further detailsCML also stimulated PDA cell proliferation FL926 was able to inhibit cell proliferation induced byMGO and GO but not CML Fig 5c Treatmentwith CML but not with MGO induced ERK activation and FL92616 was ineffective in counteractingphosphorylation status Fig 5d However the proliferating effect of both the RCS MGO and the AGECML was associated with YAP1 nuclear persistenceand activity Again FL92616 efficiently preventedeffectCMLtheofonERKthe nuclear translocation of YAP1 induced by MGObut failed to counteract the effect of CML Fig 5eConsistently FL92616 treatment reversed the MGOinduced upregulation of gene expression of CTGFWnt Family Member 5A WNT5A and EpithelialMembrane Protein EMP2three wellrecognizedYAP target genes [ ] Conversely FL92616was ineffective in preventing the modulatory effect ofCML on the mRNA level of these genes Supplementary Fig S2 in Additional file 0cMenini Journal of Experimental Clinical Cancer Research Page of CtrDiab1Diab FL1Ctr FL ± ±     ± Table Pancreatic cancer PaC incidence PancreasBodyweight Wt percent ratio and metastasisPaC NtotPancreasBody Wt Metastasis Ntot PaC ± STZnonDiab ± Cumulative incidence of PaC and PancreasBody weight Wt percent ratio incontrol Ctr diabetic Diab Diab treated with FL92616 DiabFL Ctr treatedwith FL92616 Ctr FL and streptozotocintreated nondiabetic STZnonDiab KCM mice at the end of the study weeks of diabetes weeks ofage1 The number of KCM mice with metastasis liver and or lung on thetotal number of PaC cases is also shown KCM LSLKrasG12D Pdx1Cre MITONtot number of casestotal number of mice Ntot PaC number of casestotal number of PaC PaC ductal adenocarcinoma and hepatic andor lungmetastasis were confirmed by histology P or P vs Ctr   P or  P vs Diab Statistical significance between groups forPancreasBody Weight percent ratio was calculated using oneway ANOVAfollowed by the StudentNewmanKeuls test for multiple comparisonsStatistical significance for PaC rate was assessed using the Chisquared testand Fisher™s exact test Except for three Diab and one Diab FL mice which were killed and weeks respectively before the end of the studyMechanisms underlying RCS and AGEinduced YAPactivationSilencing of YAP1 using two independent siRNAssiYAP1 and Fig 6a significantly inhibitedthe transcription activity of YAP target genes induced by both MGO and CML in PDA cells Fig6b In MGOtreated cells YAP induction was associated with a decrease in protein levels of LATS1 awellestablished negative regulator of YAP activity[] whereas CML treatmentfailed to modulateLATS1 Fig 6c Instead treatment with CML butnot with MGO was found to induce EGFR phosphorylation pEGFR Fig 6d EGFR silencing Fig6e almost completely reversed YAP1 nuclear translocation Fig 6f KRAS activation and ERK phosphorylation Supplementary Fig S3ABinduced by CMLEffects of serum from diabetic patients on proliferation ofhuman PDA cellsThe levels of AGEs were ± μgmL in thepooled sera from diabetic patients and ± μgmLin pooled sera from nondiabetic individuals The diabetic serum induced a 3fold increase in PDA cell proliferation compared to the nondiabetic serum This effectwas greatly reduced by prior selective AGE removalfrom the diabetic serum AGE levels ± μgmLand almost completely reversed by combining AGE removal from serum and FL92616 treatment of PDAcells Fig DiscussionDespite the epidemiological evidence of increased PaCrisk in both type [] and [ ] diabetestheunderlying mechanisms still remains to be elucidatedHere we showed that STZinduced type diabeteswhich is characterized by marked hyperglycaemia andinsulin ia without weight gain [] significantlyaccelerated tumour progression in a mouse model ofKras“driven PaC The absence of obesity and insulinresistance argues in favour of the hypothesis that thePaCpromoting effect of diabetes is directly related tothe adverse effects of hyperglycaemiaIn additionRCS trapping and AGE inhibition by FL92616 efficiently prevented the acceleration of PanIN progression to invasive PaC induced by diabetes Thedifference in the incidence of PaC between the twodiabetic groups ie untreated and treated with FL occurred despite similar increases of bloodglucose levels supporting the conceptthat glucosemetabolites but not glucose per se were responsiblefor PaC promotion STZtreated mice that failed todevelop or reversed hyperglycaemia showed the samePaC incidence as the Ctr group thus ruling out aneffect of STZ on invasive PaC development in DiabmiceOur finding of an association between AGE accumulation and YAP induction in PaC in

mgat5 knockout ko in hek293 cells induces metabolic changes resulting in increased intracellular udpglcnac increasedglycolysis enhanced spare respiratory capacity and higher citrate flux from the mitochondria mgat5 ko cells express constitutively high mica mainly regulated onthe transcriptional level through opening of the chromatin at the mica promoter mica expression in mgat5 ko cells is dependent on citrate turnover and histoneacetylation blocking citrate flux inhibits mica expression in numerous cancer cell lines and we propose that this is a central metabolic regulation of mica andimmune surveillanceintroductionnatural killer nk and cd8 t cells monitor autologouscells for markers of tumorigenesis and stress these immunecells express the nkg2d receptor that recognizes nkg2dligands nkg2dls upregulated on the surface of transformedcells nkg2dl expression is in many ways a doubleedged sword upregulation of nkg2dls on cancer cellsenhance nk cell ltration and promote cancer cytotoxicity conversely numerous cancer cells maintain chronicnkg2dl expression and evade immune elimination bydownmodulating and impairing nkg2d receptor signaling“cancer cells that block nkg2dl surface expression toevade immune recognition and clearance can be treated withstressinducers such as histone deacetylase inhibitors hdaci™sheatshock or shortchain fatty acids scfas that upregulatenkg2dls to date studies have primarily focused ondelineating transient nkg2dl induction whereas not much isknown about regulation of their constitutive expressionmetabolic reprogramming is a central hallmark of cancercancer cells use aerobic glycolysis that was initially believedto be a result of dysfunctional mitochondria howeverlater advances have shown that cancer cells often use aerobicglycolysis alongside mitochondrial oxidative phosphorylationoxphos mitochondria are not merely the powerhouseof the cell but also provide metabolites for anabolic pathwaysnecessary for cell growth citrate can be exported from thetricarboxylic acid tca cycle for biosynthetic purposes inthe cytosol citrate is cleaved by atp citrate lyase acly togenerate acetylcoa and oxaloacetate oaa citrateis an inhibitor of glycolysis thus to maintain high aerobicglycolysis cancer cells require low cytoplasmic citrate moreover conversion of citrate by acly is a critical regulatorof gene transcription by producing acetylcoa for histoneacetylation several of these cancerassociated metabolicfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionproperties are shared with other highly proliferating cells suchas activated t cellsexpression of nkg2dls is associated with hyperproliferation and thus with highly active metabolism two studies havelinked nkg2dl expression to active glycolysis whereasone study reports that inhibition of glycolysis increased basalnkg2dl expression in breast cancer cell lines thesestudies emphasize a link to proliferative cell metabolism andsuggest that the role of glycolysis in nkg2dl regulation iscontextspecificnkg2dls fall into two groups the ul16 binding protein ulbp16 and the mhc class i chainrelated proteins aand b mica and micb surface expression of each nkg2dlis regulated individually and at all levels of protein biogenesis we have previously shown that surface expression ofspecific mica alleles depends on nglycosylation nacetylglucosaminyltransferase v mgat5 is an oncoproteincatalyzing the formation of 16branched nglycans thatpromote surface retention of glycoproteins but it is notknown if mgat5 regulates surface expression of mica growthfactor receptors are examples of mgat5 substrates and mgat5overexpression is associated with growth adhesion invasionand metastasis of cancer “ inhibition of mgat5 reducestumor growth enhances the antitumor responses by cd4 tcells and macrophages and promotes th1 diï¬erentiation in this study we examine the metabolic regulation of thenkg2dl mica we discover that mica was increased aftermgat5 knockout ko in a metabolically dependent way anduse this as a model to investigate the regulatory mechanismsof constitutive mica expression we find that glycolysis andmitochondrial export of citrate promotes constitutive micatranscription in mgat5 ko cells a regulation that was alsoshown in several micaexpressing cancer cells in particularincreased mica transcription was associated with alteredchromatin accessibility of the mica promoter our findingssuggest that citrate drives a metabolic stress that modulateschromatin accessibility to facilitate basal mica transcription andthereby regulate immune surveillancematerials and methodsanimalsfemale nmri mice to 10weeks old taconic lille skensveddenmark were used and all studies were performed inaccordance with the danish act on animal experimentationwhich implements directive 201063eu on the protection ofanimals in scientific research the studies were approved by theanimal experimentation inspectorate ministry of environmentand food denmark license no healthmonitoring was carried out in accordance with federation forlaboratory animal science associations guidelinesreagents pharmacological inhibitorsand dna constructspharmacologicalfrom sigmaaldrich werecompoundsnacetyldglucosamine glcnac a3286 pugnac a7229carbonylcyanide2dg d61345aminoimidazole4carboxamide2deoxydglucosetrifluoromethoxyphenylhydrazone fccp c2920 uk5099pz0160 bis25phenylacetamido134thiadiazol2ylethylsulfide bptes sml0601 potassium hydroxycitrate tribasicmonohydrate hc sodium dihydrogencitrate sodium acetate s5636 oxaloacetic acid oaa o41266mercaptopurine monohydrate 6mp azaserinea4142ribonucleotideaicar a9978 nacetylcysteine nac a9165 sodiumpropionate p1880 sodium butyrate b5887 dmso d2438pbs d8537 etomoxir sodium salt was purchased fromcayman chemicals ann arbor mi united states bristol bms303141 wasunited kingdom the gfpmycmicaˆ— and micaˆ— vectors containingthe coding sequences of micaˆ— or micaˆ— alleledownstream of a generic leader a gfp cassette and a myctag were provided by dr m wills university of cambridgecambridge united kingdom pgl3basic pgl3bluciferase vector was purchased from promega promegamadison wi united states e1751 micafirefly luciferasepromoter vectors and sv40renilla luciferase promoter vectorwere provided by prof c o™callaghan university of oxfordoxford united kingdom from tocris biosciencepurification of peripheral bloodlymphocyteshuman peripheral blood mononuclear cells pbmcs wereisolated by histopaque1077 sigmaaldrich st louis mounited states separation from buï¬y coats obtainedfrom healthy blood donors the capital region blood bankcopenhagen university hospital copenhagen denmark toobtain peripheral blood lymphocytes pbls pbmcs weredepleted from monocytes by incubation with dynabeadsinvitrogen carlsbad ca united states as previouslydescribed pbls were activated in rpmi1640 withoutglucose gibco gaithersburg md united states supplemented with dialyzed fetal bovine serumfbs f9665 mm penicillinstreptomycin p4333 mmlglutamine g7513 mm sodium pyruvate s8636 and either mm dglucose g8769 or mm dgalactose g6404all purchased from sigmaaldrich pbls were activated withcd3cd28 beads invitrogen 11132d and 20uml hil2peprotech rocky hill nj united states for dayson day pbls were treated with ngml fr901228 nationalcancer institute bethesda md united states for hline pc3 and the keratinocytederived cellcell line cultivation and proliferationhuman embryonic kidneyderived hek293 cells the prostatecancer celllinehacat were purchased from american type culture collectionatcc manassas va united states nkg2d reporter cellct312 and the 2b4 parental cellline were kindly providedby chiwen chang trowsdale lab cambridge universitylines mdamb231 and mcf7 werethe breast cancer cellprovided by dr jos moreira departmentfor veterinaryfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressiondisease university of copenhagen denmark and henrikleï¬ers the state hospital copenhagen denmark respectivelythe cervical cancer cellline hela was provided by jesperjurlander the state hospital copenhagen denmark themelanoma cells skmel28 fm55m1 fm78 and fm86 andthe human colon adenocarcinoma cell lines ht29 and sw480were provided by dr per thor straten herlev universityhospital denmark hek293 mdamb231 and mcf7 cellswere cultured in dmem with glutamax gibco hela hacat pc3 fm55m1 fm78 fm86 skmel28 andsw480 were cultured in rpmi1640 sigmaaldrich r5886and ht29 were cultured in mccoy™s 5a medium sigmaaldrich m8403 media were supplemented with fbs and mm penicillinstreptomycin mm lglutamine was addedto rpmi1640 and mccoy™s 5a for longterm cell culture inglucosegalactose cells were cultured in dmem medium withoutglucose gibco supplemented with dialyzedfbs mm penicillinstreptomycin mm sodium pyruvate and mm glucosegalactose all cells were kept at culture conditions—¦c and co2 and were passaged every “ daysfor proliferation assay wt and mgat5 ko cells wereseeded in — or — cellswell for each experimentcells were counted in triplicate wells after and h usingthe biorad tc20 automated cell counter biorad herculesca united statesgene editingmgat5 ko cells were generated by zinc finger nucleasetargeting in hek293 cells and subsequent cloning and selectionwas performed as described previously hek293cells were transfected with mrna sigmaaldrich or µgof endotoxin free plasmid dna using nucleofection on anamaxa nucleofector lonza copenhagen denmark mgat5ko clones were selected by loss of reactivity with lphaand clones were confirmed to have mgat5 mutations usingpcr and sequencinglentiviralmediated gene transfer was performed with anmgat5 encoding vector constructed by inserting the mgat5sequence generated as a bluntend pcr product from a vectorfrom hw university of copenhagen copenhagen denmarkinto an entry vector system using the pentr directionaltopo cloning kit invitrogen k243520k350020 followingmanufacturer™s protocol topo clonal reaction entry vectorswere transformed into macht1 chemically competent e coliusing heatshock and soc medium followed by selectionpcr inserts were confirmed by sequencing at eurofins mwgoperons luxembourg colonies were amplified and plasmidswere purified with nucleobond xtra midi kit machereynagelduren germany mgat5 sequences were insertedinto plx302 lentiviral destination vector with lr clonase iienzyme mix invitrogen after proteinase k treatmentconstructs were transformed into dh5α using heatshock andsoc medium selected clones were amplified and dna waspurified using nucleobond xtra midi kit destination vectorswere checked for insertion using bsrgi digestion at —¦cmgat5coding lentiviral ps were packaged in hek293tcells transfected with a mix of µg pspax2 vector packagingvector µg pcmvvsvg envelope vector µg plx302vector carrying mgat5 and µl cacl2 to a final volume of µl the dna mixture was complexed with µl 2x hbsunder constant air flow and the transfection mix was addeddropwise to — hek293t cells in antibioticfree mediumcell culture medium was harvested days after transfection andviral p preparations were prepared by centrifugation at — g for min lentiviral ps were added to cells andincubated for h cells were cultivated in puromycin µgmlselection medium for weeks functional mgat5 expressionwas validated by lpha bindingtransient transfectiontransient transfections were performed as described previouslyusing amaxa nucleofector device lonza dna wasintroduced to — cells in µl nucleofector solution vlonza vca1003 and pulsed using the nucleofector programq001 for gfpmyctagged micaˆ— and micaˆ—constructs cells were transfected with µg dna and analyzedthe next day transfection with shrnas or luciferase promoterconstructs was carried out by calciumphosphate transfectionbriefly dnarna were prepared in µl cacl2 25mand adjusted to a final volume of µl dna mixture wascomplexed with µl 2x hbs hepes nacl na2hpo4and added dropwise to — cells scrambled sirnacontrol siidh1 and siidh2 ontarget plus smart poolswere purchased from ge healthcare dharmacon lafayetteco united statesfunctional assaysfor nkg2d downmodulation pbls were isolated as describedabove followed by depletion of cd4 cells using cd4 antibodyebioscience san diego ca united states anddynabeads mouse panigg invitrogen cd4depletedpbls were cultured in rpmi1640 sigmaaldrich r5886supplemented with human serum sigmaaldrich h3667 mm penicillinstreptomycin mm lglutamine and ngmlhil15 peprotech for days to enrich for nkcd8t cells nkg2d downmodulation assay was performed aspreviously described nkg2d ligands on eï¬ector cellshek293 wt or mgat5 ko cells were incubated with blockingnkg2dfc rd systems minneapolis mn united states1299nk or control igg1fc rd systems 110hg µgmlfor min at —¦c eï¬ector cells and target cells nkcd8t cells were mixed at indicated eï¬ectortarget ratios and spundown min — g to allow conjugate formation after h cocultivation nkcd8 t cells were analyzed for nkg2d surfaceexpression by flow cytometry using accuri c6 flow cytometerbd bioscience franklin lakes nj united statesfor the reporter cell assay the nkg2dreporter cell line2b4ct312 and the parental control 2b4 cell line target cells were mixed with eï¬ector cells wt or mgat5 ko cellsthat were either blocked with nkg2dfc or control igg1fc asdescribed above eï¬ector and target cells were cocultivated atdiï¬erent et ratios for “ h gfp expression of target cellswas assessed with accuri c6 flow cytometer for in vivo assaytarget cells were labeled with vybrant did celllabeling solutionfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioninvitrogen v22887 according to manufacturer™s protocol andinjected intraperitoneally together with wt or mgat5 kocells in a ratio — of each “ mice were usedper group target cells were harvested after approximately hwith peritoneal lavage and nkg2d activation of didpositivereporter cells were assessed as gfp expression with accuric6 flow cytometerwas assessed by accurate mass and retention time amrt plusfragment identification at two collision energies and evdetailed acquisition methodology has been described previously udpglcnacudpgalnac detected peak screened byexpected calculated mass could be of either compound as thesetwo sugars could not be separated chromatographically hencehas been reported as a putative metabolite pending confirmationlactate and dntp measurementsconcentrations of llactate was measured enzymatically withrandox colorimetric assay according to manufacturer™s protocolrandox crumlin united kingdom lc2389 reaction andanalysis was performed on an advia chemistry systemsiemens munich germanydntp levels were determined in — cells harvested withtrypsinization and pelleted by centrifugation for — g for min followed by resuspension of cell pellets in methanolfrozen in liquid nitrogen and boiled at —¦c for min sampleswere evaporated until dryness in a speedvac and whole cell levelsof dttp datp dctp and dgtp were determined using the dnapolymerase assay previously described lchrms metabolite profilingto determine intracellular metabolite levels cell pellets from — cells were resuspended in µl of cold methanolafter min sonication samples were prepared by svortex followed by min equilibration at room temperatureafter centrifugation at — g for min at —¦c µl supernatants were collected transferred to ultrafreemccentrifugal filter devices merck millipore ltd cork irelandand centrifuged at — g for min at —¦c from this µlwas transferred to lc vials and µl of each sample was pooledto a mixed qc samplelchrms was performed on a nity ii ultrahigh performance liquid chromatography uhplc systemcoupled to a ifunnel quadrupoletime of flight qtofmass spectrometer equipped with a dual ajs electrosprayionization source agilent technologies santa clara caunited states polar metabolites were separated on a sequantzichilic merck darmstadt germany column mm — mm µm p size coupled to a guardcolumn mm — mm µm p size and an inlinefilter mobile phases consisted of formic acid in water withsolvent a and formic acid in acetonitrile with solvent bthe elution gradient used was as follows isocratic step at bfor min b to b in min and maintained at bfor min then decreasing to b at min and maintainedfor min then returned to initial conditions over min and thecolumn was equilibrated at initial conditions for min the flowrate was mlmin injection volume was µl and the columnoven was maintained at —¦c the acquisition was obtainedwith a mass range of “ mz for where full scan highresolution data is acquired at three alternating collision energies ev ev and ev positive and negative raw lchrmsfiles were independently processed with an inhouse developedpcdl library for polar metabolites using profinder version b06agilent technologies identification of reported compoundsextracellular flux analysisthe seahorse xfe96 extracellular flux analyzeragilenttechnologies was used to measure ocr and ecar on hek293cells cells were seeded at the density — cellswell ˆ¼ hbefore the experiment one hour prior to assay run cells wererinsed and switched to xf media agilent technologies with mm sodium pyruvate and mm glucose or galactose andincubated at —¦c co2free incubator for the mitochondrialstress tests ocr was measured under basal conditions andduring sequential injection of µm oligomycin sigmaaldrich µm fccp sigmaaldrich c2920 and µmrotenone rot sigmaaldrich r8875 µm antimycina aa sigmaaldrich a8674 reported basal respiration iscalculated from the third measuring point with ocr after rotand aa subtracted atpcoupled respiration display ocr afteroligomycin subtracted from the third measuring point andmaximal respiration is ocr after fccp with ocr after rotand aa subtractedfor measuring the eï¬ect of hc ocr was assessed h after aninjection of mm hc13c6glucose tracing experiment — cells were incubated for h in dmem medium withoutglucose supplemented with fbs mm sodium pyruvateand mm uniformly labeled [u13c]glucose cambridgeisotope laboratories tewksbury ma united states clm incubation medium samples were collected and cleared bycentrifugation — g for min cells were washed and detachedsterically intracellular metabolites were extracted in ethanoland centrifuged at — g for min —¦c to separatethe soluble extract supernatant from the insoluble componentspellet cell extracts and medium samples were lyophilizedand reconstituted in water for subsequent biochemical analysesextract samples were adjusted to ph with hcl and evaporatedto dryness under nitrogen flow analytes were extracted into ananic phase ethanolbenzene followed by derivatizationwith dmf86 mtbstfa with a modified procedure from standards containing unlabeled metabolites of interest andcell extracts were separated and analyzed in a gas chromatographagilent technologies 7820a chromatograph jw gc columnhp5ms parts no 19091s433 coupled to a mass spectrometeragilent technologies 5977e the isotopic enrichment of themetabolites of interest was corrected for natural abundance of 13cusing the unlabeled standards and calculated according to data are presented as labeling of m x where m is the massof the unlabeled molecule and x is the number of labeled catomsin a given metabolite frontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionwestern blottingproteins were extracted using ripa buï¬er thermo scientificwaltham ma united states and proteinasephosphataseinhibitor cocktail thermo scientific for minon ice lysates were sonicated times for s and clearedby centrifugation at rpm for min at —¦c proteinextracts were denatured at —¦c for min in nupage samplebuï¬er and dtt sigmaaldrich proteins were resolvedusing “ sdspage gels invitrogen and transferred tonitrocellulose membranes invitrogen ib301001 using the iblotdevice invitrogen for total protein stain membranes werewashed in ddh2o and stained with revert protein stainsolution licor biosciences lincoln ne united states according to manufacturer™s protocol membranes wereblocked in tbst blocking buï¬er licor biosciences “ probed with primary antibodies in tbs w tween and bsa overnight on a shaker at —¦c and washed intbs tween secondary antibody was from licorlicor biosciences “ and signals were visualizedby the odyssey fc imaging system licor biosciencesoglcnacylation was detected with rl2 oglcnacylationantibody abcam cambridge united kingdom ab2739 atpcitrate lyase acly was detected with rabbit acly antibodycell signaling and acly phosphorylation with rabbitphosphoacly ser455 antibody cell signaling flow cytometryadherent cells were detached in pbs w mm edta invitrogen or by pipetting cell surface staining was done aspreviously described and cells were analyzed on accuric6 flow cytometer bd bioscience antibodies used for thisstudy were mica rd systems fab1300a ulbp256 rdsystems fab1298p nkg2d rd systems fab139a ulbp1rd systems fab1380p ulpb3 rd systems fab1517aulbp4 rd systems fab6285a micab bd bioscience icam1 leinco technologies c170 mouse igg1antimyctag merck millipore micb rd systemsmab1599 or igg2b isotype control rd systems mab004detected with secondary antimouse igg biolegend san diegoca united states binding of fluorescently labeledaf647lpha invitrogen l32457 and fitcepha vectorlaboratories burlingame ca united states fl1121 was usedto measure surface levels of complex nglycans all isotypecontrols were purchased from bd biosciencefor staining with mitochondrial probes neutral lipid stainsor 2nbdg uptake — cells seeded the day prior toexperiment were washed once in pbs and incubated for minat —¦c and co2 in warm growth medium containing nmtetramethylrhodamine methyl ester perchlorate tmrm sigmaaldrich t5428 nm mitotracker green fm invitrogenm7514 or for h in growth medium with µm 2nbdginvitrogen n13195 bodipy invitrogen d3922 wasdiluted in warm serumfree medium in a dilution andshaken vigorously to solubilize the lipids immediately beforeloading into the cells for min cells were washed twice inpbs fbs and detached sterically prior to analysisthe soluble nkg2d“fc receptor 1299nk rd systemsand igg1“fc 110hg rd systems were labeled with zenonalexa fluor against human igg1 z25408 invitrogen priorto staining of melanoma cellsin forwardsidescatter plotsdata were acquired with an accuri c6 instrument usingaccuri c6 software and analyzed in flowlogic v721 inivaitechnologies mentone vic australia by gating on viablecellsfollowed bysingle cell gating by areaheightscatter plots fscafsch geometric mean fluorescent intensity mfi values aredisplayed in figures as mfi or with corresponding isotype controlsubtracted as 01mfifscsscreal time pcr analysistotal rna was extracted by phase separation in trizolchlorophorm and purified on directzol spincolumns zymoresearch irvine ca united states according to manufacturer™sprotocol cdna was generated using superscript cdnasynthesis kit invitrogen under standard pcr conditionsfollowing primersequences were used for quantitativertpcr with brilliant sybr green qpcr master mixkit mica mica_f tggcagacattccatgtttctgmica_r ctcgtcccaactgggtgttg ulbp2 ulbp2_f cagagcaactgcgtgacatt ulbp2_r ggccacaaccttgtcattctidh1 idh1_f ctatgatggtgacgtgcagtcg idh1_r cctctgcttctactgtcttgccidh2 idh2_f agatggcagtggtgtcaaggagidh2_r ctggatggcatactggaagcag glut1 glut1_fctgctcatcaaccgcaac glut1_r cttcttctcccgcatcatct glut2 glut2_f tacattgcggacttctgtgg glut2_r agactttcctttggtttctgg glut3glut3_f cagcgagacccagagatg glut3_r ttggaaagagccgattgtag glut4 glut4_f tgggcttcttcatcttcacc glut4_r gtgctgggtttcacctcctrplp0_fcctcgtggaagtgacatcgt rplp0_r cattcccccggatatgaggc realtime qpcr was performed on biorad cfx96 realtime thermal cycler c1000 touch and alltranscripts were normalized to housekeeping rplp0 transcripthousekeepingand rplp0asgeneluciferase reporter assaycells were transiently transfected using calciumphosphatetransfection as described above with firefly luciferase promotervectors µg and an sv40promoter driven renilla luciferasevector µg cells were harvested and snap frozen hpost transfection pellets were lysed in dualglo luciferasereagent promega e2920 and firefly luciferase activity wasanalyzed by luminometer microbeta ii perkinelmer walthamma united states renilla luciferase activity was recorded bythe instrument after subsequent addition of volume dualglo stop glo promega e2920 to correct for transfectionefficiency firefly luciferase signals were normalized to sv40renilla luciferase signals of corresponding sampleatacseqatacseq was performed as described previously foreach cellline cells were harvested from separatefrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressioncultures and used to prepare tagmented chromatin replicatesof wt and replicates of mgat5 ko cell lines samplestotal quality of pcramplified sequencing libraries was assessedusing a tapestation instrument with high sensitivity dnascreentapes agilent libraries were sequenced as paired endreads on a single lane of an illumina hiseq4000 flow cellresulting reads were aligned to the grch37hg19 referencegenome using rsubread and alignments were filtered toremove low quality duplicate and mitochondrial reads peakswere called using macs2 on merged reads from allsamples and diï¬erential peak accessibility between cell lines wasdetermined using edger with a threshold false discoveryrate of transcription factor binding motifs enriched indiï¬erentially accessible peaks were identified using homer h3k4me3 chipseq data were downloaded from encode1 andare available under accession encff756ehfquantification and statistical analysisresults are presented as mean ± sem diï¬erences were analyzedfor statistical significance using prism or graphpad softwarela jolla ca united states statistical analysis was performed asstated in figure legends using unpaired ttest in 1a 1c 1e 3ef3h 5c 7a 7ef paired ttest in 4fg 7d multiple ttest in 1b1d 3d 4ab one sample ttest in 2ac 3c 4c 4e 7g twowayanova in 3a 5df 5hi 6a 6e 7hi or oneway anova in5g level of statistical significance was determined by ˆ—p ˆ—ˆ—p and ˆ—ˆ—ˆ—p ˆ—ˆ—ˆ—ˆ—p resultsmgat5 knockout increases nkg2dlexpression and activates nkg2d in vitroand in vivoregulation of constitutive mica expression remains largelyunknown surface expression of certain mica alleles dependson nlinked glycosylation we questioned whetherthe cancerassociated glycosyltransferase mgat5 is required formica expression to assess the role of mgat5 in regulationof nkg2dl surface expression mgat5 ko clones weregenerated in hek293 cells remarkably mgat5 ko resultedin a permanently increased surface expression of the nkg2dlsmica micb and ulbp256 compared with parental wildtypewt cells figure 1a to confirm mgat5 ko we measuredbinding of leukoagglutinin from p vulgaris lpha that bindsspecifically to mgat5modified nglycans as expected lphabinding was reduced whereas binding of erythroagglutininfrom p vulgaris epha that interacts with mgat3modifiednglycans was unaï¬ected thus verifying functional knockoutof mgat5 figure 1a modification of mgat5 expressiontherefore associated with substantial changes in constitutiveexpression of several nkg2dlsto verify the functionality of mgat5 koinduced nkg2dlswe tested nkg2d activation in a reporter cell line expressing1httpswwwencodeprojecthuman nkg2d coupled to dap10cd3ζ signaling and nuclearfactor of activated t cells nfatcontrolled gfp ultimatelyexpressing gfp in response to nkg2d activation nkg2dgfp activation was higher after cocultivation with mgat5ko cells than with wt cells figure 1b corresponding to theincreased nkg2dl expression in mgat5 ko cells figure 1athe reporter cells without nkg2d supplementary figure s1aremained inactivated indicating that the activation was nkg2dmediated figure 1b moreover blocking nkg2dls withsoluble nkg2dfc receptor impaired the activation furthervalidating nkg2d specificity supplementary figure s1bto test if mgat5 ko cells could activate nkg2d in vivowe adoptively injected nkg2d reporter cells together with wtor mgat5 ko cells into the peritoneum of nmri mice andmeasured gfp expression in reporter cells in line with ourin vitro data we observed a significant increase in nkg2dgfpactivation by mgat5 ko cells compared with wt cells theresponse was nkg2dspecific since the control reporter cellswere unaï¬ected figure 1c these data verify that mgat5 koinduced nkg2dls maintain their functional integrity in vivonkg2d is downmodulated upon activation to furtherexamine the functionality of nkg2dl expression causedby mgat5 ko we assessed nkg2d downregulation afterreceptor activation nkg2d was further downregulated oncd4depleted peripheral blood lymphocytes pbls after cocultivation with mgat5 ko cells than with wt cells and thisdownregulation was abolished by blocking nkg2dls with asoluble nkg2dfc receptor figure 1d combined these dataindicate that ko of mgat5 upregulates mica and ulbp256resulting in nkg2d activation in vitro and in vivoto ensure that the mica upregulation was a result of mgat5ko we stably transfected mgat5 into wt and mgat5 kocells lpha binding was restored within days after transfectionconfirming expression of functional mgat5 interestingly ittook multiple passages for mica expression to decrease to wtlevels figure 1e and supplementary figure s1c suggestingthat mica is regulated in response to a longterm adaptation toaltered mgat5 expressionudpglcnac upregulates micaexpressionlongterm mgat5 deficiency willlikely result in aberrantnglycosylation and an accumulation of the mgat5 donorsubstrate udpnacetylglucosamine udpglcnac to addressif mica was regulated by a change in nglycosylation inmgat5 ko cells we assessed the posttranslational regulationof mica by measuring surface expression of transgenicallyexpressed gfpmyctagged mica under a cytomegaloviruscmv promoter the mica alleles micaˆ— and micaˆ—are distinctly regulated posttranslationally and althoughmicaˆ— was upregulated in mgat5 ko cells the regulationwas minor and unlikely to account for the profound changein endogenously expressed mica figures 1a 2a micatranscripts on the other hand were highly increased in mgat5ko cells figure 2b as well as ulbp2 mrna supplementaryfigure s2a suggesting that nkg2dls are transcriptionallyfrontiers in immunology wwwfrontiersinaugust volume 0cm¸ller citrate facilitates mica expressionfigure mgat5 knockout increases nkg2dl expression and activates nkg2d in vitro and in vivo a surface expression of nkg2d ligands and binding offluorescently labeled lpha mgat5 modifications or epha mgat3 modifications on hek293 wildtype wt and hek293 mgat5 knockout ko cells or isotypecontrol staining iso analyzed by flow cytometry data are presented as histograms representative of at least three independent experiments and in bar graphsshowing mean fluorescence intensity mfi b in vitro nkg2d activation measured as gfp expression in nkg2d negative reporter cells control and nkg2dexpressing nkg2d reporter cells target cells cocultivated with wt or ko cells effector cells for “ h at indicated effectortarget et ratios c nkg2dactivation in vivo measured on reporter cells as in b after activation by wt or ko at a ratio in peritoneum of nmri mice for approximately h gfp expressionin didlabeled reporter cells signifies nkg2d activation and is shown as gfp mfi values of cells from foursix mice per group d nkg2d downmodulation wasassessed on nkcd8 t cells target cells after cocultivation for h with wt or ko cells effector cells at indicated effectortarget ratios et nkg2dls on targetcells were blocked with nkg2dfc bl or unblocked with igg1fc un the graph depicts surface expression of nkg2d presented relative to surface nkg2dexpression on target cells alone e mica surface expression left and lphaepha surface binding right after lentiviral introduction of mgat5 into wt or kocells mfi values from antibody staining were corrected for isotype background staining 01mfi statistical analysis was performed by unpaired ttests in ace andmultiple ttest with fdr comparing wt and ko in bd p p p and p regulated in mgat5 ko cells notably we found that themgat5 substrate udpglcnac although indistinguishablefrom udpnacetylgalactosamine udpgalnac tended tobe higher in mgat5

several immunotherapeutic strategies that harness the exquisite specificity of the immune systemto eliminate tumors have emerged during the past decade these include cancer vaccines immunecheckpoint blockade and adoptive cell therapy act with the potential to revolutionize thestandard of care for a range of malignanciesto a large extent the specificity of immunotherapy is dependent on the recognition of specifictumor antigens especially neoantigens neoantigens are a kind of tumor antigen derived fromtumorspecific somatic mutations and are highly restricted to tumor cells with minimal establishedimmune tolerance neoantigenbased cancer vaccines have shown promising therapeutic eï¬ectsin the clinic “ in addition a growing body of evidence indicates that neoantigenspecific tcells underlie the success of the recently emergent immune checkpoint inhibitor therapy “adoptive transfer of autologous in vitro expanded tumorltrating lymphocytes tils wasreported to achieve dramatic clinical responses in some metastatic cancer patients especially inthose with melanoma and cervical cancer “ indepth studies have revealed the criticalroles of neoantigenspecific t cells in maintaining durable responses following act “in support of these findings the adoptive transfer of selected tils targeting neoantigens led tosignificant tumor regression “ increasing research attention has been shifted to identifyingand selecting neoantigenspecific t cells “ however such a œprecise targeting strategy posesa great challenge in terms of the identification and isolation of neoantigenspecific t cells methodshave been proposed and developed for this purpose here we attempt to summarize the knownstrategies for isolating neoantigenspecific t cellsedited bycyrille j cohenbarilan university israelreviewed bymanel juanhospital cl­nic de barcelona spainrodabe n amariauniversity of texas md andersoncancer center united statescorrespondencezhenyu dingdingzhenyuscueducnspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in oncologyreceived december accepted june published august citationli q and ding zy the ways ofisolating neoantigenspecific t cellsfront oncol 103389fonc202001347frontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspecific t cellsidentification and isolation ofneoantigenspecific t cells fromtilsfor most metastatic patients this time frame is unacceptable toaddress these issues additional attempts have been made usingeither surface markers or t cell receptor tcr redundancyresearchers have long attempted to isolate neoantigenspecificsubpopulations from the of transferred tils in earlystudies an autologous tumor cell cdna library was constructedand used as a pool to screen for neoantigenspecific t cells in a study of a melanoma patient who experienced acomplete response going beyond years following adoptive tiltransfer one t cell clone specific for a mutated antigen ppp1r3bwas identified and shown to be responsible for the antitumoreï¬ects advanceshowever the timeconsuming and laborious process requiredto identify neoepitoperesponsive t cells has hindered theirextensive functional assessmentin nextgeneration sequencing have enabled the rapid assessment of themutational landscape of human cancers and made it possibleto identify immunogenic mutated tumor antigens throughin silico analysis rosenberg™s group first employed predictedneopeptides obtained by wholeexome sequencing and humanleucocyte antigen hla class i“binding algorithms for tilscreening using this approach they identified neoantigensrecognized by therapeutic bulk til cultures that mediatedobjective tumor regressions in three individuals with melanoma using a similar method neoantigenspecific cd8 tilscould also be identified in hematological malignancies such asacute lymphoblastic leukemia all prickett and stevanovic also demonstrated that neoantigenspecific t cells could be identified from therapeutic tils byscreening tandem minigene tmg libraries encoding cancermutations identified from patients™ tumors by wholeexomesequencing this finding might further facilitate the recognitionof neoantigenspecific t cells because it circumvents the needfor prediction of hla“peptide binding and synthesis of a largenumber of peptideswith the advent of these techniques the field of act took agreat leap from bulk tils to neoantigenspecific t cells a conciseflowchart showing the steps involved in identifying and isolatingneoantigenspecific t cells for act is summarized in figure tran successfully performed neoantigenspecific t celltherapy in a 43yearold woman with extensively metastatic andintensively treated cholangiocarcinoma after administration ofa bulk lymphocyte population containing a high percentage ofneoantigen erbb2ipspecific cd4t cells the patient showed alonglasting objective clinical response without obvious toxicitysubsequently neoantigenspecific t cells were identified in onecolon cancer patient and another breast cancer patient andreinfusion of these specific t cells led to a partial response inone patient and a durable complete response in another currently act with neoantigenspecific t cells is beingtested in clinical trials in both solid and hematological tumorssupplementary table howeverthe extensive expansion of neoantigenspecifict cells during preparation compromises their proliferationpotential the method involved requiressophisticated equipment and a time period of several monthsin additionapproaches based on surfacemarkerscd137 belongs to the tumor necrosis factor receptor superfamily it functions as a costimulatory molecule to promote theproliferation and survival of activated t cells cd137expression is highly restricted to transiently activated cd8 tcells but almost undetectable in resting cells upregulated cd137can be detected on stimulated cd8 t cells of all phenotypeseg na¯ve t cells as well as early and late memory eï¬ector tcells naturally occurring tumorreactive t cells stimulatedby tumor antigens also express cd137 as proven by ye in a clinical trial trial registration id nct02111863 among patients with melanoma who underwent adoptive transfer withcd137selected tils only patient achieved partial responseand the remaining progressed the study was terminatedthis approach has its pitfalls because cd137 is an activationmarker cd137 t cells obtained by largescale productionare generally overactivated and highly diï¬erentiated withlimited proliferative potential a potential solution is to obtaintcrs from these cd137t cells instead this strategy wasreported by parkhurst briefly cd8 t cells werestimulated overnight with immunogenic mutated tmg rnassubsequently the cd8 t cell population with the highestcd137 expression was sorted by fluorescentactivated cell sortingfacs and expanded in vitro then dominant tcr α and chains were sequenced in the enriched populations twentyseven tcrs from patients that recognized neoantigensexpressed by autologous tumor cells were identified howeverthis process was timeconsuming “ monthsa simplified protocol was proposed by seliktarofir here tils but not cd8 t cells were coculturedwith autologous tumor cells cd137 t cells were isolatedby magnetic bead separation and expanded no further tcrsequencing was performed the entire process took only dayst cells stimulated with neoantigens or other tumorassociatedantigens exhibit upregulated cd137 expression therefore a cd137based selection protocol was advocated forits broad antigen coverage including both neoantigen and sharedtumor antigens without prior knowledge of epitope specificityhowever the prerequisite of the establishment of autologoustumor cell lines poses a challengedirect and indirect evidence shows thatthe interactionbetween pd1 and pdl1 inhibits t lymphocyte functionleading to evasion of persistent ‚ammatory or autoimmunereactions “ howeverthis protective mechanism ishijacked by tumors to escape immune surveillance pd1 hasbeen characterized as an inhibitory receptor on chronicallystimulated tcells in the tumor microenvironment atthe tumor site tils are exposed to tumor antigensthebinding of tcr and antigen upregulates either costimulatory orcoinhibitory receptors to promote or inhibit t cell activation andfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspecific t cellsfigure the general approach of identifying and isolating neoantigenspecific tils for act the tumor cells from excised tumor tissue and matched normal cellsunderwent wholeexome sequencing wes and rna sequencing to identify nonsynonymous mutations based on the information either tandem minigenes tmgsor peptides were then synthesized these tmgs or peptides were pulsed into autologous antigen presenting cells apcs such as dendritic cells dcs or b cells andthey were processed and presented in the context of major histocompatibility complex mhc on the other side the excised tumors were minced into ˆ¼“ mm3fragments and placed in 24well plates stimulated with il2 then the tils were cocultured with these pulsed apcs the identification of the individualneoantigenspecific t subpopulation was dependent on the ifnγ enzymelinked immunospot elispot assay and the activation of the markers such ascd13741bb or cd134ox40 on the t cell surfaces when recognizing their cognate target antigen t cells with these activation surface markers would be purified byflow cytometry then the sorted t cells were subject to rapid expansion in vitro and reinfusion to the tumorbearing patientfunction respectively therefore pd1 t cell populationsamong tils may contain a large proportion of tumorspecifict cells the findings of inozume and ahmadzadeh that tumorresponsive t cells are enriched amongcd8pd1 lymphocytes from fresh melanoma specimensprovide direct support for this notionin another study gros demonstrated that pd1expression on cd8 tils in fresh melanoma tumor specimensenabled identification of a diverse patientspecific repertoireof clonally expanded tumorreactive cells including mutatedneoantigenspecific cd8 lymphocytes although pd1 is aninhibitory receptor expressed on t cells studies have shownthat il2 restored the antitumor function of t cells in vitro however on antigenexperienced terminally diï¬erentiatedeï¬ector memory temra cells pd1 is either not expressed orexpressed at very low levels therefore a pd1basedenrichment strategy may not be suitable for these cellsscreening strategies based on cd137 or pd1 expressionare suitable for cd8 t cells mainly in melanoma epithelialcancers which accountfor more than of all humanmalignancies harbor fewer mutations than melanoma theyexhibit compromised capability to induce mutationspecific tcell responses together with a limited number of ltratingneoantigenspecific tils in addition cd4t cells havebeen shown to play an important role in mediating tumorregression in animal models and patients “however cd137 or pd1 is expressed on cd8 cells as a solemarker therefore it may not be reliably used to enrich activatedcd4 cells cd134 is transiently expressed on cd4 t cells stimulated by antigens and can be used as a marker forthe classification of mutant reactive t cells recently yossef reported an approach in which thetils that expressed cd134 or cd137andor pd1 were isolatedby facs thus both cd4 t and temra cells were rescuedwhich would otherwise be missed if a single marker were usedsorted cells underwent limitingdilution in microwell plates toavoid the overgrowth of nonspecific t cells cultures were testedfor the ability to recognize a 25mer peptide pool encompassingpossible neoantigens notably the highly oligoclonal natureof these t cells makes possible the convenient applicationof single cell sequencing of their tcrs in patients withmetastatic epithelial cancer this highthroughput approach ledto the detection of cd4 and cd8 t cells targeting and neoantigens respectively whereas only and neoantigenswere identified by using the til fragment screening approachin patients in which no neoantigen was found by traditionalfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspecific t cellsscreening the novel approach identified distinct neoantigenspecific tcr clones for one patient and a highly potent mhcclass ii“restricted krasg12vreactive tcr for the other in ametastatic tumor sample from a patient with serous ovariancancer mhc class ii“restricted tcrs targeting the tp53g245shotspot mutation were identifiedtcr frequencytcr sequence analysis is used as a tool to monitor t cellresponses to specific antigens by measuring the abundance oft cell clonotypes the advent of nextgenerationsequencing has enabled identification of the full tcr repertoireof tils this valuable data for tcrs from tumorreactive tils could be used to modify t cells tcrt howeverthe lengthy expansion process and excessive stimulation wouldresult in tcr repertoire switching to avoid this problempasetto directly performed tcr sequencing of thefresh enzymatically digested melanoma tissues prior to in vitroexpansion as described earlier tumorreactive clonotypes wereenriched in cd8pd1 til subsets in melanoma the authors analyzed the tcr repertoire of tils in cd8cd8“ cd8pd1“ or cd8pd1 subsets respectivelyand found that many of the most frequently occurring tcrclonotypes present in the cd8pd1 til subset recognizedthe autologous tumor and tumor antigens included neoantigensthis report provided a much more convenient approach toefficiently identify tumorreactive t cells based solely on thefrequency of tcr and pd1 expression without prior knowledgeof the specific neoantigen however this strategy must be appliedwith caution because the isolated tcr clones may be selfreactiveand result in deleterious ontarget oï¬tumor toxicities isolation of neoantigenspecific tcells from peripheral bloodlymphocytes pblsin some situations neoantigenspecific t cells were undetectablein the til compartment possibly owing to the following factorspresentation of neoantigens in a non‚ammatory context impaired t cell ltration because of the sparse distributionof adhesion molecules on these cells and presence ofimmunosuppressive cytokines and cells eg regulatory t cellsin the tumor microenvironment furthermore the tissuefrom which tils may be obtained poses a challenge in thisregard peripheral blood is an alternative and reliable source forneoantigenspecific t cellsthe first attempt is considered to have been made by agroup led by lennerz in this study a systemof œmixed lymphocytetumor cells mltc was establishedwherein peripheral blood mononuclear cells pbmcs froma patient with metastatic melanoma were cocultured withautologous tumor cells the mtlc system could be viewed asa simplified in vitro simulation of the tumor microenvironmentfurthermore cytotoxic t lymphocyte ctls clone derived bylimiting dilution from the mltc system or mltc were subjectto autologous tumor cell cdna library screening t cell clonesreactive to mutated epitopes were obtainedthe use of mhcpeptide tetramers is a canonical methodto identify and study a certain antigenspecific t cell subset“ for act tetramers were used to isolate and expandtumor antigenspecific t cells moreoverin immunecheckpoint inhibitor icitreated cancer patients mhcpeptidetetramers have been successfully used to monitor neoantigenspecific t cells cohen used this method tosort neoantigenspecific t cells from the pbls of patients withmetastatic melanoma in brief a panel of mhcpeptide tetramersconsisting of predicted neoepitopes was synthesized and usedto screen pbls neoantigenspecific t cells targeting of the mutated epitopes identified from tils could be isolated fromautologous peripheral blood with frequencies ranging between and in cancers with intermediate mutational loadssuch as multiple myeloma the use of mhcpeptide tetramerscould also isolate neoantigenspecific t cells from the pbls however this method was only applied to cd8 t cellsand required hlabinding prediction algorithms to guide thesynthesis of hlapeptide tetramersa previous study has shown that pd1 expression couldguide the identification of neoantigenspecific cd8 t cellsfrom the tumor microenvironment the same strategycould be adopted for isolation from pbls in one study patients with metastatic melanoma were enrolled cd8pbls were expanded in vitro and cocultured with autologousdcs which were electroporated with in vitro transcribed tmgrna for mutant epitopes in out of patients neoantigenspecific lymphocytes could be isolated from the cd8pd lymphocyte subset but not the cd8pd1“ lymphocytesubset the isolation of neoantigenspecific cells from the pblsof patients with epithelial cancer is even more challengingpreexisting antigenspecific memory t cells may represent apotential solution memory t cells including central memoryt cells tcm eï¬ector memory t cells tem and temrafrom pbls were cocultured with dcs loaded with candidateneoantigens in the tmg or peptide form after coculturingmemory cells were restimulated with dcs loaded with all tmgsand then sorted by the expression of cd134 and cd137 to enrichfor neoantigenreactive t cells the resulting cells were thenexpanded and screened against all tmgs to test for neoantigenrecognition with this highly sensitive œin vitro stimulationivs method t cells targeting krasg12d and krasg12vwere successfully isolated from out of epithelial cancerpatients this new method enabled identification and isolationof neoantigenreactive t cells from the blood circulation at verylow frequenciesthe identification of neoantigenspecific t cells from na¯vet cells is also of interest a previous report showed that bothna¯ve and activated neoantigenspecific t cells could be expandedfrom the peripheral blood of follicular lymphoma patients bypriming with peptidepulsed dcs using the same methodneoantigenspecific t cells were successfully expanded fromthe peripheral blood of hlamatched healthy donors these preliminary results support the use of na¯ve t cells asfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspecific t cellsfigure a strategies of identifying neoantigenspecific t cells the limitations of current methods of identifying neoantigenspecific t cells and strategies toimprove neoantigenspecific t cells identification tils tumor ltrating lymphocytes pbls peripheral blood lymphocytes pd1 programmed cell death1 temracells terminally differentiated effector memory cells tcr t cell receptor tmg tandem minigene mhc major histocompatibility complex b the œblueprint ofisolating neoantigenspecific t cells from peripheral blood after neoantigentargeting vaccine after several rounds of immunization with neoantigen vaccines t cellsare collected from the patient™s peripheral blood and neoantigenspecific t cells are identified and isolated from these t cells then the neoantigenspecific t cellsundergo rapid expansion rep or their tcrs are exploited to modify autologous lymphocytes the expanded neoantigenspecific t cells or modified tcrt cells arereinfused to the patientan alternative source for act however their exceptionally lowfrequencies in peripheral blood and requirement for repeatedstimulation pose hurdles recentlyalargelibrarybased œminilinesscreeningapproach was proposed which aimed to identify na¯ve antigenreactive t cells from small volumes of blood “ this systembegan with a smallscale culture in 96well plates with initial t cells in each well the smallscale culture underwent arapid to 5000fold expansion miniline thousands ofsuch wellscaled cultures were conducted simultaneously eacht cell clone was maintained at a frequency of in butamplified to an absolute number of “ cells which isa sufficient number for routine detection applying this highthroughput parallel t cell culture system neoantigenspecifict cells were identified and expanded “ months prior to thefirst tumor recurrence in a patient with highgrade serousovarian cancer however the long duration of culture possiblyrendered this method more suitable as a preemptive therapeuticstrategy discussionafter decades of eï¬orts the adoptive transfer of neoantigenspecific t cells is finally close to readiness for clinical applicationhigh efficacy of this immunotherapeutic strategy has beenachieved in a number of cancer patients and the prospects arepromising however these approaches are also quite costly andhard to apply to large numbers of patients the current methodsof identifying neoantigenspecific t cells are summarized infigure 2a and supplementary table more convenient andfrontiers in oncology wwwfrontiersinaugust volume 0cli and dingisolating neoantigenspecific t cellseï¬ective screening methods for neoantigenspecific t cellsremain necessary some strategies to improve neoantigenspecifict cells identification are shown in figure 2ait is feasible to obtain neoantigentargeting t cells frompbls although their frequencies are generally much lower thantils however increasing the frequencies of thesevaluable neoantigenspecific t cells in peripheral blood remainsa challengevaccination with neopeptides has been shown to primecd4 and cd8 tcell responses in mouse models patients treated with vaccines generated neoantigenspecific tcells “ it could be reasonably inferred that the isolationof neoantigenreactive t cells from the peripheral bloodwould be more easily achieved following neoantigenspecificvaccination this neoantigenbased combo immunotherapy hasits advantages first isolation and expansion of tils in vitro isnot necessary second cancer vaccines not only elicit neoantigenspecific t cell responses and amplify existing tumorspecific tcells responses but they also increase the breadth and diversityof the tumorspecific t cell response multiclonal t cellsmay thus be obtained third the relatively easy preparation ofcancer vaccines would buy time for the isolation of neoantigenspecific t cells in maintaining the performance of patients theœblueprint is shown in figure 2bconclusionthe previous decade has witnessed theemergence ofimmunotherapy for cancer accumulating evidence suggests thatneoantigenspecific t cells underlie successful immunotherapytherefore the isolation of neoantigenspecific t lymphocytesrepresents the œholy grail for cancer immunotherapy howevera fundamental challenge is to eï¬ectively identify and isolateneoantigenspecific t cells the developments summarizedin this review and future breakthroughs are anticipated totranslate the adoptive transfer of neoantigenspecific t cells intoa powerful weapon in our armamentarium against cancerauthor contributionsql prepared the manuscript draft zyd revised it critically forimportant intellectual content and approved the final versionql and zyd contributed to the conception and design of thereview all authors contributed to the and approved thesubmitted versionfundingthis work was supported by the national clinical researchcentersichuanuniversity z2018b18for geriatrics west 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" atopic dermatitis ad is a worldwide chronic skin disease which burden public health sea buckthornsbt hippophae rhamnoides l elaeagnaceae oil as a traditional herbal medicine has been used for diseasetreatment for many years the effects of sbt oil on ad mouse model induced by repeated administration of dinitrochlorobenzene dncb in balbc mice was evaluated in this studymethods mice were divided into four groups including the normal control group ad model group ad modelgroup treated with sbt oil mlkg and ad model group treated with sbt oil mlkg same volume at differentconcentrations of sbt oil was applied daily on the latter two groups by gavage for days following ad modelinduction the function of skin barrier and the production of il4 ifnÎ tnfα and tslp were examined afteranimal sacrifice the migration and mature of langerhans cell lcs in lymph node was further assessed by flowcytometryresults sbt oil alleviated dermatitis scores decreased ear thickness prevented infiltration of mast cell reducedlymph node weight and depressed activity of th2 cells sbt oil also reduced the expression of il4 ifnÎ tnfα andtslp in ear tissue ige level in serum and mrna relative expression of il4 ifnÎ tnfα in lymph node moreoversbt oil inhibited the migration of lcs cells from local lesions to lymph node and it™s mature in lymph nodes these results suggest sbt oil had a beneficial effect either systemic or regional on dncbinduced admice via maintain the balance of th1th2 and may be a potential complementary candidate for ad treatmentkeywords atopic dermatitis sea buckthorn oil 24dinitrochlorobenzene cytokine ad is a chronic inflammatory skin disease characterizedwith eczematous pruritic rash which has high morbidityin children and could be recurrent in adulthood [ ]as a general public health disease the prevalence of adhas increased in recent years [ ] ad affects nearly correspondence hongquanguansinacom houdiandong163com xinxin wang and sijia li contributed equally to this work5college of integrated traditional chinese and western medicine liaoninguniversity of traditional chinese medicine chongshan road no79shenyang liaoning pr chinafull list of author information is available at the end of the of children and of adults worldwide and the incidents become higher and higher although thepathogenesis of ad is not explicit utterly genetic riskenvironmental factors skin barrier dysfunction and immune dysregulation are thought to play important rolesduring the pathogenesis of ad [“] as for immunedysregulation th2 skewing seems to be the key point ofad pathogenesis [ ] immunological disorder ofth1th2 balance due to strong type immune responses characterized by over infiltration of mast cellincreased production of th2 cytokines and ige level in the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang bmc complementary medicine and therapies page of serum plays crucial role in the onset and process of adthese th2 cytokines subsequently induce the release ofother proinflammatory cytokines such as ifnÎ throughactivating of th1 cells in clinical practicedue to the heavy burden ad placed on society and patients [ ] treatment approaches are needed imperativelyregional emollient andsystemic corticosteroids were generally used to cure ad[ ] however experts ofinternational eczemacouncil reached on a that although the useof corticosteroids for ad is widespread it is also discouraged due to the side effects and the risk of reboundin consideration of potential fearful side effect of topicalsteroid and immunosuppressive application [ ]there is a strong enthusiasm in seeking alternative andcomplementary medication to treat ad recently seeking new potential candidate from natural materials forad management attracted greatly attention [ “]sbt is a wild deciduous shrub or dwarf tree belongingto the elaeagnaceae family which has been used in tibetan mongolian and chinese traditional medicine extensively for disease management [“] according tomany researchers sbt has various medicinal effects suchas antitumor antistress antiinflammatory antiulcerantioxidant healing regulation of cardiovascular andimmune system [“] sbt oil which contains richfatty acids tocopherols ω3 and ω6 etc is a main bioactive part of sbt and it has been proved to have beneficial effect on skin inflammation conditions and have theability to improve the composition of fatty acid in skin[ ] therefore this study carried out to explore thebeneficial effects of sbt oil on dncbinduced admouse model and its possible mechanismmethodssbt oilsbt oil was provided by liaoning dongning pharmceutical co ltd the oil was extracted from thedried press residue including berry flesh seeds andpeel of sbt juice processing by aseptic supercriticalcarbon dioxide process analysis of samples was performed using a hp5ms capillary column m — mm μm agilentinc santaclara ca usa in a gcms 5975c agilent technologies inc sample was injected into the columnand ran using split mode split ratio the helium carrier gas was programmed to maintain atechnologiesconstantflow rate of mlmin oven was initially °c for min then finally raised to °c at °cmin fatty acids were identified by a reference standard mixture fame supelco bellefonte pa usa analyzed under the same operating conditions as thoseemployed for fame of the samples the componentsin sbt oil are exhibited in table animals and animal treatmentfemale healthy specific pathogenfree balbc miceaged weeks weighted ± g were provided byliaoning changsheng biotechnology co ltd benxichina all mice were housed in groups of mice percage waiting to be grouped in a specific pathogenfreeenvironment in h lightdark cycle and allowed free towater and food mice were acclimatized for week before ad model induction mice were randomly dividedinto groups the normal control group ad modelgroup ad model group treated with sbt oil mlkgand ad model group treated with sbt oil mlkgeach group with mice the dorsal skin of each mousewas shaved following dncb μl1 sensitizationthree times in total from day to day and the skin ofleft ear was challenged by dncb μl05 seventimes in total from day to day ad model grouptreated with sbt oil mlkg was given ml sbt oilplus ml olive oil per mice ad model group treatedwith sbt oil mlkg was given ml sbt oil permice oil was applied by intragastric administration oncea day from day15 to day olive oil ml was givenfor the normal control group and ad model group atthe same time at the same time the thickness of left earwas measured every days all animals were sacrificedat day and samples including blood left ear tissuesand submaxillary lymph nodes were collected the micewere anesthetized with avertin solution g tribromoethanol powder dissolved into ml 2methyl2butanol and ml pbs at °c which was filtered usinga nalgene μm filter thermo fisher scientific incwaltham ma usa and sacrificed via exsanguination the fullscale procedures of ad model inductionand treatment are shown in fig all experimental procedures performed were approved by the ethical committee of experimental animal careat liaoninguniversity of traditional chinese medicine shenyangpr chinatable major fatty acids and contents of sitosterol and βcarotene in sbt oilfatty acids myristic acidc140palmitic acidc160palmitoleicacidc161stearic acidc180oleic acidc181linoleic acidc182linolenic acidc183sitosterolmggβcarotenemgg 0cwang bmc complementary medicine and therapies page of fig general schematic diagram for ad model induction and sbt oil treatmentevaluation of ad severitydermatitis severity was assessed by ear thickness anddermatitis scores through the method of blind ear thickness was measured every days since day with a digitalthickness gauge mitutoyo co kanagawa japan dermatitis scores was calculated according to main characteristics drynesscrusting hemorrhageerythema erosionexcoriation and edema each one was marked on ascale from none mild moderate to severethe overall dermatitis score was consist of the sum of individual scores which range from to μm thick werehistopathological analysisthe left ear samples of mice were collected on day then fixed in formalin and embeded in paraffin thesectionseither withhematoxylin and eosin he for visualizing dermal andepidermal thickness or with toluidine blue tb for visualizing mast cell numbers the mast cells were countedin sections of power fields at — magnificationstainedimmunohistochemistryin short after deparaffinization and rehydration the eartissue slides were treated with hydrogen peroxidemethanol for inhibiting endogenous peroxidase and withhigh pressure for antigen retrieval then the slides wereincubated with sheep serum for min at °c withprimary antibodies abcam for overnight at °c withsecondary antibodies provided by zhongshanjinqiaobeijing china for h at °c at last the slides werestained with diaminobenzidine dab provided byzhongshanjinqiao beijing china for coloration resultwas analyzed by imagejrealtime polymerase chain reaction rtpcrmouse submaxillary lymph nodes were collected andweighted while sacrifice and total rna was extractedfrom lymph node tissues using trizol reagent invitrogen thermo fisher scientificfollowing theincmanufacturer™s protocols and reverse transcribed withprime scripttmrt reagent kit takara biotechnologyco ltd dalian china realtime polymerase chain reaction analyses were performed under the protocols ofsybr®premix ex taqtm ii takara biotech co ltddalian china and the primers used in this study weredesigned as shown in table relative quantities of alltargets in test samples were normalized to their corresponding gapdh levels the 2δδct method was usedto calculate relative expression quantifyflow cytometrythe antibodies used for flow cytometry were providedby bd usa and the scheme was performed follow induction about — “ — cells of submaxillarylymph node was collected in ep tube centrifuged at rpm and °c for min one hundred μl pbs wasmixed with cells after discarding the supernatant thenanother μl pbs containing fluorescent antibodieswas added for staining at °c for min and the processwas kept out of the sun five hundred μl pbs was addedand blended repeatedly for washing then centrifuged at rpm and °c for min the supernatant was discarded carefully and another μl pbs was added andmixed finally the mixture was transferred to flow tubefor flow cytometrytable primers used for rtpcrgeneil4ifnÎtnfαgapdhprimer sequenceforward ²acaggagaagggacgccat3²reverse ²gaagccctacagacgagctca3²forward ² tgagctcattgaatgcttgg ˆ’²reverse ² ggccatcagcaacaacataa ˆ’²forward ²ggaaaggacggactggtgta3²reverse ² tgccactggtctgtaatcca ˆ’²forward ²tggtgaaggtcggtgtgaac3²reverse ²actgtgccgttgaatttgcc3² 0cwang bmc complementary medicine and therapies page of statistical analysisthe data is presented as mean ± sd the significance ofdifferences of different groups was evaluated by oneway analysis of variance anova followed by the dunnett t test p005 was considered statistically significantresultssbt oil has a beneficial effect on skin against thedevelopment of dncbinduced ad models in balbcmiceto investigate the effect of sbt oil on adlike skinlesions in our model sbt oil mlkg10 mlkg wasapplied by gastric administration once a day followingthe ad model induction by dncb showed in fig topical application of dncb including sensitizationand challenge induced adlike skin lesions presenting as erythemaitching and hemorrhage companiedby abnormal scratching marks and dryness dermatitisseverity was assessed by ear thickness and dermatitisscores ear thickness was measured every days sinceday and the dermatitis scores was evaluated according to main characteristics as described previously after sbt oil administration for days theear thickness and dermatitis scores were significantlydecreased in a dosedependent manner compared tothe ad model group fig fig effects of sbt oil on ad model skin induced by dncb a examples of characteristic of adlike skin lesions b the ear thickness of the micec the dermatitis scores were summarized by the sum of scores according to various ad symtoms p p 005vs the control group p p vs the ad model group 0cwang bmc complementary medicine and therapies page of sbt oil contributed to the skin barrier repair anddecreased infiltration of mast cell in ad model miceinduced by dncbto evaluate the effect of sbt oil on adlike skin lesions histologically he and tb staining were performed on tissue slides repetitive application ofdncb induced dermal thickening epidermal hyperplasia and increased mast cellinfiltration in admodel group while according to he staining slidesthe dermal and epidermalthickness were both decreased and the epidermal hyperplasia was suppressedafter sbt oil administration for days which relatedto dosage fig 3a b according to tb staining slidesthe infiltration numbers of mast cell were also decreased in mice treated with sbt oil fig 3b csbt oil decreased the lymph node weights in ad modelmice induced by dncbthe submaxillary lymph nodes were collected andweighted after mice sacrifice to estimate whether sbtoil play a part in the process of ad induction the results indicated an increase in submaxillary lymph nodeweights in ad model group which was decreased bysbt oil administration fig sbt oil inhibited the expression of il4 ifnÎ tnfα andtslp in ear tissue in ad model mice induced by dncbin order to evaluate the effects of sbt oil on regulationof th1th2 cytokines the expression of il4 ifnÎtnfα and tslp in ad model mice was examined byimmunohistochemistry staining on ear tissue slides results demonstrated an upregulation expression of il4fig effects of sbt oil on histological ear skin tissue a he staining — thickness in hestained tissue mast cell number in tb stained tissue c tb staining — epidermis mast cell dermis b dermal and epidermal 0cwang bmc complementary medicine and therapies page of fig weights of submaxillary lymph node a submaxillary lymph node b lymph node weight a normal control group b ad model groupc treated with sbt oil mlkg d treated with sbt oil mlkg p p vs the control group p p vs the admodel groupifnÎ tnfα and tslp in ad model group which wasinhibited dosedependent by application of sbt oilfig sbt oil downregulated ige level in serum and mrnarelative expression of il4 ifnÎ and tnfα in lymphnodewe next measured ige levelin serum by elisa andmrna relative expression of il4 ifnÎ and tnfα inlymph node by rtpcr we found that ige levelinserum was increased in ad model mice induced bydncb the increase was suppressed significantly ingroups treated with sbt oil in a dosedependent manner the same as above mrna relative expression ofil4 ifnÎ and tnfα in lymph node was increased inad model group and decreased in groups treated withsbt oil fig sbt oil decreased numbers of lcs in draining lymph nodeand the expressions of cd86 ox40l and mhcii on lcsin order to assess the effect of sbt oil on the maturityand migration of lcs cell in submaxillary lymph nodewe detected cell numbers expressing cd207cd326cd86 cd80 ox40l and mhc ii by flow cytometryresults as shown in fig indicated that the expressionsof cd207cd326 cd86 ox40l and mhciion lcs cellin submaxillary lymph node were all increased in admodel groups induced by dncb and decreased ingroups treated with sbt oildiscussionad is a skin inflammatory disease induced by haptenand mediated by t cells clinically the main characteristics of ad are erythema edema papule blisterbleb bullous reaction and even necrosis the pathological changes of ad were infiltration of inflammatorystudy weand tissuecellsedemain thisemployed dncbinduced ad model using balbcmice which has been proposed as an appropriate representative of human ad because of similar symptoms including skin erosion hemorrhage epidermalhyperplasia mast cellinfiltration and increased igelevel in serum etc sbt oil as a traditional herbal extracts have been proved diversified pharmacologicalactions such as antiinflammatory relieve the pressure protecting vascular endothelial cell and immunomodulatory effects [ ] the main constituents ofsbt oilinclude fatty acids such as myristic acidpalmitic acid palmitoleic acid oleic acid etc sitosterol and βcarotene fatty acids are crucial components of cell membranes and play important role inbiologicalfunction of cells some of the fattyacids are required for innate immune activation andpathogen defense sitosterol is the main constituent of many plants and vegetables and has the abilityto modulate the functions of macrophages and antiinflammation [ ] βcarotenealso has beenshown to suppress the cellular and tissue response toinflammation [ ] in view of the immunoregulation and antiinflammatory actions of sbt oil weassessed the antiad effects of sbt oil in vivotopical application of dncb followed schedule including sensitization and challenge induced adlikeskin lesions presenting as erythemaitching andhemorrhage companied by abnormal scratching marksand dryness the ear thickness and dermatitis scoreswere all significantly increased in ad model groupcompared to control group after sbt oil administration for daysthickness and dermatitisscores in groups treated with sbt oil were significantly decreased in a dosedependent manner compared to the ad model group which indicate thatsbt oil administration suppressed the development ofadlike skin lesionsthe ear 0cwang bmc complementary medicine and therapies page of fig effects of sbt oil on expression of il4 ifnÎ tnfα and tslp in ear tissue a immunohistochemical staining of il4 ifnÎ tnfα and tslpin ear tissue b aod analysis of il4 ifnÎ tnfα and tslp p vs the control group p vs the ad model groupad is recognized as a th2midiated allergic disease withover expression of th2 cytokines and increased serum igelevel being the antibody synthesized by plasma cellsige plays an essential role in some hypersensitivity suchas ad allergic asthma and allergic rhinitis ige has thecapability of elevating the production of th2 cytokinesth2 cytokines il4 induced immunoglobulin switching inplasma cells and resulting in upregulation of serum igelevel mast cell as one of granular leukocytes can releasemany cytokines to mediate inflammatory reaction and immune regulation these cytokines also participate inpathological manifestations of many allergic disorders including ad the infiltration of mast cell which wasactivated by ige is one of the key features of adlike skin 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on ige level in serum and mrna relative expression of il4 ifnÎ and tnfαin lymph node a ige level in serum b mrnarelative expression of il4 ifnÎ and tnfα in lymph node p p vs the control group p p vs the ad model grouplesions [ ] cytokines released from activated mastcells attract eosinophils into the skin which give rise toskin tissue damage histologically according to tb staining slides the numbers of mast cell infiltration in ear skintissue of ad model mice were increased by application ofdncb and were inhibited remarkably by sbt oil the results indicated that sbt oil has beneficial effects on suppression of skin tissue mast cell accumulation in dncbinduced ad mice our tb staining results indicated thatmast cells in skin tissue were scarce in control group whileabundance in ad model group which highly conform tothe pathological changes of ad the mast cell numberwas reduced remarkably after sbt oil administration insbt oil treated group compared with ad model groupthese results suggest that sbt oil has inhibiting effect ofmast cell infiltrationaccording to studies published the over expression ofth2 cytokines go hand in hand with tslp tslp whichcan strongly promote the differentiation of th0 cells toth2 phenotype through activation of dendritic celldcs was determined as a crucial factor in the induction of th2 skewing in ad the expression of tslphas been shown to be enhanced markedly in keratinocytes of ad lesions both in ad patients and in mousemodels il4 can in turn induce the synthesis oftslp by keratinocytes importantly the migration ofdcs to draining lymph node was triggered by tslpmore interesting th1 cytokines ifnÎ which can activate keratinocytes was found also elevated in ad ifnÎand tnfα can synergistically stimulate the release ofcytokines and chemokines in chronic stage of ad inour study sbt oiltreatment reduced the increasedserum ige level which was induced by dncb application moreover sbt oil treatment also reduced dncbinduced increases in expression of il4 ifnÎ tnfαand tslp in ear tissue and mrna relative expression ofil4 ifnÎ and tnfα in lymph node these resultssuggest that sbt oil ameliorated ad symptoms partlythrough the activity suppression of th1th2 cells according to the downregulated effects sbt oil did to thetslp expression in ear tissue we speculated that sbtoil may have the ability to suppress both the activationand migration of dcs cell in order to clarify our speculationflow cytometry was used to do further studyabout lcsdraining lymph node plays an important role in thepathogenesis of ad the weight and volume of lymphnode will increase company with strengthened functionwhen it is active we investigated the local lymph nodesthrough different means first of all the submaxillarylymph node weights of dncbinduced ad model micewere increased significantly which were markedly reducedafter intragastric application of sbt oil for days in adosedependent manner we further assessed the expressions of cd207cd326 mhc class ii cd80 cd86 onlcs and ox40l on cd4 t cells in lymph node by flowcytometry because the complex immune reaction of adwas mainly taken place in lymph node langerin cd207a type ii transmembrane protein is a ctype lection oflcs lcs are virtual mediators of immune surveillance and tolerance which resided at epidermis as dc subpopulation antigens both external and internal werecaptured by lcs and presented to th0 cells within theskin draining lymph node cd207 is the only surface antigen just restricted to lcs epithelial cell adhesionmolecule epcamcd326 a cell surface protein is highlyexpressed on lcs and appears to stimulate lcs migration since cd207cd326 as the main symbol of lcs 0cwang bmc complementary medicine and therapies page of fig effect of sbt oil on the maturity and migration of lc cell a the scatter diagram which indicate the proportion of cells in lymph nodeexpressing cd207cd326 mhc ii cd86 cd80 and ox40l b the histogram of abovementioned results p p 005vs the controlgroup p p vs the ad model groupmigrated into lymph nodethe proportion changesshowed that sbt oil suppressed the migration of lcs cellfrom skin lesion to draining lymph node after degradingproteins derive from extracellular environment were takenup by endocytosis or phagocytosis and captured by mhcclass ii molecules then result in peptideloaded mhc iiand migrate to the surface of antigen presenting cell waiting for recognition by cd4 t cells finally activate adaptive immune response the increased cell proportionof mhc class ii indicated the uptrend tendency of mature 0cwang bmc complementary medicine and therapies page of lcs in lymph node in dncb induced ad model micecompared with normal control mice while this uptrendtendency was inhibited by sbt oil application in micetreated with sbt oil constant epidermal lcs are immature normally and barely express costimulatory moleculessuch as cd80 and cd86 while upon lcsmaturation the expression of these costimulatory molecules was enhanced in this study sbt oil down regulatedthe expression of cd86 on lcs in lymph node which wasenhanced by dncb in ad model mice it suggested theeffect of sbt oil on inhibiting lcs maturationox40cd134 is a transmembrane protein of tumor necrosis factor receptor superfamily member which mainlyexpressed on activated cd4 t cells and upregulatedwithin inflammatory lesions on the antigenactivated tcells [ ] tslp can stimulate the expression of ox40ligand ox40l on lcs lcs expressing ox40l migratefrom skin lesion to local lymph node and induce the transformation of th0 cells to th2 cells on one hand the increased proportion of ox40l cells in lymph node ofdncb induced ad mice was suppressed by sbt oil administration which confirmed the effect of sbt oil on activation of cd4 t cells on the other hand sbt oilconduced to the normal function of lcs through renovating the keratinocyte and suppressing tslp release as aresultthe abnormal th2 skewing inflammation wasinhibited by sbt oil administrationall in all our results suggest that sbt oil inhibitedboth the migration of lcs to lymph node and its maturation in lymph node thereby inhibited the transformation of th0 cells to th2 cells and finally limited theoccurrence of th2 type inflammatory responsein summary our study results attested that sbt oil application suppressed dncbinduced adlike symptomsby downregulating serum ige level and the productionof cytokines and chemokines and regulated th1th2balance in addition our results also indicated that sbtoil treatment inhibited the migration of lcs to draininglymph node and its maturation taken together sbt oilhas excellent therapeutic effect on inflammatory skindiseases and might be a potential complementary candidate for ad treatment in further studiesit will beworthwhile to explore the mechanism of sbt oil and itsactive constituent in the treatment of adabbreviationsad atopic dermatitis dab diaminobenzidine dcs dendritic cell dncb dinitrochlorobenzene he hematoxylin and eosin ifnÎ interferonÎige immunoglobulin e il4 interleukin4 lcs langerhans cell mhcii majorhistocompatibility complex ii rtpcr realtime polymerase chain reactionsbt sea buckthorn tb toluidine blue th1 thelper th2 thelper tnfα tumor necrosis factorα tslp thymic stromal lymphopoietinacknowledgementsnot applicableauthors™ contributionsxxw carried out the experiment and drafting of the manuscript sjl and jplassisted to accomplish the experiment dnk and xwh carried out statisticalanalysis pl and mx did the interpretation work hqg and ddh revised theresearch and manuscript ddh designed the experiment and submitted themanuscript all the authors read and approved the final manuscriptfundingthis project was supported by the national natural science foundation ofchina grant no which was granted to diandong hou the resultsindicated in this manuscript were main achievements of the projectavailability of data and materialsall data and materials are contained and described within the manuscriptethics approval and consent to participateall experimental procedures were conducted according to the guidelinesprovided by the ethical committee of experimental animal care at liaoninguniversity of traditional chinese medicine shenyang pr chinaconsent for publicationnot applicablecompeting intereststhe authors state no potential conflict of interestauthor details1liaoning university of traditional chinese medicine shenyang liaoning prchina 2basic medical and forensic medicine baotou medical collegebaotou inner mongolia pr china 3neurosurgery department northernhospital of inner mongolia baotou inner mongolia pr china 4liaoningdongning pharmceutical co ltd fuxin liaoning pr china 5college ofintegrated traditional chinese and western medicine liaoning university oftraditional chinese medicine chongshan road no79 shenyang liaoning pr chinareceived december accepted june referencesklonowska j new cytokines in the pathogenesis of atopic dermatitisnew therapeutic targets int j mol sci choopani r treatment of atopic dermatitis from the perspective oftraditional persian medicine presentation of a novel therapeutic approach jevid based complementary altern med “brunner pm guttmanyassky e leung dy the immunology of atopicdermatitis and its reversibility with broadspectrum and targeted therapiesj allergy clin immunol 20171394ss65“leung dym new insights into atopic dermatitis j clin investig “kim je molecular mechanisms of cutaneous inflammatory disorderatopic dermatitis int j mol sci kim j molecular mechanism of atopic dermatitis induction followingsensitization and challenge with 24dinitrochlorobenzene in mouse skintissue toxicol res “hou dd sea buckthorn hippophae rhamnoides l oil improvesatopic dermatitislike skin lesions via inhibition of nfkappab and stat1activation skin pharmacol physiol “campana r molecular aspects of allergens in atopic dermatitis curropin allergy clin immunol “brandt eb sivaprasad u th2 cytokines and atopic dermatitis j clin cellimmunol de vuyst e atopic dermatitis studies through in vitro models frontmed lausanne park jg tabetri tabebuia avellanedae ethanol extract amelioratesatopic dermatitis symptoms in mice mediat inflamm 0cwang bmc complementary medicine and therapies page of liu r βsitosterol modulates macrophage polarization and attenuatesrheumatoid inflammation in mice pharm biol “ vollmer d west v lephart e enhancing skin health by oral administrationof natural compounds and minerals with implications to the dermalmicrobiome int j mol sci cicero afg colletti a effects of carotenoids on health are all the sameresults from clinical trials curr pharm des “ito t il33 promotes mhc class ii expression in murine mast cellsimmun inflamm dis “ dubois a regulation of th2 responses and allergic inflammationthrough bystander activation of cd8 t lymphocytes in early life jimmunol “ girtsman t natural foxp3 regulatory t cells inhibit th2 polarizationbut are biased toward suppression of th17driven lung inflammation jleukoc biol “ wallmeyer l tslp is a direct trigger for t cell migration in filaggrindeficient skin equivalents sci rep leyvacastillo jm tslp produced by keratinocytes promotes allergensensitization through skin and thereby triggers atopic march in mice jinvest dermatol “feinberg h trimeric structure of langerin j biol chem “ zhang x tim4 is differentially expressed in the distinct subsets ofdendritic cells in skin and skindraining lymph nodes and controls skinlangerhans cell homeostasis oncotarget “kumkate s cd207 langerhans cells constitute a minor population ofskinderived antigenpresenting cells in the draining lymph node followingexposure to schistosoma mansoni int j parasitol “ gaiser mr cancerassociated epithelial cell adhesion moleculeepcam cd326 enables epidermal langerhans cell motility and migrationin vivo proc natl acad sci u s a 201210915e889“ natarajan k the role of molecular flexibility in antigen presentationand t cell receptormediated signaling front immunol weinberg ad science gone translational the ox40 agonist storyimmunol rev “kinnear g a diametric role for ox40 in the response of effectormemory cd4 t cells and regulatory t cells to alloantigen j immunol“publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsshrestha s burden of atopic dermatitis in the united states analysis ofhealthcare claims data in the commercial medicare and medicaldatabases adv ther “ arima k burden of atopic dermatitis in japanese adults analysis ofdata from the national health and wellness survey j dermatol “ megna m systemic treatment of adult atopic dermatitis a reviewdermatol ther heidelb “ glatz m emollient use alters skin barrier and microbes in infants at riskfor developing atopic dermatitis plos one 2018132e0192443 drucker a

the diagnostic platform utilizing the detection of biomarkers in various body fluids called œliquidbiopsy can revolutionize precision medicine precision medicine is aimed at attaining betterpersonalized care by the development of the latest diagnostic and prognostic methods thatconsider individual variability kaur liquid biopsy is being utilized for noninvasiveabbreviations 5hmc 5hydroxy methyl cytosine ccfdnas circulating cellfree deoxyribonucleic acids ccffetalnascirculating cellfree fetal nucleic acids ccfmirnas circulating cellfree mirnas ccfnas circulating cellfree nucleic acidsccfrnas circulating cellfree ribonucleic acids mtdna mitochondrial dnaedited byrui henriqueportuguese oncology instituteportugalreviewed bynaoko hattorinational cancer center researchinstitute japanigor kovalchukuniversity of lethbridge canadacorrespondencejyotdeep kaurjyotdeep2001yahoocoinspecialty sectionthis was submitted toepigenomics and epigeneticsa section of the frontiers in geneticsreceived february accepted july published august citationrahat b ali t sapehia dmahajan a and kaur j circulating cellfree nucleic acids asepigenetic biomarkers in precisionmedicine front genet 103389fgene202000844frontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersprognostic and predictive purposes efficient and reliable markerswithin the body fluids can help in personalized treatmentdecisions for monitoring disease and survival ccfnas haveemerged as such markers for screening diagnosis prognosismanagement and treatment of various cancers autoimmuneneurological and mitochondrial diseases prenatal diagnosisdiagnosis of pregnancyrelated complications pos diabetes ‚ammation rheumatoid arthritis stroke and traumaswarup and rajeswari an increased amount of ccfnasis observed in these disorders making liquid biopsies moresensitive rapid accurate and preferable alternatives for variousinvasive diagnostic methods pos ccfnas present in blood circulation include cellfree genomicdnas ccfdnas and cellfree mtdna kohler thierry and cellfree rnas ccfrnas includingproteincoding messenger rna mrna regulatory noncodingrnas like micrornas mirnaslong noncoding rnaslncrnas circular rnas and rnas involved in translationlike transfer rnas trnas and ribosomal rnas rrnaspos the ccfnas dnas and rnas are generally released into theblood circulation either by apoptosis necrosis or active secretionin healthy persons the origin of ccfnas is mainly attributed tolymphoid and myeloid tissues snyder while in thecase of various clinical conditions the associated or the aï¬ectedtissues would release the extra amount of ccfnas into bloodswarup and rajeswari devonshire in a patternspecific to the pathophysiological condition hunter noferesti various genetic as well as epigenetic biomarkers havebeen explored for ccfnabased liquid biopsy as geneticbiomarkers are less consistent and provide more variabilityacross studies epigenetic markers which are more generalizedbetween samples present as a promising alternative for earlydiagnosis and monitoring of the diseases these epigeneticmarks are tissue specific and reflect the pattern of diseaseprogression zeng furthermore epigeneticbiomarkers are dynamic with most techniques required foranalysis ofthese biomarkers that are already available inclinical laboratoriescare thein future precise patientthe use of epigenetic marks has revolutionized the fieldof noninvasive molecular diagnosisreplacing traditionalscreening and treatment methods these assays have greatpotentialepigeneticmarks for ccfnas reflect the pattern specific for the tissuecontributing to these ccfnas therefore the use of epigeneticmarkers can help in the diagnosis of various diseases evenbefore the onset of actual symptoms and hence help inbetter management ofthe disease precision medicine hasimproved health care by theidentification of diï¬erentstagessubsets of diseases precise diagnosis and treatmentfurthermore the development of advanced analytical softwaretechniques like machine learning and artificialintelligencecan enhance precision medicine ahlquist beltrangarcia these are used in combination withnextgeneration sequencing to identify novel ccfnabasedepigenetic markersepigenetic biomarkers in ccfnasreliable markers are required to guide personalized treatmentdecisions for monitoring disease progression and survivalthe presence of epigenetic marks on ccfnas specific toa particular clinical condition is widely being explored toadvance personalized medicine a perfect epigenetic markerfor precision medicine should be able to detect the diseasewith high sensitivity predict the risk of disease developmentand its progression and monitor the therapeutic responseofbeltrangarcia ccfdnas areassociated with various epigenetic marks schwarzenbach like dna methylation hydroxymethylcytosine 5hmcand posttranslational modifications of histones in additionnucleosome positioning and occupancy on ccfdnas haveexhibited high sensitivity and specificity in liquid biopsybasedmethods for disease detection and classificationthe patientthe5methylcytosine5mc modificationat cpgdinucleotides is the most abundant form of dna methylationit plays an important role in the regulation of gene expressionand is widely used as an epigenetic biomarker for ccfdnabasedassays dna methylation has replaced many genetic mutationor proteinbased markers these 5mc biomarkers are alsovaluable in identifying tissuespecific methylation to estimatetumor burden and tissue of origin in ccfdnas in additionto 5mc 5hydroxymethylcytosine 5hmc is also used as anepigenetic mark on ccfdnas zeng 5hmc is createdby the oxidation of 5mc by “ translocation tet proteinsalthough 5hmc is far less abundant compared to 5mc it ismore distinctly distributed among diï¬erent transcriptionallyactive regions which emphasizes its potential as a diagnosticmarker genomewide analysis of 5hmc pattern can providemore information about the potential of this epigenetic markerfor ccfdnas zeng nucleosome positioning has emerged as a recent biomarkerto distinguish the tissue of origin of ccfdna based onderived nucleosome maps snyder performed deepsequencing on ccfdnas and observed a distinct pattern ofnucleosome positioning between healthy persons and cancerpatients correlating with the tissues of origin snyder this emphasizes the use of nucleosome maps whichconsist of occupancy of transcription factor and nucleosomeas the epigenetic marks to distinguish normal versus cancerccfdnas hence nucleosome positioning can also be used toidentify various cancers that generally require invasive biopsiesfor definitive diagnosis moreover genomewide nucleosomepositioning of ccfdnas is utilized to infer pathological statesof multiple disease types a comprehensive public databasecalled cellfree epigenome atlas cfea provides the epigenomeprofile of ccfdnas from various human diseases and canhelp in a better understanding of collected data yu ccfdna are generally associated with nucleosomesand histone proteins histone proteins are posttranslationallymodified at amino acid residues located on their n andcterminal tails these modifications act as epigenetic marksthat can specifically distinguish diseaserelated ccfdnas in bloodsamples various types of histone modifications are associatedfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersarewith the development and pathogenesis of human diseaseszhao and shilatifard in addition to dna markers rna markers like mrnasmirnas lncrnas and circrnas are also getting attention in thefocus of clinical research pos most of the currently available diagnostic tests based onccfnas use either dna methylation markers or the diï¬erentialexpression of mirnas these biomarkersrelativelyeasily detected and estimated using accessible techniqueslike methylight methylspecific pcr methylationsensitivehighresolution melting and pyrosequencing garc­agimnez dna methylation specific to fetal and tumor dnahas been reported in pregnant women and cancer patientsrespectively wong poon the pattern ofthe methylation in these ccfdnas has been traced back to theirtissue of origin lun sun diï¬erentiallymethylated markers have been reported in ccfdnas like inspromoter in diabetes and reg1a and cux2 genes in pancreaticcancer lehmannwerman promoter methylationof serpinb5 rassf1a and stat5a act as epigenetic fetalmarkers in maternal blood chim chan rahat 2016atumor dna depending on the copy number mostly targetedmethylation sequencing is carried out in such cases which hasa greater potential for the detection of lower levels of ccfna inpatients with earlystage diseasechromatinbased chipseq experiments are revolutionizingour understanding of the complexes associated with chromatindynamics ongoing advances such as nanochipseq allow chipseq to be analyzed from far fewer cells necessary for embryologyand development studies nakato and shirahige theemergence of chipexo that digests the ends of dna fragmentsnot bound to protein is quite promising furey howeverthe application of these techniques to identify biomarkers islimited due to the expertise and cost associatedcriticalchipseq also providesinformation on otherchromatin modifiers such as histone marks and the enzymesthat modify these marks in diseases such as cancer chipseq has identified the role of aberrant h3k79 methylationby the methyltransferase dot1l in mixed lineage leukemiamllrearranged leukemias bernt in additionto chipseq diï¬erentlike chippcr elisabased assays or mass spectrometry are used to detect andquantify histone modifications on ccfnas in serum or plasmaadli and bernstein techniquesdiagnostic approach forepigenetic modifications in ccfnathe various diagnostic approaches to study the epigeneticmodifications in the nucleic acids include methylated cpgisland recovery assay mira and methylcap that rely onmethylcpgbinding domains mbd to capture methylateddna after dna fractionation either by restriction digestion orsonication mitchell these methods can also becombined with microarray or ngs technologies methylcapseq to identify biomarkers for cancer diagnosis and dnamethylation maps of cancer genomes simmer reduced representation bisulfite sequencing rrbs meissner is an efficient method for absolute quantification ofthe methylation status of more than one million cpg sites atsingle basepair resolution covering regions of moderate to highcpg density lee new techniques such as wholegenome bisulfite sequencing wgbs allows for an unbiasedassessment of dna methylation at singlebase resolution withfull coverage of more than million cpg sites in the humangenome and by using this technique in the clinical settingsrelevant biomarkers were identified in colorectal and breastcancers and certain types of leukemia berman some of the techniques are used in clinical settingslikeparallel shotgun sequencing and targeted sequencing norwitzand levy for noninvasive prenatal testing wgs for fetalgene detection lo and cancer personalized profilingby deep sequencing cappseq to quantify circulating tumordna newman despite the advancement of the techniques to study epigeneticmodifications the use of epigenetic biomarkers present onccfnas is limited due to their lower levels in the blood circulationin the case of cancer wgs is applied to only “ of cellfreeccfnas as epigenetic biomarkersin various diseasesthe detection and quantification of ccfnas viz rna dnafetal dna fetal rna mtdna and mitochondrial rna andmirna levels in body fluids are of clinical significance theseccfnas have the potential to act as biomarkers for diagnosisas well as prognosis of various diseases fleischhacker andschmidt breitbach such as diï¬erent cancersobstetric autoimmune neurological and mitochondrial diseasesas well as prenatal diagnosis etc kandel shaw although the most studied area of epigenetics is dnamethylation yet in the clinical setting there are only a fewmethylation markers various blood or tissuebased cohortwellpowered studies have recently shown that changes in thedna methylation are not only observed frequently in cancersbut also in other broad range of complex diseases includingneurodegenerative metabolic autoimmune and ‚ammatorydiseases although at a lower frequency tost dna methylation analysis of ccfdna might provide avaluable option in some cases when the blood“brain barrieris temporarily disrupted it was recently demonstrated by thedetection of unmethylated fragments of mbp3 and wm1 specificfor oligodendrocytes in about of patients with relapsingmultiple sclerosis zachariah cfrnas are also presentin the patient™s serumplasma in addition to ccfdnas higherlevels of circulatory rnases were observed in cancers and variousdiseases like cerebral attack preeclampsia etc and surprisinglyrna found in the circulation was found to be stable umu changes in the expression of intracellular mirnahave been causally linked with many diseases that include canceresquelakerscher and slack cardiovascular diseasesfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkersnavickas neurodegenerative diseases gupta etc such changes in expression of mirna are eithersimilar or distinct in the serum of a particular set of patientsthus enabling mirna detection in serum as biomarkers ofhuman diseases backes therefore ccfnas playa prominent role in the pathogenesis and diagnosis of variousdiseases further research is required in this field to ensure thewidespread application of these markers in clinical settingsccfnas in cancerevery year about million new cases of cancer are reportedexcluding skin cancer other than melanoma that cause about million deaths accounting for of deaths in a yearferlay an estimated number of more than million new cancer cases are likely to be diagnosed and cancer deaths are expected in the united states in whichdeciphers almost deaths per day siegel thesix major hallmarks of cancer hanahan and weinberg are uncontrolled cell growth and division programmed cell deathavoidance invasion metastasis and angiogenesis the diagnosisof cancer usually occurs following the appearance of signs orsymptoms or through screening and investigations like xraysblood tests endoscopy ct scans etc biopsy tissue examinationindicates the type of proliferating cells genetic abnormalitiesand histological grade and other characteristics thereforeadvanced measures such as estimating prognosis risk assessmentfor early diagnosis biomarkers and observing the response totherapy can lead to successful treatment positive outcomes andimprovement of the quality of life for patients the tissue biopsymatched ccfdna is considered as surrogate marker due to itsrelease from the tumor sites de mattosarruda it is proven to be a noninvasive rapid and sensitive markerfor diagnosis prognosis and therapy response monitoring indiï¬erent cancers volik in addition the integrityof ccfdna extent of ccfdna fragmentation may be utilizedas a promising biomarker for diagnosis and prognosis of cancermadhavan ccfnas as diagnostic and prognosticbiomarkers for cancerserum or plasma ccfna serves as a œliquid biopsy which is usefulfor various applications in diagnostics and avoids the necessityfor biopsy of tumor tissue the levels of ccfna in blood andlymphatic circulation are aï¬ected by degradation clearance andvarious other physiological events liver and kidney clear nucleicacids from the blood and they have a halflife of diï¬erent timeintervals in the circulation that varies from min to severalhours fleischhacker and schmidt mirnas appear to beextremely stable but their rate of clearance from the blood is notwell studied in cancer patients thus owing to the uniqueness ofthis research areaccfdnas in cancerccfdnas consists of both genomic dna gdna as well asmtdna there is a production of longer uneven fragments ofdna by necrosis in cancer patients and shorter dna fragmentsfrom apoptosis hence increased levels of longer dna fragmentsin the bloodstream have been targeted as a potential marker forthe presence of malignant tumor dna arkoboham tumor cells are the origin of ccfdna in the blood of cancerpatients stroun aberrations specific to tumors likeoncogene and tumor suppressor gene mutations wang methylation of dna fujiwara and instabilityof microsatellite dna shaw were recognized inccfdna tumorigenesis and its progression are monitored bythe change in various epigenetic modifications patients withdiï¬erent types of malignancies have methylated dna in theirserum or plasma one of the most important methods foranalyzing malignancy is by detecting the presence of methylatedccfdna in cancer patientsfor early diagnosis of colorectal cancer crc analysisof promoter hypermethylation in blood and fecal dna hasthe potential to be used as a noninvasive test and eï¬ortsare made for clinical application of these molecular markersvarious studies have observed mgmt rassf2a wif1 ngfrand sept9 as aberrantly methylated genes used as diagnosticbiomarkers in patients with crc lee powrozek several potential methylation biomarkers have beenfound that diï¬erentiate plasma from breast cancer patients andthat from control subjects hoque remarkablytwo independent studies recognized cst6 as being methylateddiï¬erentially between breast cancer and control plasma samplesradpour chimonidou for lungcancer an early focus was to search methylated cdkn2a as aplasma diagnostic biomarker studies observed hypermethylationof cdkn2a in the plasma of patients with lung cancer ascompared to cancerfree controls zhang shox2was identified as a potential biomarker in a retrospective studydone by researchers from the theracode a diagnostic firmkneip a recent study by a group as part ofthe australian pancreatic cancer genome initiative apgihas observed elevated levels of aberrant methylation in theimportant cell signaling pathways thus suggesting its possibilityas a disease biomarker they worked on a group of sixcandidate genes nptx2 sarp2 uchl1 ppenk cdkn2aand rassf1a and observed diï¬erential methylation in thepromoters of all the genes in pancreatic cancer and healthycontrols except in cdkn2a promoter which was methylateddiï¬erentially between pancreatic cancer patients and thosehaving chronic pancreatitis park epigenetic eventsin the progression of cancer include the promoter regionthe genes piclass gstp1 and apchypermethylation ofwhich are the most common somatic genome abnormalities incolorectal and prostate cancer ellinger rassf1ararb sept9 esr1 and cdkn2a are the important methylatedgenes that have shown utility in prognosis using ccfdnaassays in many patients methylation of histones is an activeprocess with vital roles in diï¬erentiation and developmenttumorigenesis also occurs due to aberrant levels of histonemethylation the promoters associated with h3k4 are primarilytrimethylated by set1a and set1b set1a plays a vitalrole in oncogenic function in breast cancer metastasis lungfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerscancer and colorectal cancer zhao and shilatifard table presents the frequently hypermethylated genes invarious cancer typesccfmirnas in cancerin various cancers mirna expression dysregulation has beenobserved that suggests its role in many processes necessaryfor the progression of cancer like proliferation cell deathmetastasis and resistance to treatmentiorio and croce during the development of the liver mirna expressionchanges dynamically mir500 is one such oncofetal mirnathat is important for the diagnosis of hepatocellular carcinomayamamoto lately in nonsmall cell lung cancernsclc mir1246 and mir1290 were recognized as tumorinitiating and cellspecific mirnas zhang mir was found to be a significant prognostic factor for osccpatients based on cox regression analysis in addition mir could serve as a valuable biomarker in oscc patientsto predict the clinical response to chemoradiotherapy lin a study by alhasan showed a serumsignature of 5mirnas mir135a mir106a mir200c mir and mir433 predicted a very highrisk prostate canceralhasan expression levels of mir21 mir23bmir200c and mir200b were upregulated in metastatic breastcancer when compared to early breast cancer patients thereforesupporting the notion that ccfmirnas presents a tool with thecrucial diagnostic and prognostic implication in breast cancerpapadaki furthermore a study discovered thatincreased mir122 expression was significantly associated witha reduction in the overall survival as well as progressionfreesurvival in breast cancer patients saleh elevationin the levels of serum mir29 mir122 mir155 and mir was observed in cholangiocarcinoma although mirnaslevels before surgery were inappropriate as survival prognosticmarker however postsurgery decrease in the serum mir122levels was significantly linked with better patient prognosisloosen ccfnas in treatment and cancerprogressionccfdna analysis is a noninvasive process that allows day to daypatient followup and monitoring of response toward treatmentges both genetic and epigenetic changes areexhibited by ccfdna stroun the study of thesechanges might provide valuable information to mold the choiceof treatment by clinicians given the limitations of the noveltargeted therapiesabnormal hypermethylation at cpg islands occurs rarely innonmalignant and normally diï¬erentiated cells so the releaseof dna from tumor cells can be found with a prominentextent of sensitivity even when the excess of dna is releasedfrom normal cells and this characterizes its potential clinicalapplication wong in this context promoter regionhypermethylation of ink4a occurs very early in the progressionof hepatocellular carcinoma hcc and henceit serves asa valuable biomarker for noninvasive diagnosis as well asprediction of response to therapy huang isatherapyimmunotherapyidentification ofrapidly developingsignificant mirnasinmany cancers because of various advantages over standardchemotherapythatprovides a foresight of response in cancer immunotherapy wouldenable better patient selection and enhancement of therapeuticefficacy and provide a novel target antonia chen mirna21 is a cellfree oncogenic mirna whichhas been known as a potential regulator of stat3 and thusit could be detected in various tumors ji thuscirculating mirna21 can act as a biomarker for response incancer immunotherapy wu in the mycnamplified neuroblastoma progression mycnis detected in circulating dna this phenomenon was found tobe associated strongly with the quick progression of tumors andpoor outcomes combaret loss of heterozygosityloh and abnormal methylation at the promoter region ofmycn were detected using ccfdna which showed elevatedlevels in patients of highgrade glioma detection of promoterregion hypermethylation of myod1 in serum may serve asa potential prognostic marker for discriminating patients ofcervical cancer at high risk for lymph node metastasis or relapsewidschwendter moreover the investigation of circulating mirnas presentsgreat potentialin revealing new insights into their role intherapy and diagnosis mirna serum signatures mir345 5pmir330 3p and mir9 3p were found to be significantlyupregulated in patients of prostate cancer pca when comparedto healthy individuals the role of mir3455p to act as anoncomir through cdkn1a targeting makes it a potential targetfor pca therapeutically tinay ccfdnasin glioma wereassociated with diï¬erentialmethylation levels of mgmt cyclindependent kinase inhibitor2a multiple tumor suppressor p16ink4a p73 and retinoicacid receptor beta rarb balana weaver wakabayashi all these studies propose acrucial role of epigenetic marks in ccfnas in cancertargetedtherapy as well as pathogenesisccfnas in cancer precision medicineprecision oncology is an approach that includes the molecularprofiling of tumors to identify eï¬ective therapeutic strategiesa clinical research program initiated by the englander institutefor precision medicine eipm in uses wholeexomesequencing of metastatic and primary tumors to identifyindividualized therapeutic options and to help guide clinicaldecision making by prospective followup of patients rennert oncology is the obvious choice for heightening theimpact of precision medicine several targeted therapies havebeen developed that have shown profound benefits recentlynovel immunological approaches produced insightful responsessnyder in addition the identification of epigenetic biomarkers leadsto more precise disease prognosis especially in therapeutic areasthat are linked with a high degree of variability concerningsurvival van neste research carried out in severalcancers like glioblastoma reveals that levels of 5hmc are criticalin the regulation of genes having a crucial role in disease andfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerstable frequently hypermethylated genes in various cancer typesgenecancer typereferencesitih5 dkk3 brca1 erbeta apc gstp1 esrbrassf1ap16arf bax bcl2 cdh1 dapk ednrb eomes faddpcdh17 pou4f2sept9 hltf nell1 cea tac1vhlrbtmeff2 prdm13ost2 mgmtapc gstp1st6galnac3 znf660brca1 rassf1a rassf2ahtertp16ink4a timp3 thbs1breast cancerprostate canceresophageal liver and pancreasbladder cancerkloten cheuk vu liu house abern wang colorectal cancerkidney tumorsretinoblastomalung cancerrenal cell carcinomaprostateovarian cancerleptomeningeal carcinomatosis in csfgliomatham semaan ma ohtanifujita palmisano lee su hauser haldrup giannopoulou lonning bougel liu show that global reduction in 5hmc over the genome leads topoor clinical outcomes in these patients johnson epigenetic changes introduced common genetic mutations inan in vitro model of lung cancer vaz epigeneticbased diagnostics can detect early disease signals and thuscan provide possibilities for clinicalintervention before theprogression of symptomsthe detection of ccfnas could be exploited by targetedtherapies approved lately and eventually benefit the patientsscrutinizing cancers by analyzing ccfna dynamics in blood orserum is an innovative and emerging research area as far as theexisting research advancement and the growth of the medicalindustry are concerned we consider that ccfna assays may beemployed for realtime personalized treatments in the future forcancer patients based on their ccfnas or ccfdna methylationlevels for diagnosis and prognosis nevertheless there is muchscope for improvement before the application of this technologyin clinical settingsuse of ccffetalnas in prenataldiagnosis and pregnancyrelateddisordersthe apoptosisnecrosis ofduring pregnancytrophoblastsarising from syncytiotrophoblast is the prime source of therelease of ccffetalnasinto the maternal blood litton the presence of ccffetalnas has pavedthe way for noninvasive prenatal diagnosisand earlylo prediction of pregnancyrelated complications the use of ccffetalnas has gradually replacedinvasive techniques like amniocentesis or chorionic villussampling serr ccffetaldna comprises “ ofthe maternal ccfdna wang andcan be efficiently detected atthe fifth week of gestationguibert the amount of ccffetaldna inmaternal blood increases progressively throughout pregnancybirch ccfnas in prenatal diagnosisprenatal diagnosis is an established practice for the managementof pregnancy as well as avoidance of prenatalneonatal deathsthe leading causes for such deaths are genetic disorderbirth defects congenital malformations and chromosomalabnormalities like trisomy down™s syndrome edward™ssyndrome and patau syndrome and sex chromosomeaneuploidies like monosomy x turner syndrome carlson andvora therefore successful management of pregnancydemands efficient and timely prenatal diagnosis to determine theoutcome of pregnancy timely detection of neural tube defectsis already providing early prenatal treatment resulting in betterneonatal outcomes adzick ccffetaldna is clinically used for the detection of fetal sexand multiple anomalies based on paternally inherited mutationsbianchi recent studies have discovered many fetalepigenetic biomarkers for ccffetalnabased liquid biopsies inclinical samples that have demonstrated high clinical potentialin disease diagnosis prognosis and pregnancy managementthese epigenetic modifications are specific to the fetus and helpto distinguish fetal nucleic acids from maternal nucleic acidsjones and takai clinical testing of recently developedfetal epigenetic markers can help in the proper managementof personalized care the first reported use of fetalderivedepigenetic marker in maternal body fluids had come frompoon who utilized an imprinted h19igf2 locusbased on parentoforiginspecific methylation and the maternaland the paternal copies of the gene were distinguished inmaternal blood poon based on the placental originof ccffetaldna having placentaspecific dna methylationpattern the genomic regions that show diï¬erential methylationbetween the placenta and the maternal blood cells can actas a marker for fetal dna in maternal blood the promoterregion of maspin serpinb5 is the first such reported universalfetal dna marker with detectable hypomethylationin thebackground of hypermethylated maternal sequences the fetalorigin of these hypomethylated maspin has been confirmedby the clearance of these sequences within h of deliveryfrontiers in genetics wwwfrontiersinaugust volume 0crahat ccfnas as epigenetic biomarkerschim the clinical use of hypomethylated maspinis limited by the required bisulfite treatment of ccffetaldna as this treatment can degrade around of the dnagrunau thus decreasing the amount of alreadylow levels of fetal dna in maternal blood such limitationwas overcome by the detection of fetalderived hypermethylatedrassf1a in maternal blood for prenatal diagnosis chan hyland tounta 2011b the maternalhypomethylated rassf1a ccfdna can be removed by treatmentwith methylationsensitive restriction enzyme digestion leavingbehind fetal hypermethylated rassf1a ccffetaldna chan various other fetalderived diï¬erentially methylatedsequences have also shown a similar potential to act as fetal dnaepigenetic markers in maternal blood table ccffetaldna methylation markers have the potential ofbeing used as both quantitative as well as qualitative markersin prenatal diagnosis as qualitative markers these are used toestimate the false positives during the determination of fetalgender rh status and paternally inherited polymorphisms chan while as quantitative markers these can estimate thelevels of ccffetaldna in maternal plasma such an applicationof ccffetaldna finds its use in the detection of chromosomalaneuploidies lun based on the location of themaspin gene on chromosome hypomethylated fetal maspinhas been used to calculate the allelic ratio to diagnose trisomy with sensitivity tong fetal trisomy was detected by analyzing chromosomal dosage via targetingof fetal hypermethylated hlcs sequences in the combinationof microfluidics digital pcr rassf1a on chromosome and zfy on the y chromosome were used as referencestong fragmentation pattern of ccffetaldnain maternal plasma has been successfully used for enrichmentmethod in size separation manner on agarose gel electrophoresisramezanzadeh various nextgeneration sequencing and highthroughputtechniques have catalyzed the identification of newer and novelfetal epigenetic markers further advancing noninvasive prenataldiagnosis the microarraybased approach has identified manyfetal epigenetic markers with diï¬erential methylation betweenchorionic villus samples and maternal blood on chromosomes and for aneuploidy detection chu combining highresolution tiling oligonucleotide array withmethylated dna immunoprecipitation medip has helped ina genomewide screen for detecting the diï¬erential methylatedsites between placental tissue and maternal blood cells it hasdetected various new fetal epigenetic markers on chromosomes and

of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified dtpa complexes21 the original substance of the modified dtpa dtpamod was synthesized in tomsk polytechnic university preparation of colloid solution dtpamod was produced using the following method a sample of modified dtpa with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of nahco3 solution by heating to a0°c after that the volume was adjusted with the same solvent up to the mark in order to reduce the p size the container with suspension was heated in water to a0°c and treated with ultrasound for a0min 1tomsk polytechnic university lenina avenue tomsk russia 2tomsk national research medical center russian academy of sciences kooperativny street tomsk russia email sadkintpuruscientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorfigure a0 the general scheme for the synthesis of 99mtcdtpamodwhich reduced the average p radius up to a0nm the general scheme for the synthesis of 99mtcdtpamod is shown in fig a0the second type of colloids is iron nanops coated with a carbon shell of fec fig a03a these ps were obtained from the institute of metal physics urb ras ekaterinburg russia in order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates adt onto the surface of these ps has been developed an effective method for the synthesis of adt followed by their application onto the carbon surface of ps was developed at the tomsk polytechnic university22 the general scheme for the synthesis of fec ps and their subsequent interaction with 99mtc is shown in fig a03bin the third type of colloids technetium99m was adsorbed on aluminum oxide powder a powder of lowtemperature cubic modification of gammaoxide al2o3 prepared from aluminum hydroxide powder aloh3 by its calcination in a muffle furnace was used the substance was synthesized in tomsk polytechnic universitya reducing agent”tin ii chloride dihydrate was used in order to obtain complexes of 99mtc with colloidsgelatin powdered ph eur uspnf pure pharma grade cas number was purchased from applichem gmbh darmstadt germanymethods method for preparation of 99mtc labeled nanocells the introduction of the radioactive label 99mtc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent sncl2ˆ™2h2o “ a0mgml in different ratios and then adding a a0ml of eluate of 99mtc “ a0mbqml to the mixtures the mixtures were incubated for a0min at a temperature of “ a0°c the preparation is ready for use after cooling at room temperature the reducing agent sncl2ˆ™2h2o was used as a hydrochloric acid solution the amount of a0g of tin chloride ii is added to the vial and a0ml of a0m hydrochloric acid hcl scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cfigure a0 a carbon encapsulated iron nanop b the general scheme for the synthesis of fec psis then added for its preparation after dissolution the volume is adjusted with distilled water to a0ml dissolution was carried out in an inert gas argon mediumdetermination of the size of 99mtc labeled colloidal ps the determination of the size of the labeled nanocolloids was carried out by spectroscopy on a nanophox p size analyzer œsympatec gmbh germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity calculations of the yield of products with different p sizes were determined according to the formulas given belowc220 avc ˆ’ a1avc c100 a1 ˆ’ a2a1 c50 a2 ˆ’ a3a2where avc is the activity of the initial suspension prior to filtration a1 is the activity measured after filtration through a a0nm filter a2 is the activity after filtration through a0nm filter a3 is the activity measured after filtration through a0nm filterin parallel determination of the radiochemical purity rcp of preparations by thin layer chromatography method was carried outthin‘layer chromatography tlc procedure to determine radiochemical purity of 99mtc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip sorbfil russia — a0 cm to determine scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0camount of sncl2ˆ™2h2o mga[sn99mtc]a atcviia table change in relative activities of the complex [sn99mtc] and 99mtc viipertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min in this system pertechnetate migrated with the front of the mobile phase rf to determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min in this system the 99mtc“nanocolloid migrated with the front of the mobile phase rf stability the stability of 99mtc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mtc“nanocolloid following by incubation at a0°c for a0h at different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using tlc24determination of the functional suitability of preparations for scintigraphic detection of sln a study to assess the functional suitability of new nanocolloid rps was performed in series of experiments involving white wistar male rats weighing “ a0g injection of rp in a dose of “ a0mbq was performed between the first and second fingers of the rat™s hind paw the animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ecam signature gamma camera siemens germany the results of scintigraphic studies determined the percentage of accumulation of rp in the lymph node and the injection site the maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œeuropean convention for the protection of vertebrates used for experiments or other scientific purposes strasbourg the experimental protocols were approved by cancer research institute biomedical ethics committee protocol number all invasive manipulations with animals were performed using inhalation or drug anesthesiastatistical analysis all mean values are expressed as idg ± sd data were analyzed statistically using methods of general statistics with a commercially available software package œstatistics for windows statsoft inc version results and discussionto carry out the labeling of colloids 99mtc extracted from a standard generator in the form of pertechnetate ions contained in a nacl solution was used it has a higher degree of oxidation vii in this chemical form and is not prone to complex formation a reducing agent”tin ii chloride dihydrate widely used for the preparation of various compounds labeled with 99mtc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 as a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess sncl2·2h2o or the additional formation of a complex of reduced 99mtc with tin26 all this required preliminary experimental studies to establish the necessary and sufficient amount of sncl2·2h2o in the reaction mixtureduring the experiments it was found that the optimal concentration of sn ii in the composition of the reaction mixture when it interacts with 99mtc should be in the range of “ a0mgml table a0it was found that when the eluate with the preliminarily reduced 99mtc vii was added to the nanops the sn ii concentration introduced in the rp was csn a0mgml almost the entire amount of 99mtc has time to enter the composition of the largesize complex with tin even before its mixing with nanops this means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the sn ii solution into the reaction mixture in this connection the reduction of 99mtc with tin ii was carried out in the presence of the selected substance in this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex the technique of applying of the 99mtc label to the surface of nanosized ps is given in the previous sectionas a result of the studies reagent compositions and conditions for obtaining three nanocolloid rps were determined table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmproceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mtc vii in the obtained preparations is “ preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in fig a0scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0ccomposition of the preparation per a0mldtpamod a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n fec a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n al2o3 a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg n colloid yield “ a0nm rcp ± ± ± table composition of reagents for production of technocium99 a0m nanocolloidsfigure a0 distribution of the preparation in the rat when the preparation is administered [al2o3 99mtc sn ii] a immediately after the administration of the drug b a0min after the administration c a0min after the administrationcomposition of preparations per a0mlal2o3 a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n dtpamod a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n fec a0mg 99mtc “ a0mbq sncl2ˆ™2h2o a0mg g a0mg n yield of colloid “ a0nm rcp ± ± ± table indicators of rcp and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsscintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mtc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed gelatin g was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mtc the results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm table a0in addition the size of these ps was determined on a nanophox p analyzer the obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in fig a0 a b c the average p size diameter is and a0nm respectivelystability test showed that complex 99mtc“nanocolloid was stable in normal serum at least for a0h radiochemical purity of the tracer at the end of the experiment was ± a study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes fig a0 table a0 displays the al2o3 99mtc dtpamod 99mtc fec 99mtc biodistribution data at different time points postinjectionthe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionas a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid rps were determined an experimental dependence of the change in the content of 99mtc vii impurities on the concentration of tin ii was established and its minimum amount a0mgml was determined to reach a rhp greater than in this case the yield of the target colloid with p sizes of ± a0nm is “ preliminary tests of the developed preparations on experimental animals showed that accumulation of rp in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml in addition there was an increase in the yield of the colloid scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0cfigure a0 change in the density of the distribution of the number of ps from their size in radiopharmaceuticals a œ99mtcal2o3 b œ99mtcfec c œ99mtcdtpamodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of scientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0cstomachtime h99mtcal2o399mtcdtpamod99mtcfec ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± figure a0 distribution of the preparation in the rat with injection of suspension [al2o3 99mtc sn ii gelatin] a immediately after the administration of the preparation b a0min after the administration c a0min after the administration d a0min after the administration the numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± bloodmlheart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± table biodistribution data up to a0h after injection of “ a0mbq of 99mtc in healthy male rats data represent the average value n accumulation in the sln thus the level of accumulation of rp œ99mtcdtpamod and rp œ99mtcfecadt in the sln is and respectively at the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityreceived march accepted july references jakobsen j k sentinel node biopsy in urooncology a history of the development of a promising concept urol oncol “ weixler b et al sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival a prospective singlecenter trial world j surg 101007s0026 beasley g m et al sentinel lymph node biopsy for recurrent elanoma a multicenter study ann surg oncol moser j et al sentinel node biopsy in melanoma a singlecentre experience with consecutive patients br j dermatol 101245s1043 “ buda a et al optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer comparison of realtime fluorescence with indocyanine green and methylene blue int j gynecol cancer “ scientific reports 101038s41598020709912vol0123456789wwwnaturecomscientificreports 0c“ sahbai s et al pericervical injection of 99mtcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in spectct clin nucl med “ hoogendam j p et al 99mtcnanocolloid spectmri fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer j nucl med “ stoffels i leyh j p¶ppel t schadendorf d klode j evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies prospective randomized clinical trial to compare icg99mtcnanocolloid hybrid tracer versus 99mtcnanocolloid eur j nucl med mol imaging “ beisani m et al initial experience in sentinel lymph node detection in pancreatic cancer rev esp med nucl imagen mol schubert t uphoff j henke r p wawroschek f winter a reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer verification in consideration of the european guidelines bmc urol “ jaukovic l et al lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions hell j nucl med “ subramanian s pandey u shah s rangarajan v samuel g an indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection nucl med commun “ ruizdom­nguez j m ibarzservio l garc­ade manuel g calaf peris© o intraoperative injection of 99mtcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery actas urol “ schauer a j specific developments in sentinel node labling using 99mtccolloids in the sentinel lymph node concept springer berlin wang y et al gasphase chemistry of technetium carbonyl complexes chem phys “ o™connor m k et al comparison of tc99m maraciclatide and tc99m sestamibi molecular breast imaging in patients with wang j zhang r evaluation of 99mtcmibi in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer ejnmmi res br j radiol costa p et al scintigraphic imaging with technetium99mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats acta cir bras “ vera d r wallace a m hoh c k mattrey r f a synthetic macromolecule for sentinel node detection 99mtcdtpamannosyldextran j nucl med “ hoh c k wallace a m vera d r preclinical studies of [99mtc]dtpamannosyldextran nucl med biol “ skuridin v et al modified dtpa moleculebased nanocolloid radiopharmaceuticals j radioanal nucl chem “ filimonov v d et al unusually stable versatile and pure arenediazonium tosylates their preparation structures and synthetic applicability lett “ lukasz k thin layer chromatography in drug analysis “ crc press london skuridin v et al radiopharmaceutical drug based on aluminum oxide indian j sci technol 1017485 ijst2015v8i36 sazonova s i et al synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging iran j nucl med “ skuridin v s et al synthesis and biological characterization of 99mtclabeled ciprofloxacin pharm chem j “ acknowledgementsthis work was financially supported by ministry of education and science of the russian federation rfmefi57514x0034author contributionsvs conducting experimental research analysis and interpretation of the data final approval for manuscript publication vs development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication en development of the concept and direction of research analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication es development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data validation of critical intellectual content final approval for manuscript publication ar conducting experimental research analysis and interpretation of the data final approval for manuscript publication nv conducting experimental research analysis and interpretation of the data final approval for manuscript publication rz conducting tests of the functional suitability of drugs preparation of the section evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and figures „– final approval of the manuscript for publication of the manuscriptcompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to vsreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020709912vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 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Journal of International Medical Research “ The Authors reuse guidelinessagepubcomjournalspermissions journalssagepubcomhomeimrCase ReportNivolumab plus gemcitabinedexamethasone and cisplatinchemotherapy induce durablecomplete remission inrelapsedrefractory primarymediastinal Bcell lymphomaa case report andliterature reviewGang Huang1 Ju Huang2 Zhili Zhang2Chongchong Xue1 and Yuan Liu2AbstractPrimary mediastinal large Bcell lymphoma PMBCL is an uncommon but aggressive type ofBcell lymphoma Patients with relapsed refractory PMBCL rrPMBCL have limited therapeuticoptions and usually have a relatively poor outcome Immune checkpoint blockade has become apotential treatment for this disease We report here a case of a female patient with rrPMBCLwho was treated with nivolumab plus gemcitabine dexamethasone and cisplatin GDP chemotherapy Complete remission was achieved after four cycles of combined therapy With continuednivolumab maintenance monotherapy she has remained in complete remission for longer than months This is the first report of nivolumab plus GDP chemotherapy inducing completeremission in patient with rrPMBCL This case supplements the limited literature and providesimplications for clinical trial designs regarding the potential use of nivolumab in the treatment ofrrPMBCL1Department of Hematology Yuebei People™s HospitalShantou University Medical College ShaoguanGuangdong Province China2Guangdong Women and Children Hospital GuangzhouGuangdong ChinaCorresponding authorYuan Liu Medical Genetic Centre Guangdong Womenand Children Hospital No Xingnan Rd PanyuDistrict Guangzhou Guangdong ChinaEmail yuanliu005163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical ResearchKeywordsRelapsed refractory primary mediastinal Bcelllymphoma nivolumab checkpoint blockadegemcitabine dexamethasone cisplatin chemotherapy programmed cell death completeremissionDate received February accepted July IntroductionPrimary mediastinal large Bcell lymphomaPMBCL is an uncommon but aggressivetumor that accounts for to of nonHodgkin lymphoma1 PMBCL is distinguished from diffuse large Bcell lymphomaby virtue of distinct clinical pathologicaland genetic features2“ Recently PMBCLwas listed as a separate entity in the latestWorld Health anization classification of hematopoieticand lymphoidtumors5 PMBCL has a similar clinical presentation as classical Hodgkin lymphomacHL and PMBCL also shares certain features at the molecular level particularly9p241 alterations and programmed celldeath protein ligand 1ligand PDL1PDL2 expression6“ At present management and outcome of PMBCL are still critical and a more serious situation is faced bypeople who are diagnosed with relapsedand refractory PMBCL rrPMBCL19The optimal salvage chemotherapy andautologousforrrPMBCL are of limited efficacy19stem celltransplantRecently agents targeting programmedcell death PD1 and PDL1 have beenimmunotherapy10developed in tumorAntiPD1 therapy with monoclonal antibodies has been approved for the treatmentof several types of solid tumor and cHLThe therapeutic potential of antiPD1 therapy in other malignancies is likely to beapproved soonIn a humanizedimmunoglobulin G1 recombinant monoclonal antibody for the PD1 receptor pidilizumab was approved by the US Food andDrug Administration FDA for treatingandpediatricpatientsthatwithadultrrPMBCL11 Another agenttargetsthe PD1 receptor called nivolumab isa fully humanized immunoglobulin G4monoclonal antibody that has been grantedapproval by the US FDA for treating several solid malignancies and cHL The therapeutic efficacy of nivolumab in patientswith rrPMBCL remains unclearWe report here a patient with rrPMBCLwho received combined treatment with offlabel nivolumab and GDP chemotherapyComplete remission CR was achievedafter four cycles of such combined treatment At the time of this submission thepatient has remained in CR for longerthan months with continued nivolumabmaintenance monotherapyCase reportA 32yearold woman presented to YuebeiPeople™s Hospital with intermittent dyspneaand chest pain A positron emission tomography PET scan showed a 10cm mass inthe anterior superior mediastinum with astandardized uptake value of Themass showed unclear margins and compressed the ascending aorta and pulmonarytrunk Small pericardial and left pleuraleffusions were also observedThe mass was diagnosed as PMBCL by asubsequent biopsy Immunohistochemicalstaining showed thatlarge lymphocyteswere positive for CD20 CD79a Pax5BCL6 CD23 CD30and multiplemyeloma1 and negative for CD10 CD3CD5chromogranin Asynaptophysin 0cHuang et alterminalencodingandincludingtwocycles positiveregion inendomysialdeoxynucleotidyltransferase cytokeratin CK CK19 andandS100 Ki67 wassituEpstein“Barrhybridization was negative She was initially treated with six cycles of frontline chemotherapyofrituximab cyclophosphamide doxorubicinvincristine and prednisolone RCHOPand four cycles of doseadjusted etoposidecyclophosphaprednisolonemiderituximabDAEPOCHR were administered Thetimeline of treatment is shown in aShe received tumor resection by thoracoscopic surgery after she continued twodoxorubicinvincristinecycles ofgemcitabine dexamethasonecisplatinumetoposide and rituximabtherapy Her first CR was achieved inDecember However monthslater a PETcomputed tomography CT scan showedhypermetabolic lesions located at the leftlung and right adrenal gland but not inthe primary mediastinal site bThe patient reported no physical symptomsand received a repeat tissue biopsy whichconfirmed a relapse with PMBCL She wastreated with each cycle of a dexamethasoneifosfamide cisplatin and etoposide regimenand ibrutinib bendamustine and cytarabine therapy A chest CT scan showedFigure Summary of treatment and monitoring the tumor response a Patient™s timeline chart with thedates of treatment and monitoring the tumor response b Positron emission tomography images Upperpanel a scan of the relapsed hypermetabolic lesions located at the left lung and right adrenal gland beforecombined treatment Lower panel complete remission was achieved after four cycles of nivolumab plusGDP chemotherapyRCHOP rituximab cyclophosphamide doxorubicin vincristine and prednisolone DAEPOCHR doseadjusted etoposide prednisolone vincristine cyclophosphamide doxorubicin and rituximab GDPERgemcitabine dexamethasone cisplatinum etoposide and rituximab CR complete remission PMBCLprimary mediastinal large Bcell lymphoma DICE dexamethasone ifosfamide cisplatin and etoposideIBC ibrutinib bendamustine and cytarabine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical Researchthat the right adrenal gland lesion had partially responded while the lesions in the leftlung had progressed After those cycles ofchemotherapy the patient showed GradeIV myelosuppression and had to receiveblood transfusion treatment Moreover acerebrospinal fluid examination showedthe presence of atypical lymphocytes andno symptoms of infection of the central nervous system were observed Intrathecal chemotherapy cytarabine mg methotrexate mg and dexamethasone mg was thenadministered and no atypical lymphocyteswere detected by repeated cerebrospinalfluid analysis These findings highly suggested a potential risk of metastasis of thecentral nervous systemtreatmentBecause the disease had progressed withsevere myelosuppression and there were nostandard chemotherapy guidelines or alternative treatment options for the patientother salvage treatments of her refractorydisease needed to be considered Aftermuch discussion with the patient and herfamily she declined autologous hematopoietic stem cell transplantation and receivedcombinedgemcitabine mg dexamethasone mg and cisplatinum mg GDPchemotherapyand the offlabel antiPD1 antibody nivolumab mg After four cycles of combined treatment a repeated PETCT scanshowed thatshe had secondary CRb She received two more cyclesof combined treatment with nivolumab andGDP chemotherapy and then continuedsingle nivolumab maintenance treatment mg Since her first dose in May she received doses of nivolumab Shereported moderate fatigue and pyrexia in to days after each administration ofnivolumab Blood tests indicated normalfunction of the liver kidney and thyroidFigure She also had normal bloodlevels of creatinine albumin globulin lactate dehydrogenase aspartate transaminasetotalaminotransferaseofalaninethey2b Neutrophilbilirubin and urea nitrogen during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the first four combined therapies because of toxicity of GDPchemotherapy butrecovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2cFurthermore no adverse signs and symptoms were observed in the lungs brainand skin At the time of this submissionshe has remained in CR for longer than months with continued nivolumab maintenance therapyEthics approval was obtained from theethicalcommittee of Yuebei People™sHospital Written informed consent wasobtained from the patient for analysis ofthe samples and publicationDiscussionTreatment and outcome are critical in managing PMBCL Because there is no established standard approachthe firstlinetreatment of PMBCL is generally thesame as that for diffuse large Bcell lymphoma including RCHOP and DAEPOCHR Relapse of PMBCL usually occurs in thefirst to months after completion offrontline therapy with a lower incidenceapproximately “than diffuselarge Bcell lymphoma19 There are varioussecondlineregimens for patients with rrPMBCL includingthe rituximab ifosfamide carboplatin andetoposide regimenthe rituximab dexamethasone cytarabine and cisplatin regimen and rituximabGDP12 Because of alack of standard guidelines or treatmentoptions for PMBCL the outcome greatlydepends on the patients™ response to theregimen Thisremains poordespite these secondline salvage chemotherapies and subsequent autologous hematopoietic stem cell transplantation912immunochemotherapyresponse 0cHuang et alFigure Blood test values during the whole treatment process since the first dose of nivolumab The firstfour cycles were nivolumab plus GDP chemotherapy and nivolumab maintenance monotherapy wasadministered since the fifth cycle a Thyroxine thyrotropin FT3 and FT4 levels b Levels of creatininealbumin globulin lactate dehydrogenase aspartate transaminase alanine aminotransferase total bilirubinand urea nitrogen c Neutrophil and platelet countsFT4 free thyroxine FT3 free triiodothyronine GDP gemcitabine dexamethasone and cisplatin 0cJournal of International Medical ResearchIn recent years strategies focusing on thecheckpoint blockade have been developedin tumor immunotherapy10 Therapeuticantibodies targeting the PD1“PDL1 axispossess clinical activity and an acceptablesafety profile in treating a growing list oflymphomas13solid tumors and BcellBased on a clinical study of patientswith rrPMBCL pidilizumab was approvedby the US FDA for treatment of adult andpediatric patients with rrPMBCL in Another antibody nivolumab has beengranted approval for treating several solidmalignancies and cHL However studiesregarding application of nivolumab forPMBCL are limited Only five reportshave described using nivolumab for treatment of PMBCLrrPMBCL Table asfollows In a phase I study published intwo patients with PMBCL wererecruited and treated with nivolumab atdoses of or mgkg every weeks afterprevious systemic treatments14 No objective responses were observed in this previous study In another phase I study onewithrefractorypatient with PMBCL received combinedtherapy of nivolumab and ipilimumaband died during the therapeutic process15Recently two reports showed the potentialtherapeutic efficiency of nivolumab forpatientsPMBCLrrPMBCL who showed failure with conventional immunochemotherapy1617 Both ofthese two cases had immunerelated adverseeffects during the antibody treatment process One patient with highgrade neutr ia had nivolumab stopped temporarilyand was treated with intravenous immunoglobulin16 The other patient with zosterreactivation was controlled by administration of valacyclovir17 Recently Zinzaniand colleagues showed that combined treatment of nivolumab and brentuximab vedotin had promising antitumor activity and amanageable safety profile in patients withrrPMBCL18 In this phase II study patients were recruited and treated withnivolumab mgkg and brentuximab vedotin mgkg every weeks The objectiveresponse rate was and achievedTable Reports regarding application of nivolumab in primary mediastinal large Bcell lymphomarelapsedand refractory primary mediastinal large Bcell lymphomaNumberof cases DoseCombinedtreatmentAdverse events or mgkg“ mgkgIpilimumab““ResponseyesnoNoNo ofpatientsReportsLesokhin AMet al Ansell S et alWright Zet al Yassin R et alZinzani“ mgkgNoNoHighgrade neutr iaYesZoster reactivationYes mgBrentuximabNeutr iaPL et al vedotinthrombocyt iaand peripheralneuropathy of patientsPresent case“ mgkgGDPMild fatigue and pyrexiaYeschemotherapyNote “ means not indicated in the report 0cHuang et alofcyclescombinedCR and achieved partial remission Of patients of them had drugrelatedadverse events and the most common wereneutr ia thrombocyt ia and peripheral neuropathy18 In the present case weattempted several available approaches intreating the patient™s relapsed disease butfailed to control the progress of the massAfter much discussion with the patient andher family we considered an offlabel nivolumab and GDP chemotherapy as salvagetreatmentfor the patient In September her second CR was achieved afterfourtreatmentCurrently with continued nivolumab maintenance monotherapythe patient hasremained in CR for longer than monthsImmunerelated adverse events that areassociated with checkpoint blockade oftenstart within the first few weeks to monthsafter treatment but can occur any time andin any an The most common immunerelated adverse events are hypothyroidismnausea diarrhea pyrexia and fatigue1920In the present case we were concernedabout immunerelated an damage sincethe first dose of nivolumab The patientreported moderate fatigue and pyrexiaafter each administration of nivolumaband soon recovered within to daysBlood testing was performed during thewhole therapeutic process and the datawere reviewed and analyzed Blood levelsof thyroxine thyrotropin free triiodothyronine and free thyroxine indicated no thyroiditisFigure 2a Our patient alsoshowed normal metabolic data during thewhole process of nivolumab therapyFigureand plateletcounts were decreased in the first four combined therapies because of toxicity of GDPchemotherapy but they then recovered tonormal levels during continued nivolumabmaintenance monotherapy Figure 2c2b NeutrophilUnlike otherarelymphomas prognosticbiomarkersinPMBCL12 Some serum molecules such aslackinglargelyCCL17 and CD163 are considered aspotential biomarkers for predicting andmonitoring responses and detection ofrelapses in patients with Hodgkin lymphoma1221 The role of serum biomarkers inPMBCL remainsinvestigatedRadiological imaging should only be usedin patients who have new clinical symptomsor signs suggestive of relapse but not inasymptomatic patients922betoTo the best of our knowledge this is thefirst reported case of nivolumab plus GDPchemotherapy that induced CR with nosevere immunerelated an damage in apatient with rrPMBCL We also reportthe longest followup observation of successful application of nivolumab in apatient with rrPMBCLThis report supplements the limited literature of nivolumab fortreatment ofPMBCLrrPMBCL and provides implications for clinical trial design regarding thepotential use of nivolumab in treatment ofrrPMBCL Further investigation needs to beperformed for potential application of singleor combined use of nivolumab for patientswith rrPMBCL who experience failure withconventional therapeutic approachesDeclaration of conflicting interestThe authors declare that there is no conflict ofinterestFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsorcid000000034880ORCID iDYuan LiuReferences Martelli M Ferreri A Di Rocco A et alPrimary mediastinal large Bcell lymphomaCrit Rev Oncol Hematol “ 0cJournal of International Medical Research Savage KJ Monti S Kutok JL et al Themolecular signature of mediastinallargeBcell lymphoma differs from that of otherdiffuse large Bcell lymphomas and sharesfeatures with classical Hodgkin lymphomaBlood “ Rosenwald A Wright G Leroy K et alMolecular diagnosis of primary mediastinalB cell lymphoma identifies a clinically favorable subgroup of diffuse large B cell lymphoma related to Hodgkin lymphoma J ExpMed “ Mottok A Wright G Rosenwald A et alMolecular classification of primary mediastinal large Bcell lymphoma using routinelyavailable tissue specimens Blood “ Swerdlow SH Campo E Pileri SA et alThe revision ofthe World Healthanization classification of lymphoid neoplasms Blood “ XuMonette ZY Zhou J and Young KHPD1 expression and clinical PD1 blockadelymphomas Blood in Bcell“ Van Roosbroeck K Ferreiro JF TousseynT et al Genomic alterations of the JAK2and PDL loci occur in a broad spectrum oflymphoid malignancies Genes ChromosomesCancer “rearrangements Twa DD Chan FC BenNeriah S et alGenomicinvolving programmed death ligands are recurrent in primary mediastinallymphomaBlood “large Bcell Cwynarski K Marzolini MAV BarringtonSF et al The management of primary mediastinal Bcell lymphoma a British Societyfor Haematology Good Practice Paper BrJ Haematol “ Ribas A and Wolchok JD Cancer immunotherapy using checkpoint blockade Science “ HematologyOncology Cancer Approvals Safety Notifications Available online URL wwwfdagovdrugsinformationondrugsapproveddrugsucm610670htmdrugsapproveddrugsucm610670htmwwwfdagovdrugsinformationon Lees C Keane C Gandhi MK et al Biologyand therapy of primary mediastinal Bcelllymphoma current status and future directions Br J Haematol “ Goodman A Patel SP and Kurzrock RPD1PDL1 immunecheckpoint blockadein Bcell lymphomas Nat Rev Clin Oncol “ Lesokhin AM Ansell SM Armand P et alNivolumab in patients with relapsed orrefractory hematologic malignancy preliminary results of a phase Ib study J Clin Oncol “ Ansell S Gutierrez ME Shipp MA et alA phase study of nivolumab in combination with ipilimumab for relapsed or refractory hematologic malignancies CheckMate Blood “ Wright Z and Brown A Highgrade neutr ia in a patient successfully treated withnivolumab for refractory primary mediastilymphoma Blood Adv nal Bcell“ Yassin R Hajeer A Alshieban S et al HLAgenotype and response to nivolumab therapy in relapsed refractory primary mediastinal Bcell lymphoma Curr Res Transl Med “ Zinzani PL Santoro A Gritti G et alNivolumab combined with brentuximabvedotin forrelapsedrefractory primarymediastinal large Bcell lymphoma efficacyand safety from the Phase II CheckMate Study J Clin Oncol “ Postow MA Sidlow R and Hellmann MDImmunerelated adverse events associatedwith immune checkpoint blockade N EnglJ Med “ Zinzani PL Ribrag V Moskowitz CH et alSafety and tolerability of pembrolizumab inpatients with relapsedrefractory primarylymphoma Bloodmediastinal “large Bcell Jones K Vari F Keane C et al SerumCD163 and TARC as disease response biomarkers in classical Hodgkin lymphomaClin Cancer Res “for Cheson BD Fisher RI Barrington SF et alinitial evaluationRecommendationsstagingofHodgkin and nonHodgkin lymphoma theLugano classification J Clin Oncol “assessmentandresponse 0c'

"Sleep disturbance is an issue reported by caregivers Waking at night is a feature of dementia and byproxy sleep disturbance among caregivers is reported to be high Little is known about the characteristics ofdementia caregivers™ sleep and the factors that may influence sleep disruptionThe purpose of this study was to investigate the sleep characteristics and disturbances of Australian caregivers of aperson living with dementia In addition it evaluated the psychological wellbeing of caregivers by evaluatingassociations between mood and sleep in this populationMethods This study used a crosssectional descriptive correlation design Participants were recruited with theassistance of Alzheimer™s Australia Dementia Australia and targeted social media advertising In total adultprimary informal caregivers of people with dementia participated completing a questionnaire on demographiccharacteristics the Depression Anxiety and Stress Scale DASS21 and the Pittsburgh Sleep Quality Index PSQIResults In this study of caregivers were female who had been caring for someone living with dementia onaverage for years of participants had two or more comorbidities namely cardiovascular disease osteoarthritisand diabetes of participants were poor sleepers with with difficulty initiating sleep and reporting havingdifficulty maintaining sleep Overall psychological distress was common with high levels of moderate to severedepression anxiety and stress Global PSQI scores were significantly positively associated with depression and anxietywith the strongest correlation seen with stress scores Depression scores were also moderately associated with daytimedysfunction Stress was identified as a significant predictor of overall sleep qualityConclusions Sleep problems are common within the population of dementia caregivers Due to the nature andduration of caregiving and the progression of dementia of the care recipient there is the potential for a decline in thecaregivers™ mental and physical health Caregivers of those living with dementia are more likely to have comorbiditiesdepression anxiety and stress Sleep quality is correlated with emotional distress in dementia caregivers although thedirection of this association is unclear Therefore sleep and psychological wellbeing may be intertwined withimprovements in one aspect resulting in a positive impact in the otherKeywords Carers Caregivers Sleep Mood Psychological wellbeing Dementia Correspondence aislingsmythecueduau1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA AustraliaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cSmyth BMC Geriatrics Page of BackgroundDementia is an inclusive term used to describe a numberof neurological conditions resulting in cognitive impairment and can include Alzheimer™s Disease senile dementia frontotemporal dementia vascular dementia LewyBody dementia Korsakoff syndrome alcohol related braininjury and younger onset dementia [“] Globally thenumber of individuals with a formal dementia diagnosisrose from million in to million in []A further million new cases of dementia worldwideare predicted each year [] as populations around theglobe continue to live longer [] These figures could bevery much under estimated as it is widely accepted thatbetween and of people affected have no formaldementia diagnosis [] This growing epidemic effects notonly the individuals living with dementia but also theirfamilies caring for them the communities they live in andthe health care systems they rely uponFamily or friends are often key informal caregivers forpeople living with dementia [ ] Globally over billion hours ofinformal care is provided to peopleliving with dementia [ ] Whilst providing this carecan be highly rewarding it has also been described as achronic stressor with caregivers reporting low qualityand quantity of sleep [] and high levels of stress anddepression [] In fact sleep disruption is prevalentamongst dementia caregivers with over of caregiversexperiencing sleep disturbances []The National Sleep Foundation recommend that olderadults ‰¥ years require seven to h sleep for optimalphysical and psychological wellbeing [] Yet numerousstudies have illustrated that dementia caregivers sleepsignificantly less than that with estimates reporting mostcaregivers sleep less than h per night [] Not only arecaregivers sleeping less than they should the sleep theydo get is of significantly lower quality than their noncaregiver counterparts []Poor sleep is associated with a myriad of negative physical and psychological outcomes including hypertensionobesity mood disorders and dementia [] A disruptedsleep pattern is also recognised as a significant factor inpredicting caregiver strain [ ] and perhaps more importantly in predicting placing an individual into long termcare [] Enabling people living with dementia to stay athome rather than transfer to longterm care is the optimaloutcome for many families However this cannot be to thedetriment of the caregiver™s own physical andor psychological wellbeing Therefore in order to support the personliving with dementia PLWD to remain in the communitymaintaining caregiver health is vital Given the pivotal rolesleep has in a myriad of physiological processes it is essential to optimise and preserve caregivers sleepDespite the important role sleep plays in dementiacaregiver health it remains an understudied populationparticularly within the Australian context In fact arecent report by Carers Australia identified noAustralian studies on sleep disruption in dementia caregivers Only sixteen international studies were identifiedwhich subjectively measured sleep in the dementiacaregivers population with the majority of studies notreporting on the causes or consequences of disturbedsleep []In order to address this paucity of Australian data thepurpose of this study was to elucidate the sleep characteristics and disturbances of Australian caregivers ofPLWD Furthermore this study will determine whetherthere is a relationship between sleep and psychologicalwellbeing among caregivers of communitydwellingpeople living with dementia Lastly we will aim to identify significant predictors of poor sleep which in turncould offer a therapeutic target of poor sleep in dementia caregiversMethodsRecruitmentThis study used a crosssectional descriptive correlationdesign One hundred and four informal caregiversof people with dementia living in the community wereenrolled in the study Participants were invited via anumber of anisations including Alzheimer™s AustraliaDementia Australia and targeted social media advertising Mail outs were conducted and online questionnairesdistributed The inclusion criterion required the participant be an adult years or older primary informalcaregiver of a communitydwelling person living withdementia Power analysis was undertaken to computeminimum number of sample size required A samplesize of at least participants is necessary to detect amedium to large effect with power assuming atwotailed ttestEthicsThis study was granted ethical approval by Edith CowanUniversity Human Research Ethics Committee No Informed written consent was obtained from allparticipants prior to participation Participants receivedno incentive for taking partMaterialsThe survey collected demographic characteristics including gender age body mass index BMI as well as information about preexisting medical history caregivinghistory and use of respite The survey also included the questions from the Depression Anxiety and StressScale DASS21 [] and the Pittsburgh Sleep QualityIndex PSQI []The DASS21 is a selfreport questionnaire assessinglevels of caregiver stress anxiety and depression over the 0cSmyth BMC Geriatrics Page of previous seven [] day period The results provide anindication of the individual™s perception of their experience of depression anxiety and stress The DASS21 isoften used as a screen for high levels of distress whenthe depression andor anxiety scores are high and as ascreen for the presence of a significant life event orproblem if the stress score is elevated DASS scorescorrespond to ranges of severity normal mild moderate severe extremely severe for depression anxiety andstress individually []The PSQI [] is a selfreport questionnaire assessinglevels of perceived quality and patterns of sleep over theprevious month across seven components subjectivesleep quality sleep latency sleep duration habitual sleepefficiency sleep disturbances use of sleep medicationand daytime dysfunction Each component has a potential score of three with a higher score indicating poorersleep related performance The total score for the sevencomponents creates a global score range to wherea score greater or equal to five ‰¥ indicates that theperson is a œpoor sleeper with severe difficulties in atleast two of the seven components or moderate difficulties in three or more components []Presence of comorbidities was determined by participantshaving one or more chronic conditions As per WorldHealth anisation WHO chronic conditions wereidentified as those requiring ˜ongoing management over aperiod of years or decades™ covering a wide range of healthproblems such as heart disease diabetes asthma immunodeficiency disorder depression and schizophreniaStatistical analysesData analysis was undertaken using IBM SPSS Version and GraphPad Prism Descriptive statistics weregenerated for demographic characteristics DASS21 andPSQI scales Spearmans rho r correlation analysis wasused to assess the relationship between categorical components of DASS21 depression anxiety and stress andboth PSQI global scores and individual componentscores subjective sleep quality sleep latency sleep duration sleep efficiency sleep disturbances use of sleepingmedication and daytime dysfunction For logistic regression all variables were categorical age in years genderBMI length of care in months comorbidities DASS21component scores An r of was considered a mediumcorrelation and an r of was considered a large correlation [] Nonpaired ttests were used to determine ifthese values were statistically significant Results wereconsidered statistically significant if p Multivariatestepwise regression analysis was undertaken to determinethe predictive factors of global PSQI scores and identify variables significantly associated with sleep quality Independentvariables included in multivariate stepwise regression analyses were age gender BMI length of care comorbiditiesand DASS21 subscale scores Subjects with missing data oneither PSQI or DASS21 were excluded from inclusion incorrelation and regression analysesResultsCharacteristics of the caregiversOne hundred and four n surveys were completedin either hardcopy n or online n Table As the surveys were widely distributed by service providers and links made available online the response ratecannot be determinedParticipating caregivers were predominantly femalen with a mean age of years Range “years The average BMI for respondents was kgm2range “ kgm2 Table which lies within theoverweight category While half of the participants n had a BMI within the healthy weight range“ twenty two participants were classified as underweight BMI less than and participants were classified as overweight The averagelength of time in the caregiving role in this study was months years ranging from months to yearsOnly n of participants reported using formalrespite services and all were female n of participants reported no comorbidities of participants n reported one comorbidity and n reported two or more comorbidities Themost commonly reported comorbidities were cardiovascular disorders n mainly hypertension and hypercholesterolemia bone and joint disorders n mainlyTable Demographic of participantsCharacteristicGendern or meanˆ’ SDMaleFemaleAllAgeBMI kgm2Caregiver role MonthsComorbiditiesNoneOneTwo TotalUse of RespiteYesNoTotal99a94b96c ± ± ± a n missingb n missingc n missing 0cSmyth BMC Geriatrics Page of osteoporosis and arthritis and endocrine disorders n mainly prediabetes diabetes and thyroid dysfunctionTable Characteristics of Caregivers™ sleepMeasuren Subjective Sleep qualityThe majority of participants n reported aglobal sleep score equal or greater than which indicated they had clinically significant sleep issues in thepreceding month with a mean global score of ± Table The highest scoring individual subcomponentsincluded sleep latency ± subjective sleep quality ± and sleep disturbances ± n of participants had a sleep latency period greater than min indicative of issues initiating sleep Table ofparticipants had sleep efficiencies less than suggestingissues with maintaining sleep while in bed of participants had taken sleep medication at least once a weekover the previous month Participants were invited to addany additional relevant comments to their PSQI sleep assessment Forty participants provided additional information regarding their sleeping issues and identified issueswhich fell broadly into three themes Sleep disruption dueto caregivers physical needs such as pain and restless legs caregivers emotional distress such as stress anxietyand worrying and responding to care recipientneeds Depression anxiety and stress in caregivers of participants reported mild depression scores of participants reported mild anxiety scores and of participants reported mild stress scores Over athird of respondents reported moderate to extremelysevere stress levels n and depression n and more than a quarter n of respondents reported moderate to extremely severe anxietylevels Table In the bivariate analyses numerous subcomponents ofthe PSQI scale were significantly associated with measures of depression anxiety and stress Table Theglobal PSQI score is significantly positively associatedwith depression r anxiety r and stressscores r Stress scores also significantly correlatedwith other PSQI subcomponents including subjectivesleep quality r sleep latency r and daytimedysfunction r Depression scores were moderatelyassociated with daytime dysfunction r Global ScoreSubjective Sleep QualityVery GoodFairly GoodFairly BadVery BadSleep Latency‰¤ mins“ min“ min minsSleep duration h“ h“ h hSleep Efficiency‰¥ ““ Sleep DisturbancesDaytime DysfunctionFrequency of SleepingMedicationNeverOnce per weekTwice per weekThree per week PSQI ComponentMean ˆ’ SD ± ± ± ± ± ± ± ± Predictors of Sleep quality in caregiversTo understand the relationship between the predictorsand global PSQI scores multivariate stepwise regressionanalyses were conducted to assess variables significantlyassociated with sleep quality The dependent variablewas the global PSQI score and the independent variableswere age gender BMI use of respite length of carecomorbidities and scores on the subscales of DASS21scale Depression Anxiety and Stress Stress was theonly significant covariate of global PSQI scores Stressscores could statistically significantly predict PSQIGlobal scores accounting for of variance Thestandardised coefficient was which was statisticallysignificant p as was the overall model F p All other variables were excluded from themodel due to nonsignificance Forward stepwise regression 0cSmyth BMC Geriatrics Page of Table Depression anxiety and stress in caregivers DASS21Clinical classificationTotalDepression n Anxiety n Stress n NormalMildModerateSevereExtremely Severe analyses were then undertaken with global PSQI scores asthe dependent variable and stress scores as a predictorwhile adjusting for the following confounders gender agecomorbidities of the caregiver and length of care Whilethese confounders were not statistically significant theywere retained within the model to adjust the effects ofstress on PSQI After adjusting for these factors that modelremains statistically significant F p andaccounts for of the adjusted variance in sleep scoresFor every one unit increase in stress scores PSQI Globalscores increases by a score of t p Thisregression model confirms that stress is a key significantcovariate of selfreported sleep issuesDiscussionA recent Australian report highlighted a significant gapin the literature around Australian caregivers of a PLWD[] Our study provides a comprehensive overview ofAustralian dementia caregiverssleep characteristicsassociations between psychological wellbeing and sleepand highlights a predictive role for stress in sleep qualityAll previous international studies identified an averagePSQI in dementia caregiver studies [] highlightingthe prevalence of the issue As expected and consistentwith previous studies [] Australian caregivers ofPLWD have a high prevalence of poor sleep with ofparticipants classified as poor sleepers The PSQI globalscore ± was higher than those found in somedementia caregiver studies [ ] but comparable withTable Correlation between PSQI subcomponent scores andDASS21 subcomponent score in dementia caregiversPSQI componentDASS21DepressionDASS21AnxietyDASS21StressPSQI Global ScoreSubjective sleep qualitySleep latencySleep durationSleep efficiencySleep disturbancesUse of sleeping medicationDaytime dysfunctionp p ˆ’ˆ’ˆ’others [] The PSQI subcomponents which greatestcontributed to the overall score were sleep latency timetaken to fall asleep sleep quality overall subjectivequality and sleep disturbances Sleep latency and sleepdisturbances have previously been identified as the mostcommon contributors to sleep quality in caregivers ofPLWD in a recent systematic review [] Given thedearth of Australian specific data this study identifiedimportant depth of detail around caregiver™ssleepcharacteristics of caregivers took longer than therecommended min to fall asleep only of caregivers slept more than h of caregivers had sleepefficiency lower that the recommended and ofcaregivers used sleep aiding medication in the previousmonth Taken togetherfindingspresent a novel and ominous overview of the poor sleephealth conditions that Australian dementia caregiversexperiencethese descriptiveCaregivers of community dwelling people living withdementia reported poor sleep quality and high levels ofdepression anxiety and stress This study found that poorsleep was correlated with subjective feelings of depressionanxiety and stress which were in keeping with previousliterature around caregiving and psychological distressMore than half of the caregivers surveyed reported symptoms of depression and stress and respectivelyand reported anxiety These findings are in keepingwith that of a recent study of dementia caregivers in ruralVictoria where of caregivers reported depression orstress and reported anxiety []Previous work has identified an association betweendepression and poor sleep among dementia caregivers[ ] Reduced quality of sleep and depression arehigher among caregivers of people living with dementia[] Furthermore caregivers of people living withdementia who were also depressed experience a greatervariation in sleep patterns []This study reveals sleep scores were significantly associated with measures of depression anxiety and stressThese findings illustrate the interplay between sleepquality and quantity and psychological wellbeing incaregivers providing care for an individual living withdementia Further exploration of predictive factors incaregiverssleep quality identified stress as a keypredictor of poor overall sleep quality whilst adjustingfor age gender comorbidities and length of care This isa novel finding and represents a potential therapeutictarget to improve sleep quality in dementia caregiversDespite the high prevalence of selfidentified poorsleep overnight respite was only used by of participants which is in keeping with previous research thatcites of Australian caregivers have never usedrespite services [] Access to respite care remains oneof the major means of easing caregiver burden and is 0cSmyth BMC Geriatrics Page of frequently identified as needed by caregivers yet remainsunderutilised Whilst a high proportion of dementiacaregivers report a need for respite services there isinsufficient awareness of and access to respite servicesfor caregivers [] Respite service availability can beinvaluable with caregivers reporting lower stress levelsand improved health after use [] Sleep disturbanceshave also been shown to be partially reversed for caregiversduring periods of respite care [] perhaps due to reversalof hyper stressed state Respite services have been cited asan important measure to allow caregivers time to attend totheir own health imperative to supporting the caregiver tocontinue in providing care for their loved one []A recent Australian parliamentary inquiry into sleephealth identified sleep as a foundation of positive healthand wellbeing alongside diet and exercise [] Furthermore the report urged government to prioritise sleephealth as a national priority given its pivotal role inmaintaining health It is clear that caregivers of PLWDhave suboptimal sleep which is associated with poorerpsychological wellbeing and potential increased risk ofdeveloping chronic health conditions such as diabetesand cardiovascular disease [] Interestingly cardiovascular disease and endocrine disease such as diabeteswere the most prevalent comorbidities in our studypopulation Previous studies have also identified highblood pressure diabetes and arthritis as the most prevalent chronic disease in the dementia caregiver groupwhich is in keeping with our findings []Managing sleep and its associated mediators will havedirect impact on both the caregiver™s own health as wellas the caregiver care recipient relationship It is imperative to provide educational support around sleep considerpractical interventions such as overnight respite and toaddress stress management interventions for caregivers inturn reinstating and preserving sleep of this critical population of informal caregiversLimitationsA limitation of this study was the small sample size despitenumerous attempts to recruit participants and involvementof national anisations Although over participantsprovided demographic details only participants completed all questionnaires and were included in statisticalanalyses Challenges related to recruiting caregivers andparticularly caregivers of people living with dementia hasbeen described in numerous publications [ ]Caregivers of people with dementia living in the community are more likely to be female and this was representedin our population Furthermore caregivers who are underthe greatest stress may also be those least likely to participate However this remains one of the larger studies ofAustralian dementia caregivers sleep Another limitationwithin this study was that no data was collected regardingthe severity of dementia or behavioural disturbances whichmay impact sleep of the caregiverConclusionSleep problems are widespread within the population ofdementia caregivers Given that the majority of peopleliving with dementia are reliant on family caregiversminimising health and psychologicalimpact on caregivers should be of major concern Furthermore sleepquality is correlated with emotional distress in dementiacaregivers so improving the sleep of caregivers may inturn improve their psychological wellbeing Converselystress is a significant predictor of poor sleep so managing stress may have a positive impact on sleepDementia caregivers are often older with coexistingmorbidities and high levels of psychological distress Withprolonged caregiving and the progression of dementia ofthe care recipient there is the potential for a concurrent decline in the caregivers mental and physical health In orderto support the caregiver in their role it is of the upmost importance that we promote and maximises their health andwellbeing By managing and minimising the negative factorsassociated with caregiving we can enhance caregiving satisfaction and gratification In turn this can ensure optimalcaregivercare recipient relationship supporting the personliving with dementia to remain at home as long as possibleAbbreviationsDASS21 Depression Anxiety and Stress Scale21 PLWD PersonPeopleliving with dementia PSQI Pittsburgh Sleep Quality Index WHO WorldHealth anisationAcknowledgementsThe authors acknowledge the statistical advice of Dr Mark Jenkins and DrPeter PalamaraAuthors™ contributionsAS was involved in study design data collection data interpretation andmanuscript preparation LW supported study design data interpretation andediting of the manuscript CV supported study design and manuscriptpreparation EQ advised on use of psychological tools interpretation of theirdata and manuscript preparation LE was involved in data collection andmanuscript preparation All authors have read and approved the manuscriptFundingThis research was supported by an Edith Cowan University ECU Early CareerResearcher Grant awarded to Dr Aisling Smyth ECU had no role in thedesign data collection and data interpretation of the studyAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was granted ethical approval by Edith Cowan University HumanResearch Ethics Committee No Informed written consent wasobtained and participants received no incentive for taking partConsent for publicationNot applicableCompeting interestsThe authors state that they have no competing interests 0cSmyth BMC Geriatrics Page of Cohen J Statistical power analysis for the behavioral sciences 2nd edHillsdale NJ Erlbaum Wilcox S King AC Sleep complaints in older women who are familycaregivers J Gerontol B Psychol Sci Soc Sci 1999543P189“Simpson C Carter P Dementia behavioural and psychiatric symptomseffect on caregiver's sleep J Clin Nurs ““Kochar J Fredman L Stone KL Cauley JA Sleep problems in elderlywomen caregivers depend on the level of depressive symptoms resultsof the caregiver“study of osteoporotic fractures J Am Geriatr Soc “Lee D Heo S Yoon S Chang D Lee S Rhee H Sleep disturbances andpredictive factors in caregivers of patients with mild cognitive impairmentand dementia J Clin Neurol “Johnson E Respite reconsidered a discussion of key issues and futuredirections for carer respite Sydney Carers Australia Phillipson L Johnson K Cridland E Hall D Neville C Fielding E et alKnowledge helpseeking and efficacy to find respite services an exploratorystudy in helpseeking carers of people with dementia in the context ofaged care reforms BMC Geriatr “ O™connell B Hawkins M Ostaszkiewicz J Millar L Carers™ perspectives ofrespite care in Australia an evaluative study Contemp Nurse “Lee D Man K Lindesay J Effect of institutional respite care on the Sleepof people with dementia and their primary caregivers J Am Geriatr Soc“ Parliament of the Commonwealth of Australia Bedtime Reading inquiry intoSleep health awareness in Australia Canberra Commonwealth of Australia [Available from httpsparlinfoaphgovauparlInfodownloadcommitteesreportrep024220toc_pdfBedtimeReadingpdffileTypeapplication2Fpdf] Accessed Mar Moon H DilworthAnderson P Baby boomer caregiver and dementiacaregiving findings from the National Study of caregiving Age Ageing“ Bristow M Cook R Erzinclioglu S Hodges J Stress distress and mucosalimmunity in carers of a partner with frontotemporal dementia Aging MentHealth “ McConaghy R Caltabiano ML Caring for a person with dementia exploringrelationships between perceived burden depression coping and wellbeing Nurs Health Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsAuthor details1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA Australia 2School of Arts and HumanitiesPsychological Services Centre Edith Cowan University Joondalup AustraliaReceived April Accepted August ReferencesAlzheimer's 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“Allen AP Buckley MM Cryan JF Ní Chorcoráin A Dinan TG KearneyPM et alInformal caregiving for dementia patients the contribution ofpatient characteristics and behaviours to caregiver burden Age Ageing“Bevans M Sternberg EM Caregiving burden stress and health effectsamong family caregivers of adult Cancer patients JAMA “ Alzheimer's Disease International Global estimates of informal care LondonUK 2018Available from httpswwwalzcoukadipdfglobalestimatesofinformalcarepdf Accessed Mar Gao C Chapagain NY Scullin MK Sleep Duration and Sleep Quality inCaregivers of Patients With Dementia A Systematic Review and Metaanalysis JAMA Netw 201928e199891Ervin K Pallant J R C Caregiver distress in dementia in rural VictoriaAustralasian J Ageing “ Peng HL Lorenz RA Chang YP Factors associated with sleep in familycaregivers of individuals with dementia Perspect Psychiatric Care “ Hirshkowitz M Whiton K Albert SM Alessi C Bruni O DonCarlos L et alNational Sleep Foundation's updated sleep duration recommendations finalreport Sleep Health “Jay S Vincent G Kovac K Dorrian J Thomas M Reynolds A Ferguson SReport Reducing Sleep Disruption in Carers Australia Carers Australia httpswwwsleephealthfoundationaufilesResearch_GrantsSpecial_reportsReport_Carers_Australia_FINALv2pdf Accessed Mar2020 Worley SL The extraordinary importance of Sleep the detrimental effects ofinadequate Sleep on health and public safety drive an explosion of Sleepresearch P T “ Rowe MA McCrae CS Campbell JM Benito AP Cheng J Sleep patterndifferences between older adult dementia caregivers and older adultnoncaregivers using objective and subjective measures J Clin Sleep Med“Schulz R Belle SH Czaja SJ McGinnis KA Stevens A Zhang S Longtermcare placement of dementia patients and caregiver health and wellbeingJAMA “ Gaugler JE Yu F Krichbaum K Wyman JF Predictors of nursing homeadmission for persons with dementia Med Care “Lovibond SH Lovibond PF Manual for the Depression Anxiety Stress ScalesSydney Smyth C The Pittsburgh Sleep quality index PSQI Insight “ Buysee DJ Reynolds CF Monk TH Berman SR Kupfer DJ The PittsburghSleep Qulaity index a new instrument for psychiatric practice and researchPsychiatry Res

craigpattersontelecare“toj rajani r perry m the reality of medical work the case for a new perspectiveon telemedicine virtual real “ bf01421807 hoek pd schers hj bronkhorst em vissers kcp hasselaar jgj the eï¬ectof weekly specialist palliative care teleconsultations in patients with advancedcancer a randomized clinical trial bmc med s1291601708669 ministero della salute telemedicina linee diavailabledocumentazionep6_2_2_1jsplinguaitalianoid2129 onlineatindirizzo nazionalihttpwwwsalutegovitportaleaccessed april aiom indicazioni aiom cipomo su covid19 per l™oncologia wwwaiomitwpcontentuploads202003accessedavailable20200313_covid19_indicazioni_aiomcipomocomupdfapril onlineat cox a lucas g marcu a piano m grosvenor w mold f cancer survivors™ experience with telehealth a systematic review andthematic synthesis j med internet res 19e11 102196jmir rogante m giacomozzi c grigioni m kairy d telemedicine in palliativecare a review of systematic reviews ann ist super sanita “ 104415ann_16_03_16 kruse cs krowski n rodriguez b tran l vela j brooks mtelehealth and patient satisfaction a systematic review and narrativeanalysis bmj open 7e016242 101136bmjopen2017 holzner b giesinger jm pinggera j zugal s sch¶pf f oberguggenbergeras the computerbased health evaluation software ches aelectronic patientreported outcome monitoring bmcsoftwaremedinform decis makfor gutteling jj busschbach jj de man ra darlington as logistic feasibilityin clinical practiceof health related quality ofresults of a prospective study in a large population of chronic liverpatients health qual life outcomes life measurement taenzer p bultz bd carlson le speca m degagne t olson k impact of computerized quality of life screening on physician behaviourand patient satisfaction in lung cancer outpatients psychooncology “ wright ep selby pj crawford m gillibrand a johnston c perren tj feasibility and compliance of automated measurement of quality of life inoncology practice j clin oncol “ 101200jco200311 jazieh ar alenazi th alhejazi a alofoncoloncologypatients“safif al olayaninfected with coronavirus101200go20a outcomejcoglobtelemed flodgren ginteractivehealthsandrevrachas afarmer ajtelemedicinecareoutcomes2015cd002098eï¬ectsoninzitari mprofessionalshepperdpracticesyst10100214651858cd0databasecochrane ye z hong y wu x hong d zhang y dong x[management of a colon cancer patientdisease ] zhejiang da xue xue bao yi xue banalinfected with corona viruset o™gorman ld hogenbirkin northern ontario astelemedicineunitstohealthcare telemed j e health “ 101089tmj20a measure of potentialjc driving distanceaccessto perri fc ottaiano acancertranslionna f longo f della vittoria scarpati g de angelisagainst head and necktherapy101016jtranon2019immunebiological mechanismsoncolresponseand“implicationonaletfrontiers in oncology wwwfrontiersinaugust volume 0ccrispo covid19 emergency and postemergency hisada y mackman n cancerassociated pathways and biomarkers of venousthrombosis blood “ 101182blood20170374 xu x lai y hua zc apoptosis and apoptotic body disease message andtherapeutic target potentials biosci rep 39bsr20180992 bsr20180992 zhao z bai h duan j wang j recommendations ofandlungtreatmentcancermedicalforepidemic thorac cancerpatientsindividualizedevents managementof covid19 “ commonduringadversetheoutbreakconflict of interest the authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestcopyright crispo montagnese perri grimaldi bimonte augustin amorecelentano di napoli cascella and pignata this is an openaccess distributedunder the terms of the creative commons attribution license cc by the usedistribution or reproduction in other forums is permitted provided the originalauthors and the copyright owners are credited and that the original publicationin this is cited in accordance with accepted academic practice no usedistribution or reproduction is permitted which does not comply with these termsfrontiers in oncology wwwfrontiersinaugust volume 0c'

" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohn™s disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cruiz castro of inflammation page of crohn™s disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties [“]alkaloids”a class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atoms”are produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinson™sdisease alzheimer™s disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimer™s disease in shsy5y cells anatabine alsoreduced the expression of betasecretase ”the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimer™s disease”through inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see œonline resource  0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days “ fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days “ b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2b“c application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine œimmune system œextracellularmatrix anization œprotein localization œmetabolism œhemostasis and œsignal transduction fig 3bof these we further explored œimmune system œextracellular matrix anization and œsignal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the œimmune system categoryincludes œadaptive immune system œcytokine signaling in immune system and œinnate immune systemin particular within these immune categories œcytokinesignaling egincluding il6 family signaling andœtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome œsignal transduction category œsignaling byreceptor tyrosine kinasesby rhogtpases œsignaling by transforming growth factortgfbeta family members œmapk family signalingcascades œintegrin signaling œsignaling by erythropoietin and œsignaling by gpcr were found to be significantly impacted online resource online resource within the œextracellular matrix anization category almost all subcategories were perturbed includingœdegradation of the extracellular matrix œelastic fiberformation and œextracellular matrix proteoglycansonline resource online resource œsignalingœdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1“pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionœfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ‰¤ note that the lower fdr values observed for the œana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large — foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect œwater dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure œanatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource œheatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined œanatabinenicotine dss effect captured by the pairwise comparisons œanatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors œdss treatment and œanatabinenicotine exposuretogether with the adjusting twofactor interaction term œanatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeœimmune system category and for the twofactor œana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ˆ’ ] whereas their statistical significance competitive q1 p values ‰¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlr“il1r“tnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvalues”from theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six œleadingedge genes”genes of the gene set with the highestœimmunedifferentialsystem œextracellular matrix anization and œsignaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 œsignaltransduction and œimmune system categoriesil6œsignal transduction and œimmune system categoriesmmp13 œextracellular matrix anization categoryserpine1 œsignal transduction and œextracellular matrixanization categories thbs1 thrombospondin œsignal transduction and œextracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase œextracellular matrix anization and œimmunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated œbeta is the slope of the best interceptfreelinear fit between microarray and qpcr values œr2 is the coefficient of determination measuring the œgoodnessoffit and œpval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [“] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including œimmune responses œsignal transduction and œextracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimer™s disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloids”in particular isoquinoline alkaloids present in traditional medicineherbs”exertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i

the heterogeneity of cancer cells is generally accepted and astem celllike subpopulation that is called œcancer stem cellscscs has been identified in various types of malignanttumors although the lack of consensus on the definitioncscs are widely recognized as a small subpopulation amongcancer cells with the properties of selfrenewal and tumor initiation as cscs play a critical role in the recurrence andmetastasis of cancer targeting the cscs is thought to bea promising approach for curing cancera large number of past studies have tried to identify andcharacterize the cscs as normal tissuespecific stem cellsare considered as the main origin of cancer the cscsare also thought to be inherited at least partially the characterization of normal tissuespecific stem cells thereforemany studies on the identificationpurification of cscs havesimply shared markers of hematopoietic stem cells includingthe most popularly used cell surface markers of cd44 andcd133 [ ] cd44 is a type i transmembrane glycoproteinthat is expressed on hematopoietic fibroblastic and glial cellsand functionally known to mediate cellcell and cellmatrixinteractions previous studies have demonstrated that thecd44 is not only a biomarker but also plays critical roles inthe maintenance of cscs the resistance to various therapiesstresses and the metastasis of cancer cells [“]cd133 is originally identified as protein expressing on thecell surface of hematopoietic stem cells and has subsequently been found to be critical in the maintenance ofœstemness of stem cells in various tissues [“] cd133has also been found in some csc [“] which contributesto therapeutic resistance through the activation of akt bcl2and mapkpi3k signaling pathways [“] although theexpressions of cd44 and cd133 in cancer cells likely associate with the resistances to radiotherapy chemotherapy andvarious stresses the diï¬erent significance between cd44and cd133 has not yet been well understoodin this study we investigated whether the expression ofcd44 and cd133 in human colorectal cancer cells hct8diï¬erently contributed to drug resistance our data indicated 0cstem cells internationalthat the expression of cd133 rather than cd44 closely associated with doxorubicin dxr resistance at least partiallythrough drug excretion and redox regulation materials and methods cell culture human colorectal cancer hct8 cells werecultured in rpmi medium fujifilm wako purechemical japan supplemented with fbs gibco°thermo fisher scientific ma usa at c in a humidifiedatmosphere of air and co2 separation of cd44 and cd133positive cells fromhct8 cells we separated the parent hct8 cells intocd44positive cd44 and cd133positive cd133 cellsby a twostep magnetic cell sorting method as described previously [ ] briefly hct8 cells were collected as a singlecell suspension by trypsinization and then incubated withmagnetic microbeadconjugated antihuman cd44 antibodymiltenyi biotec germany for min after washing cellswere separated into cd44 and cd44 subpopulations byusing the automacs„¢ pro separator miltenyi biotecaccording to the manufacturer™s instruction the purifiedcd44 cells were further expanded and then harvested as asinglecellsuspension to be incubated with magneticmicrobeadconjugated antihuman cd133 antibody miltenyibiotec for min after washing the cd44cd133 andcd44cd133 subpopulations were separated as describedabove this twostep isolation enabled us to obtain a sufficientnumber of cd44 cd44 cd44cd133 and cd44cd133 cells for our experimentsto verify the purity of each subpopulation isolated cellswere stained with pelabelled mouse antihuman cd133clone ac133 miltenyi biotec and fitclabelled mouseantihuman cd44 clone db105 miltenyi biotec according to the supplied protocols flow cytometry analysis wasperformed using a facscalibur becton dickinson asdescribed previously mouse igg1pe miltenyi biotecand mouse igg1fitc miltenyi biotec were used as a negative control cytotoxicity assays cells were seeded in 96well cultureplates at a density of — cells per well and cultured overnight the cells were then treated with various concentrationsof dxr fujifilm wako pure chemical in the absence orpresence of verapamil fujifilm wako pure chemicalcytotoxicity assays were performed using the cell proliferation kit i mtt roche applied science germany asdescribed previously the absorbance was measured at nm using a microplate reader multiskan fc thermofisher scientific analysis on the expression of abc transporters theexpressions of the atpbinding cassette subfamilies of bmember abcb1 or g member abcg2 were analyzedby flow cytometry briefly cells were incubated with mouseprimary antibodies against human abcb1 and abcg2bd biosciences ca usa and then labeled by fitcconjugated antimouse igg bd biosciences according tothe manufacturer™s instruction respective isotype controlswere used as a negative control after washing flow cytometry analysis was performed using a facscalibur analysis of cellular accumulation of dxr the intracellular accumulation of dxr was analyzed by flow cytometrybriefly cells were treated by μm dxr for hr in theabsence or presence of μm verapamil or μm buthionine sulfoximine bso sigmaaldrich mo usa cellswere then collected as a singlecell suspension and washedtwice with icecold phosphatebuï¬ered saline the accumulation of dxr within cells was evaluated by the intracellularfluorescence intensity using a facscalibur the nucleusaccumulation of dxr was analyzed by using cell pelletstreated with triton x100pbs as assay material asdescribed previously expressionlevelsanalysisimmunoblot detection of intracellular ros the intracellular roslevel based on the oxidation of ²²dichlorodihydrofluorescein diacetate h2dcfda molecular probes thermofisher scientific was measured to form the fluorescent compound ²²dichlorofluorescein dcf using a facscaliburofphosphorylatedp38 map kinase phosphop38mapk totalp38mapk and nuclear factor erythroid 2related factor nrf2 in the cells were estimated by immunoblotting brieflycell lysate μg of total protein was separated by sodiumdodecyl sulfate“polyacrylamide gel electrophoresis sdspage gel transferred to pvdf membranes biorad causa and then incubated with primary antibodies cell signaling technology ma usafollowed by appropriatehrplabeled secondary antibodies dako agilent pathologysolutions ca usa blots were developed by enhancedchemiluminescence using an ecl kit ge healthcare lifesciences pa usa semiquantitation was done by measuringthe density of bands using the image quant las minibiomolecular imager ge healthcare life sciences asdescribed previously sirna treatment smallinterfering rna sirnaspecific targeting to cd133 on targetplus sirnaand a scramble sirna on targetplus sirna negativecontrol were obtained from dharmacon horizon discovery cambridge uk cells were seeded in 6well plates — cellswell and incubated for hr transfectionswere performed using dharmafect sirna transfectionreagents dharmacon according to the manufacturer™sinstructions analyses were done at hr after sirnatransfection statistical analysis all of the results are presented as themeans ± sd statistical significance was determined by oneway analysis of variance anova followed by tukey™s testdr spss ii chicago il diï¬erences were considered significant when p results hct8 cells were separated into various subpopulationsbased on their expressions of cd44 and cd133 first we 0cstem cells internationalparent cellshlffl1hdccd44hlfhlfcd44ˆ’fl1hcd44fl1hahlfhlfcd44cd133ˆ’fl1hcd44cd133fl1hparent cd44ˆ’cd44 cd44133ˆ’ cd44133enilesab fo noitarefilorp llecparent cd44ˆ’ cd44 cd44ˆ’cd44bcfigure continued 0cstem cells internationalenilesabstnuo0ccd44cd133ˆ’ ± stnuo0ccd44cd133 ± fl2hfl2hsyad stnuo0c ± fl2hstnuo0cd ± fl2hfigure the separation of hct8 colorectal cancer cells into diï¬erent subpopulations based on the expression of cd44 and cd133 arepresentative dot plots of flow cytometry analysis show the purities of each subpopulation of isolated cells quantitative data in the dotplots are presented as the percentages of positive cells from three independent experiments b representative photos of morphologicalproperties upper and mtt assay on cell growth lower at hr after the initiation of culture data are presented as the mean ± sd fromthree independent experiments c d representative histograms of flow cytometry analysis showed the expressions of cd44 c andcd133 d at baseline and days after cell culture the dotted vertical lines through histograms indicate the diï¬erence in the expressionpeaks between the baseline and at days after culture quantitative data in the histograms are presented as the mean fluorescentintensity from three independent experimentsseparated the hct8 cells into cd44 and cd44 subpopulations and compared their sensitivity to anticancer drugs ofdxr and cisplatin cisdiaminedichloroplatine cddphowever no diï¬erence in the sensitivity to the two drugswas observed between cd44 and cd44 cells data notshown we further tried to purify a small population ofcd133 cells from these cd44 cells cd44 cells almostnegatively expressed with cd133 figure 1a as a resultwe separated hct cells into diï¬erent subpopulationsincluding cd44 cd44 cd44cd133 and cd44cd133 cells the purities of isolated cells in each subpopulation were confirmed to be around by flow cytometryfigure 1aandphenotype changein diï¬erent growthsubpopulations of cells the morphology and proliferationof these cells could not be found obviously diï¬erent amongsubpopulations figure 1b the expression of cd44 in allsubpopulations kept stable within days of reculturingfrom the frozen cells that stocked immediately after isolationinterestingly the expression of cd44 was a tendency todecrease with culture time in cd44 fluorescence intensity ± at baseline vs ± at days p figure 1c and cd44cd133 cells fluorescence intensity ± at baseline vs ± at days p figure 1c but still kept stable in cd44cd133 cells at days after reculturing fluorescence intensity ± at baseline vs ± at days p figure 1cthe expression of cd133 in cd44cd133 cells kept verystable fluorescence intensity ± at baseline vs ± at days p figure 1d and cd44cd133cells did not turn to express cd133 within days of reculturing fluorescence intensity ± at baseline vs ± at days p figure 1d therefore we usedthe cells within days after reculturing from the frozenstocked cells in subsequent experiments dxr resistance of cd44cd133 cells next we evaluated the sensitivity of cells to dxr by mtt assay withthe addition of μm of dxr in medium we foundthat the survival of cd44cd133 cells was significantlyhigher than all other subpopulations of cells after hr of 0cstem cells international ytilibaiv llecŽŽŽŽŽðœ‡mparent cellscd44ˆ’ cellscd44 cellscd44133ˆ’ cellscd44133 cellsdxrbso003hct8doxb24 hfl3h006hct8 cd44ˆ’doxb24 hfl3h009hct8 cd44doxb24 hsllec tnerapstnuocsllec ˆ’dcstnuocsllec dcstnuocdxr001hct8dox24 hfl3hstnuocadxrverapamil002hct8doxv24 hstnuocfl3h004hct8 cd44ˆ’dox24 h005hct8 cd44ˆ’doxv24 hstnuocstnuocfl3hfl3h007hct8 cd44dox24 h006hct8 cd44doxv24 hstnuocstnuocfl3hfl3hsllec ˆ’dcsllec dc006hct8 cd44cd133ˆ’dox24 h006hct8 cd44cd133ˆ’doxv24 hstnuocstnuocstnuocfl3hfl3h00hct8 cd44cd133dox24 h00hct8 cd44cd133doxv24 hstnuocfl3hstnuocstnuocfl3hbfigure continuedfl3h012hct8 cd133ˆ’doxb24 hfl3h015hct8 cd44cd133doxb24 hfl3h 0cstnuocstem cells internationalabcg2abcb1stnuocfl1hparent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cells ± ± ± ± ± parent cellscd44ˆ’cellscd44cellscd44133ˆ’ cellscd44133 cellscfl1h ± ± ± ± ± figure dxr resistance of diï¬erent subpopulations of cells a mtt assay was done to evaluate the cytotoxicity of dxr data are expressedas the percentile of baseline before dxr treatment from three independent experiments ˆ—p vs all other subpopulations brepresentative histograms of flow cytometry analysis show the accumulation of dxr in cells hr after the treatment with μm dxrin the absence or presence of μm verapamil and μm bso the dotted vertical lines through histograms indicated the mean levels ofdxr accumulation in cd44cd133 cells for comparing with other subpopulations of cells the results were reproducible in threeindependent experiments c representative histograms of flow cytometry analysis show the expression of the abcb1 or abcg2 indiï¬erent subpopulations of cells quantitative data in the histograms are presented as the mean fluorescent intensity from threeindependent experimentsculture p vs other groups at diï¬erent dxr concentrations figure2ato understand the relevant mechanism we measured theintracellular accumulation of dxr in cells by flow cytometrythe accumulation of dxr in cd44cd133 cells wasdetected as the lowest among these subpopulations at hrafter the exposure to μm dxr figure 2b we furtherfound that the intracellular accumulation of dxr in cd44cd133 cells was obviously increased by the treatment withverapamil an inhibitor for drug efflux cell membrane transporters of abcb1 and abcg2 figure 2b however theintracellular accumulation of dxr in cd44cd133 cellsdid not change by the treatment with bso a glutathione synthesis inhibitor that indirectly regulates drug efflux throughabcc1 figure 2b we also confirmed that the expressionof abcb1 p vs other groups but not abcg2 wasenhanced in cd44cd133 cells figure 2c suggestingthe probable role of abcb1 on dxr resistance in cd44cd133 cellsto further confirm the causal relationship between theenhanced drug efflux and dxr resistance we evaluatedthe cytotoxicity of dxr in the presence or absence of verapamil unexpectedly verapamil only partially enhanced thecytotoxicity of dxr in either cd44cd133 or cd44cd133 cells figure 3ait is well known that dxr interacts with nuclear dna toinhibit macromolecular biosynthesis therefore we alsoestimated the eï¬ect of verapamil on the nuclear accumulation of dxr the nuclear accumulation of dxr wasobserved obviously less in cd44cd133 than cd44cd133 cells but tended to have comparable levels withverapamil treatment figure 3b cd44cd133 cells showed better stress tolerance thancd44cd133 cells it is well known that the stress responsekinase p38mapk can be activated by various extracellularstresses and plays critical roles in cell survival and apoptosis although the basal level of phosphorylated p38mapkwas detected very similar between cd44cd133 andcd44cd133 cells p figure lower expressionwas observed in cd44cd133 than cd44cd133 cellsafter dxr exposure even under verapamiltreatmentp figure this suggests a better tolerance tostress of cd44cd133 cells independent on the accumulation of dxr 0cstem cells internationalˆ’ limaparev ytilibaiv llec limaparev ytilibaiv llec hoursŽŽŽŽ 𝜇mŽŽŽŽðœ‡mcd44cd133 cellscd44cd133ˆ’ cellsa hoursŽŽŽ ˆ’ limaparevstnuoccd44133ˆ’ cd44133𝜇mfl3hŽŽŽŽðœ‡m limaparevstnuocfl3hbfigure dxr resistance and nuclear dxr accumulation in cd44cd133 and cd44cd133 cells in the absence or presence of drug effluxinhibitor a mtt assay was done to compare the cytotoxicity of dxr in cd44cd133 and cd44cd133 cells with or without theaddition of μm verapamil data were expressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells b representative histograms of flow cytometry analysis showed the nuclear accumulation of dxr incells hr after the treatment by μm dxr with or without the addition of μm verapamil the results were reproducible in threeindependent experimentsverapamil ˆ’verapamil phosphop38 mapk totalp38 mapk noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ control hours dxr treatmentp p p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ hours dxr treatmentfigure diï¬erent expression of phosphorylated p38mapk between cd44cd133 and cd44cd133 cells representative blots andsemiquantitative data on the expression of phosphorylated p38mapk and total p38mapk in cells treated with μm dxr in theabsence or presence of μm verapamil the quantitative data are normalized to total p38mapk data are expressed as relative values tocd44cd133 cells without dxr treatment and presented as the mean ± sd from three independent experiments 0cstem cells internationaldxr ˆ’dxr ˆ’ limaparevstnuoc limaparevstnuoccd44133cd44133ˆ’stnuocfl1hfl1hstnuocfl1hafl1hverapamil ˆ’verapamil nrf2𝛽tubulin noisserpxe evitalercd133 ˆ’p p p ˆ’ ˆ’ ˆ’ ˆ’ controldxr treatment hours bp p ˆ’ˆ’control ˆ’ ˆ’ ˆ’ dxr treatment hours figure diï¬erent antioxidant capacity between cd44cd133 and cd44cd133 cells a representative histograms of flow cytometryanalysis show the intracellular ros levels hr after the treatment by μm dxr in the absence or presence of μm verapamil theresults were reproducible in three independent experiments b representative blots and semiquantitative data on the expression of nrf2in cells treated with μm dxr in the absence or presence of μm verapamil the quantitative data are normalized to βtubulin dataare expressed as relative values to cd44cd133 cells without dxr treatment and presented as the mean ± sd from three independentexperiments cd44cd133 cells showed higher antioxidantcapacity than cd44cd133 cells it is also well known thatdxr generates ros and oxidative stress due to ros generation may induce the activation of p38mapk therefore weestimated the ros levels in cells with or without dxr exposure we observed a lower level of ros in cd44cd133 0cstem cells internationalstnuocstnuocstnuocstnuocstnuoc0nm ± stnuocfl2h5nm ± stnuocfl2h10nm ± stnuocfl2h15nm ± stnuocfl2h25nm ± stnuocfl2h0nm ± fl2h25nm ± ytilibaiv llecfl2h50nm ± fl2h75nm ± fl2h100nm ± fl2hŽŽŽŽðœ‡m cd44133ˆ’ cd44133 cd44133 control sirna cd44133 cd133 sirna abfigure continued 0cstnuo0cstnuo0cexpression of abcb10nm ± 0nmstnuo0c ± fl1h5nm ± fl1h50nmstnuo0c ± fl1hfl1h25nmstnuo0c ± stnuo0cfl1h100nm ± fl1hstem cells internationalaccumulation of dxrcontrol sirna 5nm ± fl3hcd133 sirna 5nm ± stnuo0cstnuo0cfl3hcdfigure the eï¬ect of silencing cd133 expression on dxr resistance of cd44cd133 cells a representative histograms of flowcytometry analysis on the expression of cd133 in cd44cd133 cells after silencing by diï¬erent dosages of targeted sirna quantitativedata in the histograms are presented as the mean fluorescent intensity from three independent experiments b mtt assay was done toevaluate the cytotoxicity to dxr cells were treated with nm sirna for hr followed by dxr treatment for another hr data areexpressed as a percent of baseline before dxr treatment from three independent experiments ˆ—p vs cd44cd133 cells crepresentative histograms of flow cytometry analysis on the expression of abcb1 in cells after silencing by diï¬erent dosages of targetedsirna quantitative data in the histograms are presented as the mean fluorescent intensity from three independent experiments drepresentative histograms of flow cytometry analysis on the accumulation of dxr quantitative data in the histograms are presented asthe mean fluorescent intensity from three independent experimentsthan cd44cd133 cells especially under dxr exposurebut verapamil did not obviously change the ros levelsfigure 5a based on these findings we speculated thatthe enhanced antioxidant capacity in cd44cd133 cellsmight help to maintain a lower level of phosphorylatedp38mapknrf2 a transcription factor that is well known to beactivated by oxidative stress such as ros and electrophilicsubstances can protect cells against various stresses we alsocompared the expression level of nrf2 between cd44cd133 and cd44cd133 cells western blotting showeda higher expression of nrf2 in cd44cd133 than cd44cd133 cells especially under dxr exposure p figure 5b and the enhanced expression of nrf2 in cd44cd133 cells was not cancelled by verapamil treatmentp figure 5b sirna treatment to further confirm the regulatory roleof cd133 in drug resistance we tried to silence cd133expression in cd44cd133 cells by sirna and then estimated cytotoxicity of dxr although the decrease ofcd133 expression was clearly observed by targeted sirnap vs nm figure 6a dxr resistance of cd44cd133 cells only partially improved figure 6b unexpectedlythe silencing of cd133 did not change theexpression of abcb1 in cd44cd133 cells even using 0cstem cells internationalexcessive concentrations of cd133 sirna p vs nmfigure 6c we also confirmed that the silencing of cd133did not aï¬ect the accumulation of dxr in cd44cd133cells p vs control sirna figure 6dthis suggests that beyond the drug excretion and redoxregulation other complex mechanisms are also likelyinvolved in the dxr resistance in cd44cd133 cells discussionby using the wellrecognized cell surface markers of cd44and cd133 for csc identification we tried to separate thehct8 human colon cancer cells into cd44 cd44 cd44cd133 and cd44cd133 subpopulations and then investigated how the expressions of cd44 and cd133 associatedwith drug resistance actually we checked several cancer celllines on the expression of cd44 and cd133 including helacells and a549 cells however both hela cells and a549 cellsshowed almost expression of cd44 only the hct8cells showed a partial expression of cd44 about anda rare expression of cd133 therefore we only isolateddiï¬erent subpopulations from hct8 cells for this studyfirst we found that the expression level of cd44 keptvery stable in the cd44cd133 cells but gradually declinedin cd44cd133 cells during a cell passaging process on theother hand some of cd44 cells shifted to express cd44 during a cell passaging process figure 1c these findings suggested the plasticity of cd44 expression in hct8 cellsactually ohata et al reported that cd44 highexpressedcells from human intractable colon cancer patients can diï¬erentiate into cd44 lowexpressed cells and a fraction of cd44lowexpressed cells can also generate cd44 highexpressedcells in a xenograft mouse model however it is unclearwhy the cd44cd133 cells but not cd44cd133 cellsstably maintain the expression level of cd44 unlike theextensive expression of cd44 with high plasticity the expression of cd133 was only observed in very few of the hct8cells with poor plasticitya number of previous studies have demonstrated thatcscs are likely resistant to chemotherapeutic drugs thecd44cd133 cells but not the cd44 and cd44cd133cells showed dxr resistance figure 2a according to thisdata the expression of cd133 but not cd44 seems to beclosely associated with drug resistance actuallythesecd44cd133 cells showed the enhanced expression ofabcb1 and the decreased intracellular accumulation ofdxr figures 2b and 2c liu et al reported that nonsmallcell lung cancer cells treated with lowdose cddp aresufficient to enrich cd133 cells and upregulate abcb1expression through notch signaling which thereforeincreases the crossresistance to dxr however theinhibition of abcb1 by verapamil only partially improvedthe dxr resistance of cd44cd133 cells in this studyto find other potential mechanisms involving in thedxr resistance of cd44cd133 cells we investigatedseveral interesting aspects including the stress protectionand redox regulation we found that p38mapk one of themost popular protein kinases known to be activated byinflammatory cytokines lipopolysaccharide osmotic shockultraviolet light and other stresses was more obviouslyinduced by dxr in cd44cd133 cells than cd44cd133cells figure moreover the activation of p38 mapk wasnot dependent on the intracellular accumulation of dxrfigure dxr is known to insert between the base pairs of dna oftumor cells and exhibits antitumor eï¬ects by suppressing thebiosynthesis of both dna and rna through the inhibitionof dna polymerase rna polymerase and topoisomeraseii reactions furthermore it is believed that dxr has theability to generate sufficient ros to raise oxidative stressindeed we observed dxrinduced ros generation in bothcd44cd133 and cd44cd133 cells butthe dxrinduced ros generation was detected even higher in cd44cd133 than cd44cd133 cells independent on the intracellular accumulation of dxr figures 5a 2b and 3bsuggesting the enhanced antioxidant capacity in cd44cd133 cellsthe keap1nrf2 control system plays a central role in theantioxidant defense mechanisms nrf2 is known as a transcription factor to activate various genes involving in biological defense mechanisms it has been reported that nrf2 isconstantly expressed in many cancer cells [“] moreover the enhanced expression of nrf2 has been confirmedto associate with poor prognosis of cancer patients [“]in our study nrf2 expression was detected higher in cd44cd133 than cd44cd133 cells and the diï¬erence in nrf2expression was observed even clearer between cells with dxradministrationindependent on the dxr accumulationfigure 5b these findings also clearly indicate theenhanced antioxidant capacity in cd44cd133 cellsalthough the absence of direct evidence by interferenceexperiment pathways involving in the stress protection andredox regulation might at least partially contributed to thedxr resistance of cd44cd133 cellsvery strangely our data showed that the silencing ofcd133 expression in cd44cd133 cells by sirna couldonly partially increase the cytotoxicity of dxr figure 6bbut did not change the expression of abcb1 and the intracellular accumulation of dxr figure 6c other unknownmechanisms beyond the drug excretion and redox regulationare asked to be defined on the dxr resistance of cd44cd133 cellsbased on data from the present study the expression ofcd133 rather than cd44 more closely associated with theresistance of cancer cells to anticancer drug as complexmechanisms including the drug excretion and redox regulation are likely involved in the drug resistance of cscs multiple approaches may be needed to overcome the big problemof drug resistance in cancer patientsabbreviationsabcb1abcg2atpbinding cassette subfamily b member 1pglycoproteinmultidrug resistance protein1mdr1atpbinding cassette subfamily g member2breast cancer resistanceproteinbcrpcd388 0cabcc1bsocscsdxrmttatpbinding cassette subfamily c member1multidrug resistanceassociated protein1mrp1buthionine sulfoximinecancer stem cellsdoxorubicinadriamycin345dimethylthiazol2yl25diphenyltetrazolium bromidenuclear factor erythroid 2related factor nrf2p38mapk p38 map kinaserossirnareactive oxygen speciessmall interfering rnadata availabilitythe data that support the findings of this study are availablefrom the corresponding author upon reasonable requestdisclosurethe funder played no role in the study design data collectionand analysis decision to publish or preparation of themanuscriptconflicts of interestthe authors indicate no potential conflicts of interestacknowledgmentsthis work was supported by a grantinaid for the ministryof education science sports culture and technology ofjapan grant numbers and 16k15622 and thecollaborative research program of the atomic bomb diseaseinstitute of nagasaki universityreferences r c elble œthe role of cancer stem cells in relapse of solidtumors frontiers in bioscience vol e4 no pp “ j e visvader œcells of origin in cancer 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cellsnature vol no pp “ c a o™brien a pollett s gallinger and j e dick œa humancolon cancer cell capable of initiating tumour growth inimmunodeficient mice nature vol no pp “ l lin a liu z peng et al œstat3 is necessary for proliferation and survival in colon cancerinitiating cells cancerresearch vol no pp “ n haraguchi m ohkuma h sakashita et al œcd133cd44 population efficiently enriches colon cancer initiating cellsannals of surgical oncology vol no pp “ 0cstem cells international d inoue t suzuki y mitsuishi et al œaccumulation ofp62sqstm1 is associated with poor prognosis in patientswith lung adenocarcinoma cancer science vol no pp “ j q ma h tuersun s j jiao j h zheng j b xiao anda hasim œfunctional role of nrf2 in cervical carcinogenesis plos one vol no article e0133876 q yang h deng h xia et al œhigh nfe2related factor expression predicts poor prognosis in patients with lung cancer a metaanalysis of cohort studies free radical researchvol pp “ s sarvi a c mackinnon n avlonitis et al œcd133 cancerstemlike cells in small cell lung cancer are highly tumorigenicand chemoresistant but sensitive to a novel neuropeptideantagonist cancer research vol no pp “ q zhang s shi y yen j brown j q ta and a d le œasubpopulation of cd133 cancer stemlike cells characterized in human oral squamous cell carcinoma confer resistanceto chemotherapy cancer letters vol no pp “ s ma t k lee b j zheng k w chan and x y guanœcd133 hcc cancer stem cells confer chemoresistance bypreferential expression of the aktpkb survival pathwayoncogene vol no pp “ s bao q wu r e mclendon et al œglioma stem cells promote radioresistance by preferential activation of the dnadamage response nature vol no pp “ c yan l luo c y guo et al œdoxorubicininduced mitophagy contributes to drug resistance in cancer stem cells fromhct8 human colorectal cancer cells cancer letters vol pp “ s goto y ihara y urata et al œdoxorubicininduced dnaintercalation and scavenging by nuclear glutathionestransferase ϝ the faseb journal vol no pp “ h ohata t ishiguro y aihara et al œinduction of the stemlike cell regulator cd44 by rho kinase inhibition cont

" healthcare is an essential service at any time more so in the crisis like covid with increase in numberof cases and mortality from covid the primary focus is shifted to the management of the covid crisis and otherhealth emergencies thus affecting normal health services and routine treatment of other diseases like cancermethods this reviews the published literature and guidelines on covid and cancer and discusses them tooptimize the care of cancer patients during covid pandemic to improve treatment outcomesresults the results of the review of published literature show a twofold increase in probability of getting cov2infection by the cancer patients and a fourfold increase in chance of death on the other hand if left untreated a increase in cancer death is expected data further show that none of the medicines like remdesivir hydroxychloroquin dexamethasone or azithromycin improves survival and response to covid in cancer patients surgicalresults too show similar outcome before and after the pandemic though most of these report on highly selectedpatients populationss the covid pandemic places cancer patients in a very difficult situation wherein if they seektreatment they are exposing themselves to a risk of developing cov2 infection and if they do not the probabilityof dying without treatment increases hence for them it is a choice between the devil and deep sea and it is forthe healthcare providers to triage patients and treat who cannot wait even though the data from the carefullyselected cohort of patients show no increase in mortality or morbidity from treatment during covidkeywords covid cancer cov2 coronavirus treatment chemotherapy surgeryintroductionwith the onset of covid19 pandemic the situation forcancer patients has become a nightmare most of the patients who were on treatment in march had to miss outtheir further treatment due to lockdown and closure ofhospitals and all modes of transport both public and private those who developed cancer during lockdown toocould not reach doctors for the same reason surgerieswere postponed radiotherapy was postponed and there correspondence manojpandey66gmailcomdepartment of surgical oncology institute of medical sciences banarashindu university varanasi indiawas uncertainty as far as chemotherapy and immunotherapy were concerned every society and anization cameup with their own guidelines however none of these addressed the logistic problems that patients faced reachingthe treatment centers this lead to an increased pool ofuntreated patients an analysis from england estimatesadditional deaths from cancer over next monthsthat is increase proposed due to covid pandemic those who were willing to travel and take the risk ofgetting infected with covid19 too faced challenges dueto travel restrictions though the indian government issued special passes to allow travel for cancer treatment the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cchakraborty and pandey world of surgical oncology page of however the rider was that he or she has to travel withhis own private transport and not more than two peoplewere allowed in a four wheeler this meant that apartfrom the driver only the patient could travel withoutany caregiver if the patient does not own the car andcaregiver does not know how to drive these passeswere issued district or state wise as there was a ban oninterdistrict and interstate travel so the patients comingto bigger institutes like ours faced special problemswherein despite having a travel pass some of them hadto pay fines most of our patients come from neighboring states of bihar and madhya pradesh and even fromwest bengal and nepalapart from that at the beginning of the pandemicthings were not clear and there was no data on the incidence and mortality from covid19 in cancer patientsfurther though there were several guidelines [“] theywere not based on evidence as the evidence did not existat that time and it was not clear as to what treatmentmodalities are safe giving rise to uncertainties in themind of patients and health care workers the testing facilities were limited the availability of ppe was limitedand these were prioritized for suspected cases and contacts while other diseases were kept on hold or receivedlow priority this reviews the current literatureon various aspects of covid and cancermethodsa review of literature was carried out using the pubmedfor the s published using the string œcovid[allfields] and œcancer s[all fields] or œcancerated[all fields] or œcanceration[all fields] or œcancerization[all fields] or œcancerized[all fields] orœcancerous[all fields] or œneoplasms[mesh terms]or œneoplasms[all fields] or œcancer[all fields] orœcancers[all fields] following filters were applied clinical study clinical trial comparative study controlledclinical trial metaanalysis observational study pragmatic clinical trial randomized controlled trialthe s that discussed the incidence prevalencemortality treatment of cancer during covid or morbidity of treatment were included in the review as the obtained data was very heterogeneous no attempt atpooling or metaanalysis was doneresultsa total of s were extracted the of allthe s was read and after excluding reviews s were included in this review including one metaanalysis as for the prevalence the initial data from amulticentric study comparing cancer with noncancerpatients infected with covid found higher incidence ofsevere outcome in cancer compared to normal the outcome was worse in lung cancer and metastatic disease a study evaluating fatality in patients withcovid found a times risk of death in cancer patientscontracting covid this fear and uncertainty had affected the quality oflife and metal health of cancer patients caregivers andhealth care workers there were no socializing no outlets no markets or movies or dining out even the placesof worships were closed an italian survey of young cancer patients showed that they feel more vulnerable tocontract cancer and risk of higher complications ahigher burnout has been reported in healthcare workersworking in covid hospitals over a period of time things have started clearing upand preliminary data on incidence severity and complications of covid19 is now available a retrospectiveanalysis of patients across mainland china reported incidence of severe cases of these had at least one comorbidity with hypertension being most prevalent followed by diabetes in thisstudy the cancer was seen only in patients ofwhom had severe illness another study from chinaon patients with covid had patients with cancer they too found higher severity in cancer patientshowever the number of cancer patients in this study toowas very small similarly the study of chen had patients in total of covid19 patients andonly three of these had a severe disease similar resultshave been reported by others [ ]a pooled metaanalysis of studies reporting oncovid prevalence in cancer patients found a pooledprevalence of prevalence was higher at insmaller series reporting on less than patients between march and june over patients wereseen at our center of which undergoing surgery orthe interventions were tested for covid of these patients were found to be positive the prevalenceof cancer is high among covid19 patients in europehowever these do not seem to convert into higher mortality of cancer patients a study from uk reporting on deaths did not find increased mortality in cancer except in patients receiving chemotherapy however ametaanalysis involving patients thatincluded cancer patients showed a significant increase inhazard of death in cancer patients infected with covid hr the study also showed increased admission to icu hr however when patients abovethe age of were considered no difference in deathrate was observed this increase in cancer mortalityin covid19 has been attributed to advance age additional comorbidities diagnosis of lung cancer use ofchemotherapy etc [ “]recent data from the usa canada and spain fromthe covid19 and cancer consortium ccc19 database reported on patients with cancer of these 0cchakraborty and pandey world of surgical oncology page of patients had died advance age smokingmale gender more than one comorbidity and ecogmore than were found to be associated with increasedmortality these results and those detailed above clearlyshow that prevalence of covid in cancer is higher andmortality in these patients too is higher especially in patients with additional comorbidities and active diseaseand one needs to exercise full precaution while takingtreatment decisionsdiscussionprecautions taken at our center to prevent spread incancer patientsas cancer patients are at a higher risk of contractingcovid19 than the general population special precautionsare taken when patients are seen in the outpatient clinicopd and surgery no patient or the caregiver is allowedto enter the opd without a face mask all patients arescreened for body temperature and those found to havefever are immediately sent to covid opd this is followedby filling up of a symptom checklist and questionnairebased screening for all patients those with covid symptoms are referred for rtpcr testing before being allowedto see doctor and get the treatment this is similar to allothers coming to the hospital for treatment as there areno separate guidelines for cancer apart from that stricthand hygiene is followed and ppe has to be worn byhealth care workers those planning to undergo procedures and surgery are screened for covid19 using rtpcr testing they are kept in the holding area and afterbeing reported are shifted to wards any patient foundpositive is treated as per guidelinesplanned surgeryelective surgeries for cancer patients are avoided ifpossible this is usually a collective decision taken bythe oncologist in consultation with the patient if it isdecided to do a surgery a special covid19 consent istaken all emergency surgeries are performed withproper written covid consent and after covid testingfor even asymptomatic patients other than emergencies patients who cannot wait for weeks for surgeryas the disease may progress and become inoperableare considered for surgery other than that patientswho were started on neoadjuvant therapy before theonset of covid and have now completed neoadjuvanttherapy are also candidates for surgery as the opportunity of window period should not be lost any patient where the multidisciplinary team think can waitfor weeks is postponed patients undergoing surgeryare reported to be at higher risk than those treatedby radiation planned radiotherapyin the beginning all patients were postponed after discussion with patients or caregivers however as it is notpossible to postpone radiation forever selected patientsare now being taken up for radiation patients are informed that whatever little evidence that is available suggest higher chances of covid infection in this subset ofpatients recent guidelines have suggested omitting radiation for patients older than years using fast andfast forward omitting boost and hypofractionation[ ] recent s also suggest modification of thedelivery technique to reduce treatment time [ ]immunosuppressive therapythe current evidence does not support changing orwithholding chemotherapy targeted therapy and immunotherapy in cancer patients some of the studiessuggested stopping of immunotherapy for patients whohave a complete response or prolonged response ofmore than years a report on immune check pointinhibitors suggested that as there is not much immunecompromise and minimal hematological toxicities theycan be used with caution as patients are not devoid ofits benefits [ ] at our center we have continuedwith chemotherapy and immunotherapy and have notfound any increase in covid19 infection or mortality inthis sub groupstem cell transplantationcurrent guidelines recommend delaying the plannedallogenic stem cell transplant [ ] this is a uniquesituation where the donor and recipient both are at riskand a careful decision making keeping all factors into account be made no stem cell transplants took place atour center during this periodcoronavirus therapy in cancerthere is no evidence of benefit of using various therapies to treat covid19 in cancer patients remdesivir hasbeen approved by the fda for treatment of covid casesunder emergency use authorization the ccc19 studyfailed to show any benefit of azithromycin hydroxychloroquine alone or in combination in fact use of acombination of the two was found to be an independentfactor predicting mortality with hazard ratio of which was statistically significant there are occasional reports on the use of lopinavirritonavir [ ]in lung cancer patient with covid another dataset fromccc19 study reported on patients treated withhydroxychloroquine n azithromycin n remdesivir n highdose corticosteroids n tocilizumab n andother therapy n showed no benefit of anytreatment apart from slight activity of remdesivir no 0cchakraborty and pandey world of surgical oncology page of other drug showed any benefit [ ] patients also relowmolecular weightceived dexamethasone aspirinheparinabovetreatmentsanticoagulants besidesand otherresearch on covid2019 and cancera recent bioinformatic study using the geo databaseshowed that angiotensinconverting enzyme ace2 iselevated in uterine corpus endometrial carcinoma andrenal papillary cell carcinoma this increase also correlated with immune infiltrate present in the tumor theauthors suggested that these tumors may be more proneto covid19 infections however the authors failedto present any evidence to suggest this hypothesis similar bioinformatics study by fu also showed similarresults and they suggested that testes may also get affected by covid19 infections other tumors thatmay be affected are pancreas and colon howeverall these studies are based on geo databases and thereis no experimental evidence presented till datesthe literature on cancer and covid is fast expanding withmore and more information pouring in the literature sofar is clear in indicating that cancer patients are at highrisk of developing covid and when developed the severityand mortality is higher than the normal population therecent data also suggests a possible role of remdesivir inthe treatment of covid19 in cancer patients however theevidence to support this is very weak this absence of conclusive evidences has led to development of strategies totreat cancer safely most of these are based on reducingthe hospital visits and avoiding immune compromise theevidence is still evolving and more practice changes areexpected in the days to come and that may continue evenin post covid eraacknowledgementsnoneauthors™ contributionsmc prepared the draft and mp conceived and designed the and edited the final version the authors read and approved the finalmanuscriptfundingnoneavailability of data and materialsnot applicableethics approval and consent to participatethis does not report on human and animal experiments ethicalapproval and publication of consent is not applicableconsent for publicationnot applicablecompeting intereststhe authors declare that there are no conflicts of interestreceived june accepted august references wise j covid19 cancer mortality could rise at least because ofpandemic study finds bmj 2020369m1735 httpsdoi101136bmjm1735m1735civantos fj leibowitz jm arnold dj stubbs vc gross jh thomas gr sargiz casiano rr franzmann ej weed d perez c samuels m goodman kwgoodwin wj ethical surgical triage of head and neck cancer patientsduring the covid19 pandemic head neck coles ce aristei c bliss j boersma l brunt am chatterjee s hanna g jagsir kaidar po kirby a mjaaland i meattini i luis am marta gn offersen bpoortmans p rivera s international guidelines on radiation therapy forbreast cancer during the covid19 pandemic clin oncol r coll radiol “cortiula f pettke a bartoletti m puglisi f helleday t managing covid19in the oncology clinic and avoiding the distraction effect ann oncol “elkaddoum r haddad fg eid r kourie hr telemedicine for cancer patientsduring covid19 pandemic between threats and opportunities futureoncol “finley c prashad a camuso n daly c aprikian a ball cg bentley jcharest d fata p helyer l o'connell d moloo h seely a werier j zhongt earle cc guidance for management of cancer surgery during the covid pandemic can j surg 202063s2“finley c prashad a camuso n daly c earle cc lifesaving cancer surgeriesneed to be managed appropriately during the covid19 pandemic can jsurg 202063s1hanna tp evans ga booth cm cancer covid19 and the precautionaryprinciple prioritizing treatment during a global pandemic nat rev clinoncol “dai m liu d liu m zhou f li g chen z zhang z you h wu m zheng qxiong y xiong h wang c chen c xiong f zhang y peng y ge s zhen byu t wang l wang h liu y chen y mei j gao x li z gan l he c li zshi y qi y yang j tenen dg chai l mucci la santillana m cai h patientswith cancer appear more vulnerable to sarscov2 a multicenter studyduring the covid19 outbreak cancer discov “ deng g yin m chen x zeng f clinical determinants for fatality of patients with covid19 crit care “ casanova m pagani be silva m patriarca c veneroni l clerici ca spreaficof luksch r terenziani m meazza c podda m biassoni v schiavello echiaravalli s puma n bergamaschi l gattuso g sironi g massimino mferrari a how young patients with cancer perceive the covid19coronavirus epidemic in milan italy is there room for other fears pediatrblood cancer 2020e28318 wu y wang j luo c hu s lin x anderson ae bruera e yang x weis qian y a comparison of burnout frequency among oncologyphysicians and nurses working on the front lines and usual wardsduring the covid19 epidemic in wuhan china j pain symptommanag guan wj ni zy hu y liang wh ou cq he jx liu l shan h lei cl huidsc du b li lj zeng g yuen ky chen rc tang cl wang t chen pyxiang j li sy wang jl liang zj peng yx wei l liu y hu yh peng pwang jm liu jy chen z li g zheng zj qiu sq luo j ye cj zhu syzhong ns clinical characteristics of coronavirus disease in china nengl j med “ cai yc wang w li c zeng df zhou yq sun rh jiang h guo h wang sxjiang j treating head and neck tumors during the sarscov2 epidemic to sichuan cancer hospital head neck chen atc courafilho gb rehder mhh clinical characteristics of covid19in china n engl j med pii 101056nejmc2005203sa2 doi 1056nejmc200520310fong d rauch s petter c haspinger e alber m mitterer m infection rateand clinical management of cancer patients during the covid19pandemic experience from a tertiary care hospital in northern italy esmoopen 20205e000810 yu j ouyang w chua mlk xie c sarscov2 transmission in patients withcancer at a tertiary care hospital in wuhan china jama oncol 0cchakraborty and pandey world of surgical oncology page of zhang h xie c huang y treatment and outcome of a patient eith lungcancer infected with severe acute respiratory syndrome coronavirus2 jthorac oncol 202015e63“ rivera dr peters s panagiotou oa shah dp kuderer nm hsu cy rubinsteinsm lee bj choueiri tk de lima lg grivas p painter ca rini bi thompsonma arcobello j bakouny z doroshow db egan pc farmakiotis d fecher lafriese cr galsky md goel s gupta s halfdanarson tr halmos b hawley jekhaki ar lemmon ca mishra s olszewski aj pennell na puc mm revankarsg schapira l schmidt a schwartz gk shah sa wu jt xie z yeh ac zhu hshyr y lyman gh warner jl utilization of covid19 treatments and clinicaloutcomes among patients with cancer a covid19 and cancer consortiumccc19 cohort study cancer discov 2020cd0941 yang j li h hu s zhou y ace2 correlated with immune infiltration servesas a prognostic biomarker in endometrial carcinoma and renal papillary cellcarcinoma implication for covid19 aging albany ny “fu j zhou b zhang l balaji ks wei c liu x chen h peng j fu jexpressions and significances of the angiotensinconverting enzyme genethe receptor of sarscov2 for covid19 mol biol rep “ chai p yu j ge s jia r fan x genetic alteration rna expression and dnamethylation profiling of coronavirus disease covid19 receptor ace2in malignancies a pancancer analysis j hematol oncol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations desai a sachdeva s parekh t desai r covid19 and cancer lessons from apooled metaanalysis jco glob oncol “ httpsdoi101200go2000097557559lee lyw cazier jb starkey t turnbull cd kerr r middleton g covid19mortality in patients with cancer on chemotherapy or other anticancertreatments a prospective cohort study lancet giannakoulis vg papoutsi e siempos ii effect of cancer on clinicaloutcomes of patients with covid19 a metaanalysis of patient data jcoglob oncol “ httpsdoi101200go2000225799808 gosain r abdou y singh a rana n puzanov i ernstoff ms covid19 andcancer a comprehensive review curr oncol rep “ addeo a obeid m friedlaender a covid19 and lung cancer risksmechanisms and treatment interactions j immunother cancer e000892 yang k sheng y huang c jin y xiong n jiang k lu h liu j yang j dongy pan d shu c li j wei j huang y peng l wu m zhang r wu b li ycai l li g zhang t wu g clinical characteristics outcomes and risk factorsfor mortality in patients with cancer and covid19 in hubei china amulticentre retrospective cohort study lancet oncol “ mehta v goel s kabarriti r cole d goldfinger m acunavillaorduna apradhan k thota r reissman s sparano ja gartrell ba smith rv ohri ngarg m racine ad kalnicki s perezsoler r halmos b verma a casefatality rate of cancer patients with covid19 in a new york hospitalsystem cancer discov “ meng y lu w guo e liu j yang b wu p lin s peng t fu y li f wang zli y xiao r liu c huang y lu f wu x you l ma d sun c wu p chen gcancer history is an independent risk factor for mortality in hospitalizedcovid19 patients a propensity scorematched analysis j hematol oncol“kuderer nm choueiri tk shah dp shyr y rubinstein sm rivera dr shetes hsu cy desai a de lima lg jr grivas p painter ca peters s thompsonma bakouny z batist g bekaiisaab t bilen ma bouganim n larroya mbcastellano d del prete sa doroshow db egan pc elkrief a farmakiotis dflora d galsky md glover mj griffiths ea gulati ap gupta s hafez nhalfdanarson tr hawley je hsu e kasi a khaki ar lemmon ca lewis clogan b masters t mckay rr mesa ra mans ak mulcahy mfpanagiotou oa peddi p pennell na reynolds k rosen lr rosovsky rsalazar m schmidt a shah sa shaya ja steinharter j stockerlgoldstein kesubbiah s vinh dc wehbe fh weissmann lb wu jt wulffburchfield exie z yeh a yu pp zhou ay zubiri l mishra s lyman gh rini bi warnerjl clinical impact of covid19 on patients with cancer ccc19 a cohortstudy lancet huang sh o'sullivan b su j ringash j bratman sv kim j hosni a bayleya cho j giuliani m hope a spreafico a hansen ar siu ll gilbert r irishjc goldstein d de aj tong l xu w waldron j hypofractionatedradiotherapy alone with gy per fraction for head and neck cancerduring the covid19 pandemic the princess margaret experience andproposal cancer “ vordermark d shift in indications for radiotherapy during the covid19pandemic a review of anspecific cancer managementrecommendations from multidisciplinary and surgical expert groups radiatoncol “ davis ap boyer m lee jh kao sc covid19 the use of immunotherapy inmetastatic lung cancer immunotherapy “kattan j kattan c assi t do checkpoint inhibitors compromise the cancerpatients' immunity and increase the vulnerability to covid19 infectionimmunotherapy “ bersanelli m controversies about covid19 and anticancer treatment withimmune checkpoint inhibitors immunotherapy “ ardura mi hartley dm dandoy c lehmann l jaglowski s auletta jjaddressing the impact of the coronavirus disease covid19 pandemic onhematopoietic cell transplantation learning networks as means for sharingbest practices biol blood marrow transplant mahmoudjafari z alexander m roddy j shaw r shigle tl timlin c culosk american society for transplantation and cellular therapy pharmacyspecial interest group position statement on pharmacy practicemanagement and clinical management for covid19 in hematopoietic celltransplantation and cellular therapy patients in the united states biol bloodmarrow transplant 0c"

"gut microbiota composition influences the balance between human health and disease increasing evidencesuggests the involvement of microbial factors in regulating cancer development progression and therapeuticresponse distinct microbial species have been implicated in modulating gut environment and architecture thataffects cancer therapy outcomes while some microbial species offer enhanced cancer therapy response othersdiminish cancer treatment efficacy in addition use of antibiotics often to minimize infection risks in cancer causesintestinal dysbiosis and proves detrimental in this review we discuss the role of gut microbiota in cancerdevelopment and therapy we also provide insights into future strategies to manipulate the microbiome and gutepithelial barrier to augment therapeutic responses while minimizing toxicity or infection riskskeywords intestinal dysbiosis cancer development cancer therapy microbial therapy human intestinal microbiota is essentialfor microbialhomeostasis regulation of metabolism and immune tolerance intestinal dysbiosis occurs when there are alteredratios of healthy microbial flora along with changes in theirdiversity and density such changes may lower mucus layerthickness reduce antimicrobial defense and disrupt theepithelial tightjunction barriers to allow increased translocation of intestinal bacteria and bacterial products intothe systemic circulation and trigger inflammation andimmune responses circulating bacterial products such asendotoxin genotoxin and trimethylamine oxide have beenimplicated in many human disorders including metabolicsyndrome cardiovascular complications atherosclerosisand thrombosis and various neoplastic conditions intestinal dysbiosis may also affect adaptive immunity by correspondence seahhlimyahoocom1division of hematology and oncology suny downstate health sciencesuniversity clarkson avenue room b5495 brooklyn new york usa2division of hematology and oncology department of medicine new yorkmedical college valhalla new york usamodulating the functions of t lymphocytes and promotingtumor immune escapewhile increased translocation of intestinal luminal content is associated with carcinogenesis and poor therapeuticresponse the causeeffect relationship is often bidirectionalin this review we will discuss the role of gut microbes inmodulating tumor immunity intestinal permeability andcancer development next we will highlight the effects ofintestinal dysbiosis and increased permeability in cancertherapy finally we will explore the options to improve guthealth to enhance the efficacy of cancer therapyintestinal immunity and permeabilitythe intestinal architecture and microbiota regulateinnate and adaptive immunity disruption of the architecture andor microbiota affects these functions therelationships between the different players in the intestinal microenvironment is summarized in fig the composition of microbes in the gut dictatesmucus layer thickness and production of antimicrobialsignals in germfree mice mucus layer and effector t the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cdutta and lim biomarker research page of fig interplay between different factors involved in gut immunity and permeability a the intestinal epithelial cells containing paneth cellsgoblet cells enterocytes and enteroendocrine cells coordinate with intraepithelial lymphocytes to generate a functional immune responsepaneth cells secrete antimicrobial peptides and goblet cells produce mucus to cover the epithelial layer this mucus layer prevents adhesion ofmicrobes to the epithelial cells lamina propria situated under the mucus layer contains peyer™s patches and immune cells including antigenpresenting cells apcs like dendritic cells dcs t cells and b cells pattern recognition receptors prrs such as tolllike receptors tlrs onepithelial cells interact with microbederived pathogenassociatedmolecular patterns pamps such as lipopolysaccharide lps to activatemyd88dependent signaling dcs travel to mesenteric lymph nodes mln and promote the differentiation of na¯ve t cells to regulatory t tregcells that migrate to other sites treg cells secrete il10 to elicit an antiinflammatory response b dysbiosis decreases mucus layer thickness andshortchain fatty acid scfas production this affects the secretion of antimicrobial peptides and allows microbes to come in close proximity tothe epithelial cells reduction in scfas influences gut barrier dysfunction as a result the gut luminal content also translocated and spreadedthrough the systemic circulation to trigger local and systemic immune responses in addition to pamps damps released from damaged intestinalepithelium interact with prrs to facilitate expression of macrophages and maturation of dcs mature dcs promote the differentiation of na¯ve tcells to effector t cells such as t helper cells th1 th2 th17 th1 release tnfα and ifnÎ and th17 secrete il17 to recruit polymorphonuclearneutrophils pmns these cytokines create a proinflammatory condition 0cdutta and lim biomarker research page of cells are absent [ ] microbes secrete shortchain fattyacids scfas such as propionate and butyrate thatprevent microbial binding to the epithelial cells and helpmaintain barrier function and immune homeostasisbutyrate promotes tightjunction formation [ ] andactivates peroxisome proliferatoractivated receptor gammapparÎto enhance epithelial oxygen consumptionresulting in reduced emanation of oxygen from the mucosalsurface it helps in maintaining an anaerobic condition inthe gut lumen needed for colonization of obligate anaerobes this intestinal microenvironment determines thecomposition of resident bacterial species for example onlyclostridium lactobacillus and enterococcus are enrichedon the epithelial surface and in the mucus layer whereasbacteroides bifidobacterium streptococcus enterobacteriaceae enterococcus clostridium and lactobacillus are allpredominant in the intestinal lumen dysbiosis increases inflammatory signals that shiftthe metabolism of enterocytes epithelial hypoxia iseliminated and increased oxygenation results in therelease of more oxygen from the mucosal surfacesince only facultative anaerobes can respire oxygendysbiosisinduced shift in epithelial oxygenation altersgut microbial community from obligate to facultativeanaerobes intestinal pathogens such as proteobacteria produce genotoxins like colibactin and cytolethaldistending toxin cdt to induce inflammation andhost deoxyribonucleic acid dna damage that initiates tumor formation dysbiosis also decreasesmucus layer thickness reduces scfa production anddamages mucosal barrier allowing pathogenassociatedmolecular patterns pamps to interact with pattern recognition receptors prrs and activate tolllike receptortlr 24myeloid differentiation primary response protein myd88 signaling pathways in addition changes inmicrobial composition and density triggers epithelial releaseof damageassociated molecular patterns damps such asextracellular adenosine triphosphate atp cytoplasmiccalreticulin high mobility group box hmgb1 proteinsendogenous nucleic acids and intracellular proteins tointeract with prrs prr engagementtriggers a proinflammatory condition that causes tissue damage and localinflammation microbiotadriven tlr immune signalinghas been implicated in cancer formation and modificationof treatment efficacy [“] for example cpg oligodeoxynucleotides that mimic bacterial dna acts as a pamp totrigger a tlr9dependent tlr4 activation and tumor necrosis factor tnfα production by tumorinfiltratingmyeloidderived cells mice bearing el4 lymphomamc38 colon carcinoma and b16 melanoma when treatedwith cpg oligodeoxynucleotides show reduced tumrowth and enhanced survival rate the beneficial effects ofcpg oligodeoxynucleotides were positively associated withthe abundance of alistipes shaii in the gut effects of intestinal microbiota on cancerdevelopmentintestinal microbes can influence local and distantcarcinogenesis through infection and microbial productsor by modulating tumor immunosurveillance this isaccomplished via altering the balance between the rateof cell proliferation and apoptosis triggering chronicinflammation andor immunosuppression or changingthe metabolism of the products produced by host andmicrobes in this section we will discuss how intestinaldysbiosisrelated permeability may contribute to tumorigenesis in different anscolorectal cancerfusobacterium nucleatum a gramnegative mucosaadherent anaerobic bacteria has been implicated in theinitiation and progression of colorectal cancer crc[ ] fada an adhesion molecule on f nucleatumbinds to host ecadherin to enter epithelial cells this activates the wntβcatenin pathway leading toan increased secretion of inflammatory cytokines including il6 il8 and tnfα and upregulation of nuclearfactor kappa light chain enhancer of activated b cellsnfκb that facilitates crc development in addition itattracts myeloidderived suppressor cells and the autotransporter protein fap2 interacts with the human inhibitoryreceptor t cellimmunoreceptor with ig and itimdomains tigit to create a tumor immunosuppressivemicroenvironment f nucleatum may also induce chemoresistance by modulating the tlr4myd88 signalingpathway following 5fluoruracil treatment in crc patients an increased abundance of f nucleatum along with clostridium difficile and species ofstreptococcus campylobacter and leptotrichia has beendemonstrated in tumor tissue and fecal materials [“]f nucleatummediated colorectal carcinogenicity occursdownstream of apc introduction of f nucleatum resultedin rapid onset of colonic tumors in mice deficient in onecopy of adenoma polyposis coli apc apcmin gene both intestinal dysbiosis and loss of apc disruptepithelial tightjunctions and mucus layer [ ] andallow increased infiltration of f nucleatum and other nonresidential microbes to drive crc development the roleof defective gut barrier in crc has been confirmed inmucin 2knockout muc2ˆ’ˆ’ mice in which the lack ofgastrointestinal mucin resulted in spontaneous crc development therefore dysbiosisinduced gut permeabilitymay play an important role in tissue enrichment of fnucleatum and increased risks for crchepatobiliary cancerthe liver is chronically exposed to intestinal microbiotaand its products via the portal vein intestinal dysbiosisand increased permeability enhance translocation of gut 0cdutta and lim biomarker research page of microbiota to trigger inflammation and chronic liver disease that predisposes patients to the development of hepatocellular cancer alteration in bile acid metabolism due tochanges in clostridium spp suppress anticancer immunity in mice eradication of grampositive bacteria by oralvancomycin inhibits secondary bile acid conversion resulting in the upregulation of chemokine cxc motif ligandcxcl16 in liver sinusoidal endothelial cells cxcr16recruits natural killer t nkt cells in the tumor microenvironment and kill tumor cells in a cd1ddependentmanner in addition gut microbiotaderived lipopolysaccharides lps promote tumor progression in liver cancerby activating the tlr4 signaling in a study involving cholangiocarcinoma patients bile ducttissues haddistinct dominance of dietziaceae pseudomonadaceae andoxalobacteraceae members pancreatic cancergut microbiota influences the development of pancreaticcancer through activating tlr4 signaling the stromain pancreatic tumor harbors an abundance of microbiotaespecially bifidobacterium pseudolongum compared tonormal pancreas this helps in creating an immunosuppressive environment by differentially activating distincttlrs in monocytes pancreatic adenocarcinoma has anenrichment of proteobacteria synergistetes and euryarchaeota longer survival is observed in patients with amore diverse intratumor microbial composition primarilyof sachharopolyspora pseudoxanthomonas streptomycesand bacillus clausii tumoral colonization with mycoplasma hyorhinis and gammaproteobacteria is associatedwith gemcitabine resistance antibiotics diminishmyeloidderived suppressor cells and increase antitumorm1 macrophages to promote th1 differentiation of cd4t cells and cd8 t cell activation in the tumor cotreatment of gemcitabine with ciprofloxacin abrogatedgammaproteobacteriainduced chemotherapy resistance the efficacy of immune checkpoint inhibitors icistherapy is also enhanced by antibiotics lung cancerwhile local microbiota is important there are reportsthat gut microbiome may also contribute to lung cancerdevelopment lung cancer patients demonstrated an abundance in intestinal enterococcus and depletion in bifidobacterium and actinobacteria they are also enrichedwith veillonella bacteroides and fusobacterium depletedof dialister enterobacter escherichiashigella fecalibacterium and kluyvera in nonsmall celllung cancernsclc patients butyrate producers such as faecalibacterium prausnitzii clostridium leptum clostridial cluster iruminococcus spp clostridial cluster xiva and roseburiaspp were significantly reduced since butyrate isessential for preserving mucosal homeostasis reduction ofintestinal butyrate producers may imply a compromised intestinal barrier in these patientshematologic malignanciesdysbiosisinduced intestinal permeability affects mucosaassociated lymphoid tissue malt and plays a significantrole in hematologic malignancies composition of intestinalmicrobiota is responsible for maintaining the pool of bonemarrow myeloid cells preleukemic myeloproliferationis driven by microbial signals in teneleven translocation2tet2deficient mice [ ] these mice show increasedinfiltration of inflammatory cells disrupted mucosal barrierand increased translocation of bacteria [ ] it wassuggested that dysfunction of small intestinal barrier andleakage of microbes can occur due to tet2 mutation in occurrence of tet2hematopoietic compartmentmutation intestinal dysbiosis and leaky gut is common inleukemia and lymphomaacute myeloid leukemia aml and acute lymphoblasticleukemia all patients have a compromised intestinalbarrier [“] fecal microbiota in all patients showedlower microbial diversity they were enriched in enterococcaceae porphyromonadaceae and bacteroidetes mainlyb fragilis and depleted in blautia erysipelotrichiales lachnospiraceae and clostridiales members [ ] abundanceof staphylococcaceae and streptococcaceae have also beenreported in pediatric all and adult aml [ ]helicobacter pylori is associated to malt lymphoma and chamydophila psittacito ocular maltlymphoma while borrelia burgdorferi was linked tocutaneous bcell nonhodgkin lymphoma two studiesdid not find significant risk of borrelia burgdorferi in thedevelopment of nonhodgkin lymphoma [ ] abundance of proteobacteria is a predictor for neutropenicfever and enrichments of enterococcaceae and streptococcaceae are strong predictors of infectious complications inall similarly higher gut microbiota diversity inmultiple myeloma is associated with reduced risk fordisease relapse all patients with infectious complications have an abundance of brevundimonas diminuta andagrobacterium tumefaciens whereas faecalibacteriumprausnitzii producer of scfas is completely absent similar findings have been reported in nonhodgkinlymphoma with infectious complications effects of intestinal microbiota on cancer therapythe efficacy of cancer treatment is in parts dependenton normal immune function since gut microbiota playsa crucial role in modulating immune response it is notsurprising that dysbiosis affects treatment outcomesprophylactic antibiotics are commonly used for cancerpatients undergoingallogeneichematopoietic stem cell transplantation allohsct toreduce the risk of neutropeniaassociated infectionchemotherapyand 0cdutta and lim biomarker research page of however antibiotic use causes intestinal dysbiosis thatresults in negative outcomes including poor treatmentresponse and toxicity and the development of clostridium difficile infection cdi in addition to antibioticsopioid analgesics for cancer pain management may alsotrigger dysbiosis opioid analgesics impair intestinal motility and promote bacterial overgrowth resulting in dysbiosisand gut permeability intestinal dysbiosis induces mucosal injury and triggers the release of damps damps have a dual andbidirectional effect on cancer although damps exertimmunosurveillance and immunemediated cell death toeliminate tumor cells and protect against cancer development chronic inflammation induced by damps maypromote tumor initiation damps released by apoptoticcells from cancer therapy may also induce chemoresistance and promote metastasis for example tlr78expressed on tumor cells may bind damps loxoribinefor tlr7 and poly u for tlr8 and promote chemoresistance through the activation of nfκb and the upregulation of bcl2 damps may also activate tlr9on human breast prostate and lung cancer cells to trigger tumor invasion and metastasis [ ] given theclinical significance of dysbiosismediated mucosal injuryand permeability in cancer we will in this section discusshow the treatment outcome by various cancer therapymay be affected by intestinal microflora and permeabilitychemotherapy and radiation therapyintestinal microbial composition and mucosal barrier function influence chemotherapeutic outcome and the effect isbidirectional while dysbiosis can exacerbate chemotherapy drug toxicity and reduce its efficacy chemotherapy canitself cause dysbiosis although prevalence of certain intestinal microbes in the gastrointestinal tract offer beneficialeffects others contribute to chemoresistance and drug toxicity this multiplepathway effect is best covered by timer mechanisms “ translocation of microbes immunomodulation metabolism and enzymatic effects on drugsand reduced microbial diversity these mechanistic effectsalter chemotherapy efficacy and toxicity and risks forinfections for example translocation of microbes due tochemotherapy induceddysbiosis and disruption of mucosal barrier can increase the risk of infection howevercertain chemotherapy drugs such as cyclophosphamideand doxorubicin damage intestinal barrier for the translocation of commensal bacteria into secondary lymphnodes to elicit antitumor immune response vancomycin prophylaxis inhibits antitumor effects of cyclophosphamide in fibrosarcoma inoculated mice irinotecanused for crc treatment is transformed into its active formsn38 by tissue carboxylesterase it is detoxified in theliver by host udpglucuronosyltransferases into inactiveglucuronide sn38g and excreted into the gut via bileducts in the gut bacterial βglucuronidases reconvertssn38g into active sn38 which causes severe intestinaltoxicity and diarrhea streptomycin inhibits irinotecanabsorption and reduces epithelial carboxylesterase activityand diarrhea ciprofloxacin inhibit βglucuronidases and low dose amoxapine βglucuronidases inhibitorsuppress irinotecanassociated diarrhea in rats table provides a selection of chemotherapeutic agents affectingand affected by intestinal microbial composition andpermeabilitylocal pelvic irradiation damages intestinal epitheliumand barrier integrity and produce reactive oxygen speciesirradiation increase alistipes and decrease prevotella inmice in gynecologic cancer patients receiving pelvicradiotherapy firmicutes and fusobacterium were significantly decreased in addition to reduced diversitysignificant enrichment of clostridium iv roseburia andphascolarctobacterium was associated with radiationenteropathy in pelvic cancer patients the effects oftotal body irradiation which is a preparative regimen forallohsct that causes dysbiosis and gastrointestinaltoxicity is discussed in more details in the allohsctsection belowimmunotherapycancer cells often create an immunosuppressive microenvironment to mediate tumor immune escape this immune escape mechanism may be reversed by icis directedat cytotoxic t lymphocyteassociated antigen ctla4programmed death receptor pd1 or pd1 ligandspdl1 since intestinal microbes influence local and systemic antitumor immune reaction by modulating prrspamps and dampsintestinal dysbiosis may impacttreatment outcome figure illustrates how the potentialmechanisms ofthe antitumor immune responses aredownregulated by intestinal dysbiosis the effects of intestinal microbiome on responses to icis have been discussed previously [ ] broadspectrum antibioticsbefore during or after icis therapy alter intestinal microbiome and resulted in lower tumor response rate inferiorprogressionfree survival and reduced overall survival responses to inhibition of ctla4 by ipilimumab inmouse models of mca205 sarcoma ret melanomaand mc38 colon carcinoma were inferior in germfreeor in broadspectrum antibiotic treated mice poorresponses were associated with decrease in intestinalbacteroides thetaiotaomicron bacteroides uniformis andburkholderia cepacia and increase in clostridiales suchdysbiosis was also associated with mucosal damage andcolitis oral feeding with either bacteroides thetaiotaomicron or bacteroides fragilis individually or with acombination of bacteroides fragilis and burkholderiacepacia restored the antitumor effects of ctla4 blockade through augmentation of th1 responses in tumor 0cdutta and lim biomarker research page of table selection of chemotherapeutic agents and the bidirectional effects between the chemotherapy and intestinal microbiotachemotherapy drugcisplatintoxicity infectioncdi ototoxicity effects on gut changes in microbiotadamages mucosal barrier by impairing dnareplication of rapidly proliferating epithelialcells facilitates translocation of gut bacteriacommensal gut bacteria influencesgenotoxicity by inducing reactive oxygenspecies ros production and recruitment ofpathogenic th17 cells in the tumormicroenvironment independently ofimmunity elicited by immunogenic celldeath microbial interventionantibiotics against grampositive bacteriaabrogate antitumor chemotoxicityincrease tumor size and decrease survivalratecisplatin alone show better responsecompared to a combined treatment ofcisplatin and antibiotics in mice with lungcancer the combination treatmentincreased tumor size and decreasedsurvival ratelactobacillus acidophilus restores antitumorefficacy following antibiotic treatment[ ]restoration of gut microbiota andepithelial integrity by fmt andtreatment with dmethionine [ ]prevent infections and ototoxicity withoutaffecting tumor chemotoxicityfmt increases a muciniphilaabundance and reduces cipn oral butyrate supplementation improvesgut barrier by reducing inflammation andmucositis antibiotics reduce mucositis and cytokineproduction but also diminish antitumorefficacy and promote chemotherapyresistance antibiotics against grampositive bacteriareduce th17 responses and subsequentdevelopment of cyclophosphamideresistancereestablishment of e hirae alone restoresantitumor activity e hirae decreases tumorinfiltrating tregsbarnesiella intestinihominis accumulates inthe colon and increases the number ofintratumoral ifnÎproducing Îδt cellse hirae and b intestinihominissynergistically stimulate local and systemicimmunity to improve anticancer effects nod1ˆ’ˆ’nod2ˆ’ˆ’ mice having abundant bintestinihominis demonstrate increased Îδtcells in tumor beds and enhancedcyclophosphamide efficacy paclitaxel5fluoruracilincreases gut permeability as indicated by5fold elevation in circulating lpsbindingprotein and systemic inflammation reduces abundance of roseburiaporphyromonadaceae and akkermanisamuciniphila [ ]reduces clostridium spp and increasesmembers of proteobacteria mainlyenterobacteriaceae damages mucosal barrierchemotherapyinducedperipheral neuropathicpain cipn cdi [ ]mucositis along the entiregastrointestinal tract cdi [ ]cyclophosphamidecdi triggers disruption of gut barrier by alteringbacterial compositiongrampositive bacteria such asenterococcus hirae lactobacillus johnsoniiand l murinus translocate from gut intomesenteric lymph nodes and spleen this enhances immune responses by theproduction of interferon gamma ifnÎ andactivation of th17 cellsdraining lymph nodes and promotion of maturation oftheintratumoral dendritic cells dcscombination treatment of bacteroidesandburkholderia cepacia prevented intestinal damage andrefractory colitisin additionfragilisfecal microbiota analysis of melanoma patients beforeand after ipilimumab treatment showed a change in therelative proportions ofenterotypeclusters cluster a was dominated by prevotella spwhereas clusters b and c by different bacteroides sppfecal microbiota transplantation fmt from patientsinto tumorbearing germfree mice showed that onlyfecal material from cluster c resulted in colonizationthree dominantwith bacteroides thetaiotaomicron or bacteroides fragilisand enhanced ipilimumab response in another study ofipilimumab in mice vancomycin treatment resulted in amore severe manifestation of colitis whereas oraladministration of bifidobacterium ameliorated the sideeffects similarly melanoma patients with increasedabundance of bacteroidaceae rikenellaceae and barnesiellaceae members responded better to ctla4 antibodies however a different study in ipilimumabtreated melanoma patients found that bacteroides spp were associatedwith decreased response whereas faecalibacterium andother firmicutes members improved clinical outcome 0cdutta and lim biomarker research page of fig potential antitumor immune mechanisms induced by intestinal dysbiosis a in the presence of intact mucosal barrier and signals fromcommensal microbiota effector t cell activation is modulated by t cell receptor tcr ligation with major histocompatibility complex mhc classi and costimulation of cd80cd86 and cd28 binding of cytotoxic t lymphocyteassociated antigen ctla4 receptor to antictla4 antibodyon treg impairs its effector tcell inhibitory function it also downregulates ctla4 expression on apc ligation of repressive receptorprogrammed death receptor pd1 and its ligand pdl1 to antipd1 and antipdl1 antibodies respectively activate effector tcell proliferationand function activated effector t cells interact with tumor cells and release cytokines to induce tumor cell death b signals from unfavorablemicrobes due to dysbiosis upregulates ctla4 pd1 and pdl1 expression to inhibit tcell activation ctla4 on treg binds to cd80cd86 onantigen presenting cell apc cd80cd86 on apc also disengages from cd28 and binds to ctla4 on effector t cells pdl1 the ligand of pd1is expressed on antigen presenting cell apc and tumor cells pd1 on effector t cells ligates to pdl1 on apc and tumor cells these activitiesinhibit effector tcell activation reduces immune checkpoint inhibitor ici efficacy and causes tumor escape 0cdutta and lim biomarker research page of patients with higher abundance of faecalibacteriumand improved response to ctla4 antibodies showedhigher incidence of enterocolitis and lower level of treg inperipheral blood thus the beneficial effects of specificand isolated gut microbes may depend on the commensalassociation with other microbial species and may differ between humans and micepd1 blockade may also be modulated by intestinalmicrobiota melanoma patients who responded to pd1blockade had increased abundance of enterococcus faeciumcollinsella aerofaciens bifidobacterium adolescentis klebsiella pneumoniae veillonella parvula parabacteroidesmerdae lactobacillus sp and bifidobacterium longumwhereas in nonresponders the intestinal microbiome wasenriched in ruminococcus obeum and roseburia intestinalis another study found higher abundance of faecalibacterium species in responders and enrichment with bacteroides thetaiotaomicron escherichia coli and anaerotruncuscolihominis in nonresponders clinically nonsmallcell lung cancer nsclc and renal cell carcinoma rccpatients experienced increased resistance to pd1 blockadeafter antibiotic treatment these patients had shorterprogressionfree survival as well as overall survival in thisstudy response to pd1 blockade correlated with higherfecal abundance of akkermansia muciniphila fmt fromresponders to germfree or antibiotictreated mice improved the outcome of pd1 blockade administration ofa muciniphila after fmt from nonresponders restoredresponsesimilarly intestinal microbiota may influence the outcome of chimeric antigen receptor t cell car t therapypatients with complete response to cd19 car ttherapyexhibited enrichment of oscillospiraceae ruminococcacaeae and lachnospiraceae in their intestinal microbiomewhereas patients who did not attain a complete responseshowed increased abundance of peptostreptococcaceae effectiveallogenic hematopoietic stem cell transplantationalthough allohsct issomein treatinghematological malignancies the immunosuppressive agentsbroadspectrum antibiotics and chemoradiation used withthe transplant often induce intestinal dysbiosis gut permeability and impaired systemic immune response highermicrobiota diversity is associated with longterm survivaland lower diversity in gut microflora is associated with reduced overall survival and higher transplantrelated mortalityfollowing allohsct [ ] severe infections that occurdue to intestinal dysbiosis such as cdi and vancomycinresistant enterococci vre infections are also associatedwith higher treatmentrelated mortality [“] allohsctdisrupts the equilibrium of bacterial composition in feceswith a dominance of enterococcus streptococcus and proteobacteria members [ ] and reduces beneficial bacteriasuch as faecalibacterium and ruminococcus higherabundance of blautia was found to be associated with improved overall survival moreover allohsct patientswith reduced risk of relapse had an enrichment of eubacterium limosum ofgraftversushost diseaseone of the major complications of allohsct is thedevelopmentgvhdoccurrence of cdi during allohsct increases the riskof gvhd besides the loss of overall microbial diversityreduction in beneficial faecalibacterium blautia lactobacillus and ruminococcus and increased abundance ofenterococcus and clostridiales was observed in gvhd[ “] patients without gvhd had increasedabundance of parabacteroides and bacteroides in theirpretransplant feces in a preclinical study reducedgvhd and improved overall survival was observed afterthe administration of the probiotics lactobacillus rhamnosus gg alone or in combination with ciprofloxacindue to the preservation of gut mucosal integrity in therecipient mice restoration of normalintestinalmicrobiome by fmt has been found to benefit patientswith steroidrefractory gvhd [ ] multipleclinical trials are currently ongoing to investigate howmanipulation of gut microbiota using dietary intervention and fmt might reduce the risk of gvhdmanipulation of intestinal microbiome and barrierto improve outcome of cancer therapeuticsifintestinal dysbiosis and its associated increased gutpermeability are associated with cancer development andtherapyrelated complications and treatment outcomes itfollows that intervention of the intestinal microbiomeandor gut barrier may alter cancer outcome in thissection we will explore three broad approaches fig that might be investigated nonselective modificationof intestinal microbiome using fmt semiselectivemodification of intestinal microbiome using antibioticsand biologic modification of intestinal barrier we willdiscuss the challenges and obstacles each of the approaches may encounternonselective modification of intestinal microbiome usingfmtmodification of the intestinal microbiome is theoreticallybest accomplished by fmt unmanipulated fmt will notonly replete the dysbiotic intesti

introduction postoperative ileus poi a common complication after surgery severely affects postoperative recovery it is unclear whether pretreatment with transcutaneous electrical acupoint stimulation teas can improve recovery from poi this trial will evaluate the effects of pretreatment with teas on poimethods and analysis this will be a prospective randomised controlled trial american society of anesthesiologists asa physical status classification i“iii level patients aged “ years and scheduled for laparoscopic colon surgery will be included in the study it is planned that subjects will be randomised to the teas and sham teas steas groups the groups will undergo two sessions of teassteas daily for days before surgery with a final teassteas treatment min before anaesthesia the primary endpoint of the study will be time to first defaecation secondary endpoints will include time to first flatus time to tolerance of oral diet gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to independent walking length of hospital stay postoperative pain visual analogue scale score on the first days after surgery analgesic requirements complications and plasma concentrations of interferon ifn ifnÎ interleukin6 il6 and il1 multiple linear regression will be used to identify independent predictors of outcome measuresethics and dissemination this study has been approved by the chinese registered clinical trial ethics review committee no chiecrct20170084 the results of the trial will be published in an international peer reviewed trial registration number this study has been registered with the chinese clinical trial registry no chictr inr17013184trial status the study was in the recruitment phase at the time of manuscript submissionintroductionpostoperative dysfunction ileus poi of gastrointestinal is a transient gi strengths and limitations of this study –º this study aims to evaluate whether pretreatment with transcutaneous electrical acupoint stimulation teas can prevent postoperative ileus poi –º teas is a safe non invasive and easily accepted adjunctive intervention –º this study will provide deeper insights into the mechanism by which teas pretreatment reduces the inflammatory response –º this is a single centre study which is a potential limitationpropulsion that often occurs after abdominal surgery and may also occur after surgery at other sites1 the main symptoms of poi include abdominal pain and distention nausea vomiting difficult defaecation and intolerance to solid food poi is usually temporary but if prolonged may lead to surgical incision dehiscence intestinal anastomotic fistula abdominal cavity infection intestinal ischaemia aspiration pneumonia and other serious complications2“ a retrospective cohort study involving nearly hospitals in the usa showed that poi is a key reason for prolonged hospitalisation and increased medical costs for patients undergoing abdominal surgery1 the usa spends more than billion treating poi every year5 at present the most common methods used to treat poi include rational perioperative use of narcotic drugs and opioids eating as soon as possible after surgery avoidance of nasogastric tubes after the operation early ambulation postoperative epidural analgesia restriction of fluid intake the use wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access of minimally invasive surgery such as laparoscopic drug therapy and the use of chewing gum despite the numerous treatment strategies poi remains a difficult clinical challenge that compromises the rapid recovery of postoperative patients it is therefore necessary to find more effective convenient and economical treatment methods6“the main mechanism underlying poi may be activation of macrophages in the external muscular layer during the surgical procedure11 intestinal manipulation during surgery can activate macrophages in the outer muscle layer of the small intestine leading to release of inflammatory factors interleukin6 il6 il1 and the chemokine mip1α together with increased expression of the adhesion molecule icam1 on endothelial cells and induction of neutrophils and monocytes in the circulation into the small intestine muscle layer these cells and activated macrophages can release a large amount of inducible nitric oxide synthase and prostaglandin which inhibit the movement and contraction of the gi tract12 transport of these inflammatory mediators in the bloodstream causes activation of macrophages in the distal gi tract leading to poi over the entire intestinal tract14 it has been confirmed by a large number of animal experiments that reducing the inflammatory response is an effective way to treat poi15“there is a long history in traditional chinese medicine tcm of using acupuncture to treat functional gi diseases and in recent years there has been significant global interest in the beneficial effects of acupuncture on poi the positive effect of electroacupuncture ea on poi has been clearly demonstrated ng used ea to treat poi in patients undergoing laparoscopic colon surgery18 defaecation time and length of hospital stay were significantly shortened in patients who received ea compared with those who did not receive the treatment in patients undergoing hepatic resection you found a significant reduction in the incidence of poi in patients treated with a combination of acupuncture and chinese herbal medicine the length of hospitalisation was also significantly shortened in the treated group ± days vs ± days p001419in the previous studies we proved that pretreatment with acupuncture could reduce excessive activation of the innate immune system and inhibit the inflammatory response this effect may be achieved by activation of the vagal nervous system20 other studies have shown that transcutaneous electrical acupoint stimulation teas and ea have similar effects in the treatment of pain and alleviating the inflammatory response22 tcm holds that the best treatment for disease is prevention based on all of the above studies we hypothesise that the use of teas as a preoperative treatment may reduce the incidence of poi there have so far not been any studies that address this questionwe have therefore designed a randomised controlled trial to investigate whether pretreatment with teas can reduce the incidence of poi in patients undergoing laparoscopic colon resection the study is also designed to verify that the anti inflammatory effect is associated with the immunomodulatory function of teasmethods and analysisstudy objectivethe primary objective is to assess the effect of teas on clinical recovery of bowel function after laparoscopic colon surgery the secondary objective is to verify that suppression of overactivation of the innate immune system and reduction of the inflammatory response are the mechanisms underlying the ability of pretreatment of percutaneous acupuncture to prevent poistudy locationa prospective single centre double blinded randomised controlled trial will be conducted at shuguang hospital which is affiliated to the shanghai university of traditional chinese medicine chinastudy populationparticipants will be recruited according to the inclusion and exclusion criteriainclusion criteria male and female patients aged “ years patients undergoing elective laparoscopic colonic surgery and upper rectal resection such as left collect right colectomy and anterior resection of the upper part of the rectum and lower part of the sigmoid body mass index “ kgm2 asa classification i“iii patients provide signed informed consent the consent form can be viewed in online supplementary appendix exclusion criteria middle and lower rectal resection totalproctocolectomy or the need for complex endoscopic surgery need for abdominal wall fistula gi fistula fistula surgery or stoma creation history of abdominalpelvic operations or complications patients receiving epidural anaesthesia or epidural analgesia patients with skin infections surgical incision or scar at the point of application of acupuncture patients have a history of limb surgery spinal surgery or nerve injury patients who participated in other clinical trials or received other acupuncture therapy in the previous weeks patients with cardiac pacemakers patients have one of the following conditions before surgery chronic pain drug addiction or alcohol dependence patients with preoperative combination of severe central nervous system disease and severe mental illnesswang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cendpointsprimary endpointfirst defaecation time h that is time to first anal defaecation after laparoscopic surgerysecondary endpointstime to first flatus h time to tolerance of solid oral diet h gi2 composite outcome of time to first defaecation and time to tolerance of oral diet time to walk independently h length of hospital stay defined as the number of days from operation to discharge d criteria for hospital discharge include stability of vital signs with no fever achievement of flatus or defaecation ability to tolerate solid food without vomiting control of postoperative pain absence of other postoperative complications and ability to function at home independently or with home care provided pain will be assessed using the visual analogue scale vas on postoperative days and scale of to where represents complete absence of pain and represents the worst pain intensity postoperative requirements for analgesia will also be assessed inflammatory mediators interferon ifn ifnÎ interleukin6 il6 and il1 in blood will be measured before teassteas intervention and on days and after the operation postoperative complications will be recorded using the clavien dindo classification for complication assessment24 the follow up period will be at least monthswe add gi2 as a secondary outcome to the original protocol after recruitment of the study had already begun gi2 is a time indicator which will be calculated from two existing outcomes time to first defaecation and time to tolerance of oral diet there will be no harm to subjects no additional cost and no more workopen accessrandomisation and blindingpatients will be randomised to receive either teas or steas by stratified randomisation according to sex in a ratio figure using a computer generated random sequence a sealed envelope will be opened to determine to which group the patient has been assigned the acupuncturist will be aware of the treatment group patients as well as the outcome investigator nurse anaesthetist will be blinded to the treatment allocationcurrent sample size justificationaccording to wang jian and song jiangang™s preliminary study of teas pretreatment for prevention of poi in patients undergoing laparoscopic colon surgery in shuguang hospital the mean time to first defaecation following laparoscopic colon surgery was ± hours m±sd working on the assumption that a clinically meaningful difference in mean time to first defaecation between the teas and steas groups is day or hours patients would be needed in each group to reach a power of and a type i error rate if the dropout rate is a total sample size of patients for the two groups is needed for this studystatistical analysisdata for continuous variables ie first defaecation time first passage of flatus time to tolerance of oral diet time to walking independently length of hospital stay will be reported using the mean and sd m±sd for normally distributed data or median range for skewed data data for categorical variables will be expressed as a number percentage intergroup differences will be assessed using the student™s t test or mann whitney u test intergroup differences in inflammatory mediators at time points of pre teassteas treatment and on figure flowchart of the study protocolwang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access figure acupoints selected in this trial a hegu il4 and neiguan p6 b zusanli st36 and shangjuxu st37 c han™s acupoint nerve stimulatorpostoperative days and were assessed by two way repeated measures analysis of variance with bonferroni post hoc test the significance level will be set at all data will be analysed using spss v170 software or other appropriate statistical software packagespretreatmentpatients randomised to the teas and steas groups will undergo two treatment sessions daily for three consecutive days before surgery the patients will then be treated for a final time min before anaesthesiafor patients in the teas group the zusanli st36 shangjuxu st37 hegu li4 and neiguan p6 acupoints will be identified before electrical stimulation with surface electrodes figure selection of these acupoints is based on a consensus between the acupuncturists carrying out the study the acupuncturist will stimulate these acupoints using a han™s acupoint nerve stimulator hans200a nanjing jisheng medical technology nanjing china at a frequency of hz the intensity will be adjusted for each individual to maintain a slight twitching of the regional muscle and achieve de qi sensations such as soreness numbness distention and heaviness the steas group will receive a strong but comfortable current for s and the current will then gradually vanish over the next s25 the participants of both groups will be told that they are receiving current stimulation each session of acupoints treatment will last for min during the application of teas patients will be required not to change the current settings themselves a prompt beep at the end of teas will indicate the end of treatmentall surgery will be carried out under general anaesthesia using standardised anaesthetic procedures patients will be fasted for hours before surgery right upper extremity venous access will be established before the patients entering the operating theatre ringer™s lactate solution mlkg will be administered by intravenous infusion for compensatory expansion before induction of anaesthesia patients will then receive midazolam mgkg fentanyl µgkg vecuronium bromide mgkg and propofol “ mgkg intravenously for induction of anaesthesia anaesthesia will be maintained using a cp600 anaesthesia delivery system slgo medical technology beijing china the dose wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0cof propofol will be adjusted to maintain the bispectral index in the range of “ after surgery all patients will remain in the post anaesthesia care unit and then return to the ward for recovery until dischargethe perioperative management of all patients will be standardised early ambulation will be encouraged and oral feeding will be resumed as early as possible all patients will be followed up for at least months after discharge from the hospitaladverse eventsall adverse reactions will be closely monitored through spontaneous reports by patients or direct observation by clinicians or by asking the patients about adverse events using open questions all adverse reactions will be recorded and appropriate treatment will be provided if necessary serious adverse events will be reported to the ethics committeedata collection and managementdemographic variables and clinical data will be collected from all patients during the study blood pressure heart rate and oxygen saturation will also be monitored any adverse events will be recorded data will be collected throughout the study and will be securely managed under conditions of confidentiality data collection will be performed by a nurse anaesthetist the participants will be referred to by their participant number rather than by their name throughout the study unless otherwise specified all relevant documents and files will be archived for years the data will be accessible only by investigators who sign the confidential disclosure agreement and by institutional or governmental auditors during the study data without patient identifiers will be publicly accessible after the study data collection and management will be monitored by the institutional ethics committee for clinical research of shuguang hospitalpatient and public involvementthis study is currently in the recruitment phase patients andor the public were not involved in study design or conduct of the study the participants will be able to access the study results through social mediadiscussionpoi continues to represent an important cause of morbidity after colon surgery the prevention of poi is thus of great importance in reducing perioperative complications and reducing hospitalisation costs although it has been shown that ea can shorten the duration of poi18 the effectiveness of teas which is a similar technique in preventing poi has not been investigated it is therefore important to assess the effectiveness of teas in preventing poi through a clinical studythis study has several strengths first the intervention strategy of the protocol will be pretreatment with teas previous studies have shown that pretreatment open accesshas a prophylactic effect for example pretreatment with teas has been shown to improve pain treatment26 and to improve resuscitation after anaesthesia with reduction of postoperative nausea and vomiting28 it is however unclear whether preoperative teas can prevent poi studies suggest that early preoperative intervention may be more beneficial in regulating physiological functions and preventing poi29 in an extension to these findings the present study will help to determine whether teas pretreatment could improvement poisecond the effectiveness of teas will be evaluated by assessing clinical function and by serological examination in this randomised controlled trial of patients undergoing laparoscopic colorectal surgery our aim is to assess the effects of preoperative teas on poi using relevant clinical parameters associated with bowel function these include time to first defaecation time to first flatus time to tolerance of oral diet and gi2 importantly we will also measure serum concentrations of inflammatory mediators associated with poi such as ifn ifnÎ il6 and il1 our findings may thus provide deeper insights into the mechanisms by which teas improves poithere are also limitations to this protocol various clinical indicators have been used in studies for the diagnosis of poi but there is no consensus on which clinical parameter is the best for assessment of gi transit9 two indicators that are widely used to assess bowel movement will be used in this study time to first defaecation will be the primary outcome and time to first flatus will be one of the secondary outcomes there is a possibility that we may observe conflicting results ie significant improvement in time to flatus but not defaecation because flatus can vary considerably between patients clinical trials support the time to tolerance of oral diet and gi2 defined as the later of the following two events time to first tolerance of solid food and time to first bowel movement as supplementary secondary outcomes to measure the recovery time of gi function and these will be used in this study32 other limitations of these indicators are that they require objective measurement of motility and are time consuming to measure34 recently this situation has been improved by the use of in vivo monitoring techniques to assess the function of gi movements innovative devices such as sitz markers have been used to evaluate postoperative recovery of small bowel movement by counting the number of sitz markers that did not pass through the ileocecal valve but remained in the small intestine using radiography36 the smartpill is a swallowable device that record parameters within the gi tract indicators such as ph temperature and intracavitary pressure can be collected to analyse gi transit times in vivo37 these devices acquire objective parameters to evaluate bowel movement and could save time research into the satisfaction of both doctors and patients with these device needs to be carried out furthermore this study is a single centre trial and because the therapeutic effect of teas may be affected by ethnicity and region it will wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0copen access be necessary to conduct multicentre and large sample studies in the futurenotwithstanding its limitations this study can clearly indicate the overall effects of teas on postoperative recovery we hypothesise that pretreatment with teas could improve recovery of gi function in patients undergoing laparoscopic surgery if this study provides positive results it will be possible to recommend this pretreatment strategy for patients undergoing abdominal surgery relevant cost effectiveness studies are also worthy of considerationauthor affiliations1anesthesiology shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai china2anesthesiology wenzhou medical university the sixth affiliated hospital lishui china3research institute of acupuncture anesthesia shuguang hospital affiliated to shanghai university of traditional chinese medicine shanghai chinaacknowledgements we thank dr stanley tao from shanghai ruihui biotech for his valuable assistance in the statistical design of this studycontributors jw conceived the study dl wt jg and gf participated in its design and coordination wc yy ws and jg collected references and developed the protocol gy and ly will perform statistical analyses rf will follow up with patients and record data jw lf and js drafted the manuscript all authors have read and approved the final manuscriptfunding the present study is supported by the project of the national natural science foundation of china nos and and the commercial sponsorship of sinch pharmaceuticals techcompeting interests none declaredpatient consent for publication obtainedprovenance and peer review not commissioned externally peer reviewedopen access this is an open access article distributed in accordance with the creative commons attribution non commercial cc by nc license which permits others to distribute remix adapt build upon this work non commercially and license their derivative works on different terms provided the original work is properly cited appropriate credit is given any changes made indicated and the use is non commercial see a0http creativecommons org licenses by nc orcid idlihua a0fan http orcid org references iyer s saunders wb stemkowski s economic burden of postoperative ileus associated with colectomy in the united states j manag care pharm “ boelens pg heesakkers ffbm luyer mdp et a0al reduction of postoperative ileus by early enteral nutrition in patients undergoing major rectal surgery prospective randomized controlled trial ann surg “ melis m fichera a ferguson mk bowel necrosis associated with early jejunal tube feeding a complication of postoperative enteral nutrition arch surg “ moghadamyeghaneh z hwang gs hanna mh et a0al risk factors for prolonged ileus following colon surgery surg endosc “ goldstein jl matuszewski ka delaney cp et a0al inpatient economic burden of postoperative ileus associated with abdominal surgery in the united states p and t “ bragg d el sharkawy am psaltis e et a0al postoperative ileus recent developments in pathophysiology and management clin nutr “ wolthuis am bislenghi g fieuws s et a0al incidence of prolonged postoperative ileus after colorectal surgery a systematic review and meta analysis colorectal dis 201618o1“ nguyen dl maithel s nguyen et et a0al does alvimopan enhance return of bowel function in laparoscopic gastrointestinal surgery a meta analysis ann gastroenterol “studies and clinical aspects of gadolinium salts and chelates cardiovasc drug rev “ koscielny a kalff jc t helper cell type memory cells and postoperative ileus in the entire gut curr opin gastroenterol “ mikkelsen hb thuneberg l opop mice defective in production of functional colony stimulating factor1 lack macrophages in muscularis externa of the small intestine cell tissue res “ van bree shw nemethova a cailotto c et a0al new therapeutic strategies for postoperative ileus nat rev gastroenterol hepatol “ hilton wm lotan y parekh dj et a0al alvimopan for prevention of postoperative paralytic ileus in radical cystectomy patients a cost effectiveness analysis bju int “ wehner s behrendt ff lyutenski bn et a0al inhibition of macrophage function prevents intestinal inflammation and postoperative ileus in rodents gut “ wehner s straesser s vilz to et a0al inhibition of p38 mitogen activated protein kinase pathway as prophylaxis of postoperative ileus in mice gastroenterology “ schwarz nt kalff jc türler a et a0al prostanoid production via cox2 as a causative mechanism of rodent postoperative ileus gastroenterology “ engel dr koscielny a wehner s et a0al t helper type memory cells disseminate postoperative ileus over the entire intestinal tract nat med “ adding lc bannenberg gl gustafsson le basic experimental ng ssm leung ww mak twc et a0al electroacupuncture reduces duration of postoperative ileus after laparoscopic surgery for colorectal cancer gastroenterology “ you x m mo x s ma l et a0al randomized clinical trial comparing efficacy of simo decoction and acupuncture or chewing gum alone on postoperative ileus in patients with hepatocellular carcinoma after hepatectomy medicine 201594e1968 song jg li hh cao yf et a0al electroacupuncture improves survival in rats with lethal endotoxemia via the autonomic nervous system anesthesiology “ zhang j yong y li x et a0al vagal modulation of high mobility group box1 protein mediates electroacupuncture induced cardioprotection in ischemia reperfusion injury sci rep balogun ja biasci s han l the effects of acupuncture electroneedling and transcutaneous electrical stimulation therapies on peripheral haemodynamic functioning disabil rehabil “ jiang y wang h liu z et a0al manipulation of and sustained effects on the human brain induced by different modalities of acupuncture an fmri study plos one 20138e66815 dindo d demartines n clavien p a classification of surgical complications a new proposal with evaluation in a cohort of patients and results of a survey ann surg “ rakel b cooper n adams hj et a0al a new transient sham tens device allows for investigator blinding while delivering a true placebo treatment j pain “ huang l pan y chen s et a0al prevention of propofol injection related pain using pretreatment transcutaneous electrical acupoint stimulation turk j med sci “ zhang q gao z wang h et a0al the effect of pre treatment with transcutaneous electrical acupoint stimulation on the quality of recovery after ambulatory breast surgery a prospective randomised controlled trial anaesthesia “ zheng lh sun h wang gn et a0al effect of transcutaneous electrical acupoint stimulation on nausea and vomiting induced by patient controlled intravenous analgesia with tramadol chin j integr med “ stakenborg n labeeuw e gomez pinilla pj et a0al preoperative administration of the ht4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons gut “ vather r trivedi s bissett i defining postoperative ileus results of a systematic review and global survey j gastrointest surg “ wu z boersema gsa dereci a et a0al clinical endpoint early detection and differential diagnosis of postoperative ileus a systematic review of the literature eur surg res “ deng g wong wd guillem j et a0al a phase ii randomized controlled trial of acupuncture for reduction of postcolectomy ileus ann surg oncol “ van bree shw bemelman wa hollmann mw et a0al identification of clinical outcome measures for recovery of gastrointestinal motility in postoperative ileus ann surg “wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c maffezzini m campodonico f canepa g et a0al current perioperative management of radical cystectomy with intestinal urinary reconstruction for muscle invasive bladder cancer and reduction of the incidence of postoperative ileus surg oncol “ bungard tj kale pradhan pb prokinetic agents for the treatment of postoperative ileus in adults a review of the literature pharmacotherapy “open access chae h d kwak m a kim i h effect of acupuncture on reducing duration of postoperative ileus after gastrectomy in patients with gastric cancer a pilot study using sitz marker j altern complement med “ vilz to pantelis d lingohr p et a0al smartpill® as an objective parameter for determination of severity and duration of postoperative ileus study protocol of a prospective two arm open label trial the pidusa study bmj open 20166e011014wang a0j et a0al bmj open 202010e030694 101136bmjopen2019030694 0c'

"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a

" while the impact of family caregiving has been welldocumented many of such studies center oninvestigating external factors such as socioeconomic status accessibility to resources and availability of socialsupport as the primary causation of caregiver wellbeing outcomes this paper explores the motivations that drivefamily caregivers in supporting their family members at the endoflife and critically examines how internalappraisal processes of such motivations can both positively and negatively impact their wellbeingmethods this study adopted an interpretative phenomenological analysis ipa to investigate the motivations andinternal appraisal processes of asian family caregivers in singapore who were tending to a dying family memberqualitative dyadic interview data n was drawn from a larger randomized controlled trial for a novel familydignity intervention fdi for palliative care patients and their families the sampling population consisted ofparticipants aged and above who were identified to be the primary caregivers of older palliative care patientswith a prognosis of less than months data collection was conducted in the homes of patients and familycaregiverscontinued on next page correspondence andyhyhontuedusg1psychology programme school of social sciences nanyang technologicaluniversity singapore singapore2centre for population health sciences lee kong chian school of medicinenanyang technological university singapore singaporefull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0ctanho bmc palliative care page of continued from previous pageresults findings revealed six themes that could either nurture or diminish caregiver wellbeing honoringfidelity caregivers were motivated to commit to their caregiving roles in order to avoid regret alleviatingsuffering caregivers were motivated to relieve their family member™s pain enduring attachment caregiverswere motivated to spend time together with their family member preserving gratitude caregivers weremotivated to express their appreciation to their family member by caregiving navigating change caregiverswere motivated to adapt accordingly to changes in the illness trajectory and reconciling with mortalitycaregivers were motivated to respond accordingly to their family member™s prognosis the final theme of thewellbeing determinant is posited as an indication of selfdetermination and is conjectured to influence howcaregiving motivations are appraised by the caregiver fulfilling and enhancing one™s sense of selfdetermination appears central to infusing one™s caregivingmotivations with positive meaning and consequently nurturing one™s wellbeing in the endoflife caregivingjourney these findings are discussed with recommendations for healthcare professionals working with familycaregivers of palliative care patientskeywords palliative care caregiver motivations wellbeing meaning burnout resilience selfdetermination endoflife qualitative research the experience of an endoflife eol family caregivercan be likened to a paradox “ what could evoke a senseof pleasure appreciation and gratitude could also bringabout feelings of anxiety distress and pain while somehave related the caregiving journey to the metaphor ofascending a mountain the expedition of an eol family caregiver usually spans beyond merely a couple ofdays weeks or months they must navigate the peaks ofdiagnosis to prognosis and eventually death and bereavement in what often unfolds into a lifelong climbthe role of the eol family caregiver is often multifaceted and interminable daily duties involve managingmedical regimes traversing the healthcare system andtaking charge of other dependents alongside providingphysical mental and emotional support throughout theillness trajectory [ ] many family caregivers are rarelyequipped with formal or adequate training and nor dothey possess sufficient resources and skills before theyfind themselves embroiled in eol caregiving responsibilities family caregivers must also process and manage amultitude of thoughts and emotions as they come toterms with the changes and sometimes losses in theirpersonal lives this complex experience is not limited to a minorityof people despite strong global advancements in medical technology and healthcare systems older populations remain highly susceptible to chronic and terminalmorbidities that are incapacitating in the unitedstates alone over million caregivers tend to their ailing family members annually while an estimated of patients in europe requiring longterm care areattended to by informal caregivers with the anticipated number of older adults in the world soaring to billion by the year there will certainly be a surging demand for family caregivers to relieve the ensuingresource strains on healthcare settingsthe impact of family caregiving stressors has beenwelldocumented [“] with various studies exploringcaregiver burnout and its effects on the community andsociety complementing these studies are literature thatreveal characteristics of resilience and transformationalgrowth displayed by family caregivers in adversity [“] the common thread that impacts both caregiverburnout and resilience appears to be a lack of or adequate coping a process that requires the individual toconstantly change efforts in both thoughts and behaviorsin order to manage internal or external demands thatare considered stressful despite this indication thatone™s psychological resources are key to maintainingone™s wellbeing many studies often consider externalfactors such as socioeconomic status accessibility toresources and availability of social support as the primary causation for the degree of caregiver wellbeingthus these studies often recommend pragmatic interventions that focus on improving external circumstancesaccordinglyperception emotion and motivation of the familycaregiverwhile it is undoubtedly beneficial to mitigate tangiblestressors one must not lose sight of the magnitude of aperson™sthedemands of caregiving this perception and appraisalembody one™s subjective caregiver burden “ observed inreviews of over caregiver studies to pose deepseatedimplications on caregivers™ quality oflevels ofdepression and anxiety and stress coping [ ]internal perception and appraisal oflife 0ctanho bmc palliative care page of campbell later confirmed this observation in astudy that evaluated multiple variations in the caregiverexperience subjective caregiver burden was repeatedlyidentified as the primary indicator for caregiver stressit was around the same time that folkman andmoskowitz discovered that caregivers experiencepositive emotions alongside negative emotions duringstressful events they put forth the tenet of meaningfocused coping in which caregivers derive mental andemotional sustenance thus effecting positive emotionsin challenging circumstances by deferring to their beliefsvalues and existential goals folkman and moskowitzfound that meaningfocused coping is an intrapsychicprocess of discovering benefits in caregiving reminding oneself of such benefits setting goals thatinspire a sense of mastery and competence realigningpriorities in view of changes and infusing ordinaryevents with positive meaning folkman furtherattested that meaningfocused coping exists alongsidenegative appraisals in a caregiver™s stress process inorderand psychologicalresources during stressful eventsto reinstate physiologicalgiven the importance of psychological appraisal it isreasonable to postulate that gaining insight into a caregiver™s beliefs values and goals that stimulate them incaregiving defined as motivations in this paper wouldyield valuable information that could be used to enhancemeaningfocused coping such interventions would target the essence of a person™s selfconcept “ that is theindividual™s perception of who they are and what theybelieve in “ and transcend current cursory social mentaland emotionalfor caregiverstresssymptom management[ ] a studybridging the research gap in asian caregivingwhile the multidimensional nature of caregiving burdenand coping is not confined to any specific culture thereare distinctive elements that bear influence on the asiancaregiving experience family takes precedence in asiansocieties with strongly inculcated values and expectations of filial piety and filial responsibility placed uponfamily membersconducted insingapore a multiethnic society that is predominantlychinese found that family caregivers who internalizedand prioritized societal expectations as motivations overtheir personal wellbeing most often faced internalconflict and were highly likely to experience difficultiesin maintaining their mental health familial ties and caregiving duties this is corroborated by the evolvingattitudes towards such confucian rules “ younger asiangenerations no longer perceive absolute submission orcomplete obedience to the family as instrumental valuesin a modern and globalized society and it would bevaluable to further understand how these complexitiesmight manifest in a family caregiver™s motivationsthis paper aims to contribute to and grow the currentbody of knowledge for asian eol family caregivers byanswering the following research questions what arethe internalized motivations defined here as unconone™ssciously assimilated beliefs and valuesattitudes or behaviors of asian family caregivers how might these motivations affect the way they respond to caregiving and impact their wellbeing howcan an understanding of these motivations be integratedinto psychosocial interventions to enhance and sustaincaregiver wellbeingintomethodsresearch design and proceduresthe current study draws qualitative dyadic interviewdata n from a larger randomized controlled trialfor a novel family dignity intervention fdi for asianpalliative care patients and their families n thesampling methods inclusion criteria interview procedure and study protocol for the fdi are comprehensivelydescribed by ho briefly the fdi is developedbased on an integrative body of empirical investigationthat focuses on dignified endoflife care in both western and asian contexts [ ] it integrates elements oflogotherapy and narrative life review to provide psychosociospiritual support to patients and families facingmortality and has been piloted for acceptability andfeasibility before being fully adapted into the intervention study in practice fdi comprises a recorded dyadicsemistructured interview with a patient and a familycaregiver conducted in their homes the fdi therapistuses a guided question framework to facilitate joint conversation on shared memories and living wisdoms thatlead to meaningmaking and the expression of appreciation and reconciliation this is done with the ultimategoal of creating a legacy document that tells the life storyof the patient and is bestowed to the rest of the familythrough an open reading each interview lasted between and min and was conducted in english malaymandarin or a chinese dialect hokkien teochew orcantonese these recorded interviews were transcribedverbatim translated into english by a native languagespeaker where applicable and edited into legacy documents transcripts and legacy documents were reviewedand finalized by patients and caregivers to ensure accuracy and authenticitysamplingthe sample drawn for this study consisted of primaryfamily caregivers of older palliative care patients aged and above with mainly a cancer prognosis of lessthan months eleven were spousal caregivers seven 0ctanho bmc palliative care page of were adultchildren caregivers and two were siblingcaregivers the majority were female aged between to with a mean age of years see table for caregiver demographics they were recruited through theinpatient daycare and homecare hospice service unitsof hca hospice care dover park hospice tan tockseng hospital singapore cancer society and methodistwelfare services the inclusion criteria required familycaregivers to be above years old and identified by thepatient to be their primary carer patients and familycaregivers came from varioussocioeconomic s and were predominantly of chinese ethnicityas the fdi focused on patient narratives transcriptsbearing a moderate to sizeable amount of input from thefamily caregiver were selected for data analysisdata analysisstudy adopted an interpretative phenomenothislogical analysis ipato investigate the internalizedmotivations of family caregivers in tending to a dyingfamily member the ipa is an approach in qualitativeresearch that aims to provide insights into how an individual makes meaning out of a phenomenon itis important to note that as the current study drawsqualitative data from the larger fdi study no explicitinternalized caregiver motivationsquestionsaboutwere asked reflections on motivations towards caregiving occurred anically throughout the interviewtranscripts and were identified by the researcher usingipa through a process of data reduction and datareconstructionfirst authors and screened all transcripts andselected those that had adequate inputfrom familycaregivers to be used for analysis authors and thenconducted linebyline coding to develop descriptivethemes and analytical categories conceptualizing newinterpretation of the data this was followed by regularmeetings among all authors for the further refinement ofthemes and categories to encapsulate the meaning andcontent within the cluster of similar codes with theemergent themes and subthemes created via a summarychart all authors reviewed and defined the emergentthemes once consensus was reached operational definitions were created finally relationships between categoriesthemes and subthemes were proposed andmapped with supporting quotes from transcripts to address issues of trustworthiness and credibility emergentthemes were constantly compared and contrasted withinand across groups during regular meetings the finaltheme categorization and definitions were agreed uponby the entire research team and data saturation and investigator triangulation were achieved table demographics of family caregiversidentifierdph14caregiver relationshipchildcaregiver ethnicitychinesepatient™s diagnosislung cadph19dph34dph42dph53dph59dph68hca12hca68hca75hca81hca87hca109hca114hca116hca117mws004scs18ttsh61ttsh65spousespousesiblingspousechildspousespousechildchildchildspousespousespousespousespousesiblingspousechildchildchinesechinesechinesechinesechinesemalayeurasianchinesemalaychinesemalaychinesechinesechinesechinesechinesechinesemalaychineseprostate calung casigmoid calung cagynaecological malignancylung caprostate cacolon cabreast caendometrial carenal caendometrial cabrain canasopharyngeal capancreas cacopdliver calung cagynaecological capatient™s prognosis months“““““““““ 0ctanho bmc palliative care page of didn™t i do this why didn™t i do that™  dph19spousein some instances family caregivers displayed poignant emotion and dedication to their family membersconveying the great extent to which they would goto give theirthe best care andcomfortfamily membersœi wish to care for him till the very end¦ i want thebest for him and i will do what™s best for him i amwilling to sacrifice my soul to make that happen ortake his place if i could dph68 childalleviating suffering n family caregivers displayed awareness and empathytowards their family member™s physical and emotionalsuffering tied in with a desire to relieve their painœthis morning she was upset with me for forcing herto drink the bitter medicine i told her ˜i love you iwouldn't do this if i had a choice i want you todrink this for your own benefit not mine i'm just encouraging you from thedph53spousesidelines™underlining this motivation to alleviate suffering was thefamily caregivers™innate compassion for their familymember an empathic bond so strong that witnessingtheir family member™s suffering caused them deep emotional distressœ¦ it hurts a lot to drain the fluids i™m heartbrokenwhen i see how much pain she is in especially wheni see the tubing being inserted it must hurt somuch hca109 spousefindingsfigure presents the six caregiving motivations and onewellbeing determinant generated from data to form theblessings or burdens of endoflife caregiving bobecmodelthe six caregiving motivations honoring fidelityalleviating suffering enduring attachment preservinggratitude navigating change and reconciling withmortality represent the beliefs values and goals that areassimilated into the eol family caregiver™s daily life eachmotivation is posited to affect the way a caregiver makesmeaning of their role hence leading to a nurturance ofcaregiver wellbeing termed in this paper as blessings ora diminishment in caregiver wellbeing termed in thispaper as burdens the wellbeing determinant which ischaracterized by the caregiver™s sense of control selfempowerment and kinship derived from their experiences serves as an indication of selfdetermination andis theorized to have a positive influence on how the sixcaregiving motivations are appraised by the caregiverthese themes are described in greater detail and demfrom study participantsonstrated by direct quotesbelowhonoring fidelity number of transcripts theme hasoccurred in n family caregivers expressed their faithfulness and commitment to attend to the needs and wishes of their family members for the remainder of their lives fearingregret to see things through till the end this motivatedthem in fulfilling their sense of duty to the utmost oftheir abilitiesœi shouldn™t regret anything whatever i can do forhim i will do my best and [instead of waiting till]he™s in the coffin you know [and then say] ˜oh whyfig the blessings or burdens of endoflife caregiving bobec model 0ctanho bmc palliative care page of enduring attachment n family caregivers experienced a prevailing attachment totheir family member that motivated them in spendingcherished time together and doing all they could toensure that their family member was well taken care ofœi think i try to make him as comfortable as he canbe every medical checkup every appointment wewill keep to it and i will always be there for him[there will never be] any appointment that i am notgoing with him hca117 spousesome family caregivers found that the motivation tosustain this attachment was also driven by feelings ofanxiety about their family members™ wellbeing thesecaregivers felt the need to be within their family members™ physical proximity as much as possibleœi get worried when she™s lying there and sleepingbecause i™m not sure if anything has happened toher i™m much happier when she™s sitting here withme when she™s just lying there i would think ˜ohno what if something has happened to her™ and i™dbe worried dph59 childpreserving gratitude n family caregivers described past circumstances and beliefs that motivated present feelings of gratitude to theirfamily members this consequently influenced their efforts and responses in caregivingœshe was constipated for as long as a week and shedidn™t tell me when she eventually relieved herselfshe made a mess on the bathroom floor as i wascleaning the mess i thought about how she hadcleaned me up when i was little so i didn™t mindhca81 childwhile many family caregivers reported feelings of gratitude stemming from how their family member hadtreated them in the past some indicated that religiousand cultural beliefs had indoctrinated a sense of indebtedness to their family membersœmy mother says i was indebted to my brother in mypast life this is why i have to settle my debt in thislifetime [by caregiving] because he is here to get hiscompensation mws004 siblingnavigating change n family caregivers reflected on their perceptions of thechanges that had taken place in their lives and that oftheir family members throughout the illness trajectorysome caregivers found motivation in helping their familymembers adaptenergy to liftsupportto changes by dedicating time andtheirspirits and provide emotionalœi would bring my father food when i visit while myhusband would share words of encouragement andtalk to him to cheer him up we just want him to behappy so that he wouldn't spend the whole day innegativity ttsh65 childothers saw the changes as a temporary setback andfound motivation in steering their family member backto their previous condition if possibleœsometimes i will move his legs a little to give himthat exercise i hope that he can walk again but itdepends on how strong his willhca116spouseisreconciling with mortality n the knowledge of their family member™s prognosis motivated some family caregivers to make the most of thetime left with their family members “ creating treasuredmemories and remembering their legaciesœall of us just want to cherish the time that we haveleft with her and we want her to help us spend moregood times together we [want to] learn about mygrandmother learn about my mother so that wecan pass on to the next generation share with themthe traits and the role models to look up tottsh61 childother family caregivers perceived their family member™sprognosis to be unacceptable choosing to push for further treatment in order to stall death™s journey to theirdoorsœmy grandmother lived past years old so ithought my mother would live till atleast without any problems i felt really shocked becausei always thought œshe still has more than years istill have time ¦ so we felt that if it was possibleshe should extend her life dph14 childthe wellbeing determinant n family caregivers reflected on the discovery of positivetheir family member™schanges amidstillness one such change was found within strengthenedkinshipthe trials ofœas we grow up it™s a bit harder [to have familygatherings] because we are all working so when thedisease came even though it™s not a good thing not 0ctanho bmc palliative care page of something you will ask for it united us again maybewithout it [we] would have been a bit more separated ttsh61 childœi feel like we are more united now maybe in thepast we didn't really chat with each other¦ theamount of communication we had increased i feelthat our unity has become stronger dph14 childthey expressed pride in overcoming initial fears by taking charge of and learning to perform unfamiliar taskswith a newfound confidence in their abilities to faceboth practical and emotional difficulties family caregivers also demonstrated a sense of selfempowermentand strengthbased reflection in their sharingœi know nothing about going to visit the government¦ or to do this do that but somehow i findmy way there [i am a] much stronger person so ifanything happens to me i think i know i can faceup to it dph19 spouseœthis is how you grow i learnt to grow because of[my husband] you have to face the insurmountablechallenges that come your way i learnt how toshoulder my responsibilities on my own scs18spousediscussionthis is the first known study that investigates and bringsattention to the internalized motivations of eol familycaregivers while the family dignity intervention studyquestions did not specifically query family caregivers ontheir motives these motivationcentred responses occurred spontaneously and abundantly throughout the interviews “ an indication that internalized motivationsare profoundly espoused within eol caregiving attitudesand behaviors the bobec model fig illustrates theduality ofthese caregiving motivations in regard tomeaningfocused coping and intrapsychic strains as well as identifies an important influence on caregiver wellbeingmotivations with cultural influencessome themes revealed cultural undertones that reflectedthe internalization of asian values into family caregivermotivations in their motivation for alleviating sufferingfamily caregivers displayed the desire to do so by practical means such as administering medication to theirfamily member and experiencing distress when suchmethods were not feasible this is in line with the asianculture of preferring to show concern for their familymembers through pragmatic ways in their motivation for preserving gratitude family caregivers demonstrated the significance of filial piety as well as culturalbeliefs about karma and pastlife within theirattitudes towards caregiving finally in their motivationfor reconciling with mortality family caregivers indicated the importance placed on close intergenerationalconnections as well as longevity for their elders motivations as blessings meaningfocused copinghonoring fidelityall eol caregiving motivationsalleviating suffering enduring attachment preservinggratitude navigating change and reconciling with mortality were found to embody the tenets of meaningfocused coping fuelled by these motivationsfamilycaregivers displayed the propensity for benefitfindingand benefitreminding even in witnessing their familymember™s suffering and imminent mortality adaptivegoal processes in adjusting their expectations and aspirations in accordance with their family member™s physicalcondition and prognosis reordering priorities in hopesof making the most of the time left with their familymember and infusing ordinary events both past andpresent with positive meaning that allowed them to feelaffirmed encouraged and grateful in their daily caregiving as such the capacity for imbuing stressful caregiving events with positive meaning and responseswould make these motivations advocates of perceivedœblessings in the eol caregiving journeymotivations as burdens intrapsychic strainsparadoxically the authors found that these eol caregiving motivations also ran parallel to the intrapsychicstrains as postulated by pearlin and colleagues intheir seminal stress process model intrapsychic strainsoccur when the caregiver™s selfconcept is diminisheddue to the chronicity of providing care intrapsychicstrains unique to caregivers were defined as role captivity in which the caregiver feels entrapped within hisor her role whether or not by personal choice the lossof self in which the caregiver experiences a loss of identity and sense of personhood as enmeshment with thepatient ensues perceived low competencein whichthe caregiver does not see the value and skill of the workthey do leading to a sense of helplessness and perceived lack of gain in which the caregiver does not findpersonal growth orcaregivingprocessenrichmentin theshould their eol caregiving motivations personifythese strains it is only a matter of time before familycaregivers experience outcomes of mental and emotionaldistress such as depression anxiety and irritability aswell as a decline in physical health and a disengagementfrom their caregiving roles in short these motivations would most certainly bludgeon the family caregiverwith great ˜burdens™ within the eol caregiving journey 0ctanho bmc palliative care page of the crucial factor selfdeterminationselfdetermination theory suggests that people needto feel a sense of competence gaining mastery of tasksand having selfefficacy relatedness feeling like theybelong and mutually relating to others and autonomycontrol over their own choices behaviors and goals inorder to fuel high quality motivation that helps one tothrive the theme of the wellbeing determinant alignswith this concept in a caregivingcentric phenomenonfamily caregivers contributing to this theme displayedconfidence in carrying out previously unfamiliar caregiving tasks competence affirmed a stronger sense of kinship relatedness with the patient and their families andtook ownership of their caregiving responsibilities andchallenges autonomy encouragingly numerous studieshave proposed that high quality motivations stemmingfrom selfdetermination can elicit outcomes of greaterfortitude higher commitment and more positive emotions and selfconcepts [“]building on said studies the authors propose that family caregivers who feel a sense of competence relatedness and autonomy within their caregiving motivationswould be further inclined to meaningfocused copingsuch as deriving perceived benefits from their caregiving even in difficult events reminding themselves ofthese benefits when faced with similar circumstances setting their own goals in caregiving having the competence and confidence to be flexible and adaptive and finding positivity in normal everyday situations possessing a sense of selfdetermination in the caregivingrole would in essence safeguard one™s caregiving motivations from the intrapsychic strains of perceived entrapment a sense of disempowermentineptitude andfruitlessnessas such the bobec modelidentifies the wellbeingdeterminant as an indicative element of caregiver selfdetermination and a crucial factor as to whether the eolfamily caregiver perceives their journey as one lined withblessings or laden with burdens competencetargeted mediators apart fromgeneral psychoeducation on symptom managementmedical care and selfcare interventions could incorporate mediums to develop and improve selfefficacy these can take the form of personalstrengths journaling facilitating peer support between new and experienced caregivers rolemodelling and goalsetting such interventions canbe created in the form of structured support groupsonline platforms or mobile applications autonomytargeted mediators in addition toproviding adequate and appropriate eol caregivereducation autonomytargeted interventions suchas those involving mindfulness practice and thearts can serve to give caregivers a sense of control as well as help them make meaning out oftheir thoughts emotions and circumstances todate the mindfulcompassion art therapymcat for eol care professionals showsgreat potential to be converted into a programfor eol family caregivers relatednesstargeted mediators dyadic or familyprojects that recall shared memories expressappreciation seek fiveness impart wisdom andcreate generativity such as the fdi are valuable incrafting the bond of relatedness among familycaregivers and their family members such projectsshould be implemented as a foundation ofpsychosocial interventions at the endoflifeinterventions should be offered to family caregiversin a culturallyrelatable manner this can be donethrough emphasizing how these mediators will helpfamily caregivers enhance their practical caregivingwith increased competencesense of control andmeaningmaking as well as enrich their intergenerational familial bonds with conversations that focus onlegacy creationimplications and recommendationsfindings from a number of studies show that fulfillingone™s sense of selfdetermination appears central to sustaining one™s motivation and innate satisfaction in caregiving [“] the findings from this paper indicatethat caregivers are driven by motivations that couldequally contribute to wellbeing nurturance or diminishment at the same time our findings also indicate thatcaregivers possess a caregivercentric sense of selfdetermination the wellbeing determinant that is theorized to have positive affect on their motivations thusthis paper recommends that caregiver support interventions should comprise all of the following mediators inorder to fulfil the need for selfdeterminationlimitations and future directionswhile the anically occurring responses emphasize thesignificance of motivations within eol caregiving theseresponses were not examined further during the fdi interviews due to other pri

range of diseases including malignancies and autoimmune disordersIts high eï¬ectivenessprice ratio also won extensive application in ophthalmology On the other hand although MTX has anexcellent pharmacological efficacy MTX associated side eï¬ects in clinical use which vary from patient to patient are nonnegligible ere is no comparatively systematic review on MTX associated side eï¬ects and its risk factors is review aimed toreveal novel clinical approaches of MTX and its adverse eï¬ects in order to provide a reference for ophthalmic scholars in clinicalapplication of MTX IntroductionMethotrexate MTX is an antifolate metabolite that inhibitsDNA synthesis repair and cellular replication It was firstlyused as one of the essential treatments of pediatric leukemia[ ] According to previous studies MTX has also beenused to treat rheumatoid arthritis RA and psoriasis as anti‚ammatory and immunomodulatory agent [] as MTXcould not only optimize the efficacy of biological diseasemodifying antirheumatic drugs DMARDs [ ] but alsomake the therapeutic goals via lower doses in comparisonwith other conventional synthetic DMARDs [] Figure shows the pathway of folate in DNA synthesis the cellularpathway of MTX and how MTX works inside the cell Whileimmediate and lowdose MTX is used to treat nonmalignantand immunemediated disorders highdose MTX HDMTX more than mgm2week is widely used to treatmalignancies Until now HDMTX with or without radiation therapy is still the backbone of most modern chemotherapy regimens [] as well as the prevention ofsystemiccentral nervous system CNS lymphoma recurrence at a dose of gm2 per week []MTX has also been widely applied in ophthalmic diseases systemically and locally Recently published spay more attention to new clinical applications routes ofadministration and newly discovered side eï¬ects which arefoci of this review Clinical Applications in OphthalmologyAs one of the known corticosteroidssparing agents MTXhas been widely used in the treatment of anterior intermediate posterior or pan uveitis scleritis and ocularmucous membrane pemphigoid [] as well as advancedproliferative diabetic retinopathy [] However followedresearches reveal that MTX works with a significant difference in eï¬ectiveness ratings by anatomic location of‚ammation [] with treatment success achieved mostcommonly in patients with anterior uveitis and scleritis []In the treatment of noninfectious intraocular ‚ammationoral and intravenous are the most common routes with ausual dose range of mg to mg weekly e typical doseobserved was mgweek [ ] which is in the range oflowdose MTX e median time to achieve the success of 0cJournal of OphthalmologyFigure e cellular pathway of folate and MTX Dietary folate enters the cells through RFC1 as well as MTX In lowdose MTX treatmentMTX inhibits enzymes of the folate pathway Ultimately MTX leads to an increase in intracellular adenosine level which would cause anti‚ammatory eï¬ects RFC1 � reduced folate carrier ABC family � adenosine triphosphatebinding cassette ABC family DHF � dihydrofolate THF � tetrahydrofolate GGH � cglutamyl hydrolase FPG � folylpolyglutamate synthase MTXPG � methotrexate polyglutamate DHFR � dihydrofolate reductase MTHFR � methylene tetrahydrofolate reductase AICAR � aminoimidazole carboxamideribonucleotide ATIC � AICAR transformylasetreatment defined as control of ‚ammation with theability to taper corticosteroids to mg or less daily rangesfrom months to months for MTX [ ]Intravitreal MTX injection with or without systemicchemotherapy and radiotherapy has already been used totreat primary intraocular lymphoma patients [ “]According to Larkin [] intravitreal MTX injectioncould achieve remission in a proportion of patients withprimary intraocular lymphoma What is particularly noteworthy is that although MTX has a slow rate of onset ofeï¬ect when it was used to treat intraocular lymphoma viaintravitreal injection it prolonged local remission of oculardisease even with an aggressively growing tumor []erefore it has been taken as a relatively firstline choice forthe treatment of recurrent intraocular lymphoma [] although the treatment for primary intraocular lymphoma islacking solid justification because of the limited retrospectiveand prospective case series [] Local treatment via intraocular injection provides a consistent therapeutic MTXconcentration to reduce the systemic MTX associated sideeï¬ects [] erefore intraocular MTX injection is worthtrying especially for unilateral ocular diseases New Approaches of Applications of MTX MTX Used against Epithelial Downgrowth Previousstudies have already demonstrated safety of intravitrealMTX [] It has been used to treat intraocular lymphomaand proliferative vitreoretinopathy because ofits antiproliferative properties [] ere is a novel use of intravitreal MTX for recurrent epithelial downgrowth which wasnot treated by surgical and medical methods Lambert et al[] administered intravitreal MTX to patients with refractory proliferative membrane after cataract surgery whilemembrane peel and endolaser treatment failed e injectionof MTX was administered alone based on previous protocols and the presumed halflife of drugs in vitreous cavityAfter injections totally there was no membrane recurrence is case suggests that intravitreal MTX plays a role intreatment against epithelial downgrowth MTX Used in Conventional erapyResistant DiseasesGenerally the antivascular endothelial growth factor antiVEGF therapy has dramatically improved the prognosis ofneovascular agerelated macular degeneration nAMDHowever there are still some patients who remain refractoryto antiVEGF therapy which is termed as treatmentresistant nAMD As there is evidence that MTX has eï¬ects ininterrupting the angiogenesis cascade at various levels []Kurup oï¬ered intravitreal MTX to patients who wererefractive to standard antiVEGF therapy [] Although itwas an oï¬label use the patients™ visual acuity improved atfollowup visit while ophthalmic imaging examinationsshowed significantly reduced cystoid macular edema usFolatefolic acidRFC 1Folatefolic acidDHFTHFDHFR5MTHFMTHFRPurine and pyridine synthesis DNA synthesisMTXMTXABC familyFPGGGHMTXFGAICAR5FormylAICARATICAdenosine †‘AdenosineCell membraneCell membrane“““ 0cJournal of Ophthalmologypatients who are refractory to traditional antiVEGF therapymight benefit from intravitreal injection of MTXis approach is not alone Khalil [] had μg01 ml of MTX intravitreal injection once monthlyadministrated to adult Behcet™s disease BD patientssuï¬ering from BDassociated ocular ‚ammation withposterior segment involvement eir results prove thatintravitreal MTX improves visual acuity reduces posteriorsegment manifestations associated with Behcet™s disease andallows the reduction of corticosteroids and immunosuppressive drugs [] ese results also supported Taylor andassociates who conducted trials on patients with unilateral uveitis andor cystoid macular edema [] eirclinical trials suggest that intravitreal MTX may help patients with uveitisassociated posterior segment involvementto regain normal anatomical structure and then allowed thereduction of immunosuppressive therapy The Pharmacogenetics of MTXWith molecular sequencing and highthroughput technology large numbers of genetic polymorphisms can now bedetected accurately and rapidly [] Researchers pay moreattention to pharmacogenetics the study of genetic polymorphisms in drugmetabolizing enzymes and the translation of inherited diï¬erences to diï¬erences in drug eï¬ects[] e genes encoding transporting proteins and metabolizing enzymes for MTX are also known to harborfunctionally significant SNPs e SNPs may ‚uence theefficacy of MTX and have been suggested as potential riskfactors for enhanced MTX toxicity even in lowdose regimens based on previous researches []e research of pharmacogenetics of MTX could bedivided into genetic polymorphisms aï¬ecting MTX transport and SNPs that‚uence enzymes in the cellularpathway of MTX []Once taken MTX enters the cell through an activetransport which is mediated by the reduced folate carrier RFC1 e loss of RFC1 gene expression might lead toeï¬ects of uptake and intracellular levels of MTX A G80ASNP of RFC1 was proposed [] making a decreasing [] orincreasing [ ] eï¬ect on intracellular level of MTXerefore a significant association between RFC1 SNPs andMTX toxicity should be considered Chango state thatthese SNPs strongly impact the overall MTX associated sideeï¬ects by resulting in altered cellular MTX concentrationbut with no ‚uence on MTX efficacy [] However someresearchers argue that these SNPs have no definite eï¬ect[] us it remains controversial whether SNPs of RFC1aï¬ect the transport of MTX Moreover Pglycoprotein amembrane transporter that has ‚uences on the dispositionand bioavailability of MTX [] was studied SNPs ofABCB1 including C3435T SNP and C1236T SNP werebelieved to have eï¬ects on the expression of Pglycoprotein[] Gervasini speculate the C1236T SNP of ABCB1aï¬ects the administered doses of MTX and the incidence ofhematological toxicity [] However just like G80A SNPthere are disputes about the ‚uences of these SNPs asdiï¬erent studies had diï¬erent outcomes []Metabolizing enzymes were also being analyzed giventhe critical role of transporters in disposition of MTX and itsactive products as well as the folate metabolism MTXpharmacogenetics mostly focused on the SNPs in theMTHFR gene e present study shows that genetic polymorphisms in the folate metabolic pathway and in MTXtransporters ‚uence the toxicity but not the efficacy of thelowdose MTX treatment in patients with autoimmunediseases [] For example C677T and A1298C are knownin MTHFR gene to result in a lower enzyme activity []Windsor and associates reported that MTHFR A1298C andC677T were associated with MTX related nephrotoxicityand anemia [] ese SNPs might be associated withdecreased activity of methylenetetrahydrofolate reductaseelevated plasma homocysteine levels and altered distribution of folate [] us patients with this genotype weremore vulnerable to potential MTX induced toxicity sincethese reactions above may lead to slower folate metabolismand slower cell repair [] Weisman used univariatelogistic regression to reveal that the MTHFR C677T alsoincreases the occurrence of side eï¬ects in central nervoussystem manifested as headache and lethargy [] HoweverLambrecht argued that MTHFR C667T was not apredictive factor for toxicity [] Berkani found noassociation between A1298C polymorphism and MTXtoxicity [] Interestingly Grabar claimed that thepatients with MTHFR 1298C genotype have a lower risk forMTX toxicity than the carriers of MTHFR 1298A allele []To date the study of pharmacogenetics of MTX continues An increasing number of SNPs have been found to bepossibly associated with the efficacy and toxicity of MTXe newly discovered genotypes include C347G in ATICand ²UTR 28bp repeat and ²UTR 6bp deletion inTYMS which may ‚uence both efficacy and toxicity ofMTX similarly factors that may aï¬ect MTX associatedtoxicity are for example A2756G in MS and A66G in MTRR[ ] e genes and their SNPs that might beassociated with the eï¬ects and side eï¬ects of MTX aresummarized in Table Growing evidences suggest that asingle genetic factor is unlikely to adequately predict theefficacy and toxicity of MTX in polygenic disease such as RAand autoimmune associated ocular disease Given the impactof MTX in several metabolic pathways a complex of multiple risk genotypes examination would help to predict theefficacy of MTX and to identify patients who may haveadverse eï¬ects from MTX administrationTaken together the efficacy and toxicity of MTX mayremain associated with the genetic markers in the patientserefore although this remains a controversial subject it isreasonable to believe that pharmacogenetics may be able topredict who is at risk of MTX associated adverse eï¬ects andmay help in maximizing the benefitrisk ratio of MTX The Side Effects of MTXe doselimiting toxicity of MTX mainly includes hepatotoxicity and nephrotoxicity [“] but mortality hasoften been reported due to either pneumonitis or secondaryinfections [] 0cJournal of OphthalmologyTable Summary of genes and their SNPs which might have possible clinical eï¬ects towards MTXTransportingproteinsABCfamilyGeneRFC1 [“]ABCB1 [ ]ABCC1 []ABCC2 []ABCC4 []MetabolizingenzymesMTHFR [“]ATIC [“]TYMS [ ]GGH [ ]DHFR [ ]MS [ ]MTRR [ ]SNPsG80AC3435TC1236Trs246240Srs3784862A2412GG1249AG1058AC934AC677TA1298CC347G²UTR 28bp²UTR 6bprepeatdeletionC452TC401TT721AC830TA2756GA66GSome experts divided MTX associated pulmonarycomplications into ‚ammatory infectious and lymphoproliferative [] In the authors™ opinion all MTX relatedside eï¬ects can be classified into these three categoriesaccording to the pharmacological eï¬ects of MTXMajor adverse events for MTX are related to the folateantagonism and primarily aï¬ect highly proliferative tissuessuch as bone marrow and gastrointestinal mucosa []Given the immunosuppression eï¬ect of MTX pancyt iawas one of the most frequent severe toxicities of methotrexate [] Meanwhile the risk of developing an infectiousprocess is increased all along the treatment and the severityof the infected disease would be worsen [ ] includingcommon bacterial infections herpes zoster eruptions andopportunistic infections According to previous studies therisk is larger than that with other disease modifying antirheumatic nonbiological drugs DMARDsSecondly the MTX acts as the hapten [] and is likely toreact directly with nucleophilic groups present in proteins ieto combine with endogenous protein [ ] e proteinadducts thus act as an antigenic signal to direct the eï¬ector armof the immune response [] e provoked immune responsesare most commonly type I immediate hypersensitivity andtype III immune complex reactions [] Hypersensitivitypneumonitis is the most common severe and unpredictablecomplication with a mortality of up to almost []Moreover a few studies have shown that longterm MTXuse can lead to lymphoproliferative disorders LPDs in bothnodal sites and extra nodal sites such as the skin lungsepipharynx thyroid gland nasal cavity spleen and kidneysespecially for patients who are positive for EBV infection[“] e reported frequency of EBV positive in MTXassociated LPDs patients is “ [] Although themechanism of onset is not fully understood it is believedPossible clinical eï¬ectsIncreasingdecreasing intracellular MTX levelAï¬ecting efficacy of MTXAï¬ecting the distribution of MTX and incidence of hematological toxicityAssociation with MTX related toxicityLeading to accumulation of MTX to nephrotoxic levelsAssociation with MTX related gastrointestinal toxicityAssociation with MTX related hepatotoxicityAssociation with MTX related hematological toxicityAï¬ecting the toxicity but not the efficacy by resulting in a lower enzymeactivity association with related nephrotoxicity anemia and neurologicside eï¬ectsAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy and toxicity of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAï¬ecting efficacy of MTXAssociation with MTX associated toxicityAssociation with MTX associated toxicitythat the combination of immunodeficiency and the immunosuppressive eï¬ect of MTX has been implicated in thepathogenesis of MTX associated LPDs e World HealthOrganization WHO has classified MTX associated LPDs aslymphoid neoplasms whether iatrogenic or immunodeficiency associated diseases [ ] MTX associated LPDsoften take a spontaneous remission which tends to completemostly within weeks after the discontinuation of MTX[] But there are a few reports showing that the lymphoidneoplasms occur even after stopping using MTX [] e Eï¬ects of Administration Routes Generally the sideeï¬ects of MTX depend on the route of administration Dosedependent [] gastrointestinal side eï¬ects are the mostfrequent side events with orally administered MTX as oraladministration is the most common delivery method[ ] More than of MTX is excreted by therenal system thus MTX associated nephrotoxicity is common among patients taking MTX Fortunately the resolution usually occurs after discontinuation of therapy andsalvage treatment with highdose corticosteroids[]erefore to achieve treatment with less side eï¬ects theappropriate route of administration and dose of MTX arenecessary During the treatment monitoring of patients™general condition mattersAdverse eï¬ects of intravitreal injections of MTX occuronly within the eyeincluding hyperemia keratopathycataract iridocyclitis vitreous hemorrhage retinal detachment maculopathy and endophthalmitis []Splitting doses of MTX rather than intravenous administration is a new attempt to avoid MTX associated sideeï¬ects MTX is split and given twice or thrice in a week toachieve higher bioavailability and better clinical response 0cJournal of Ophthalmology[ ] thus providing us with a novel method of oraladministration of MTX with less adverse eï¬ects Is LowDose MTX Safer Based on clinical cases observation side eï¬ects which can lead to discontinuation ofMTX are rare during the typical ophthalmology treatmentbecause of the lower dose of MTX required [] e application of lowdose MTX regimen has also become one ofthe main therapies of a variety of immunemediated diseasesbecause of its efficacy and an acceptable safety profile asmost lowdose MTX associated toxicity has been describedin case reports and relatively small case series []However although welltolerated and mostly reversibleeven a lowdose regimen of MTX can result in clinicallysignificant toxicity with substantial death rates about according to Kivity™s cohort study [] e lowdose MTXassociated severe adverse eï¬ects include major centralnervous system complications [] mucositis pulmonaryinvolvement hepatotoxicity [] and myelosuppression Is MTX Safe to the Pregnant and Fetuses As one of thelipidsoluble and low molecular weight drugs MTX could bereadily transferred across the placental membrane duringpregnancy and adversely aï¬ect the fetus [] In addition MTXmight take longer time for elimination in fetal tissues []Regarding pharmacogenetics mutations caused by MTXlead to severe decrease of the expression of folate and nucleobaseenzymes which are critical for cellular homeostasis [] Inpractice MTX aï¬ected formation of the blastocyst and causeddysmorphic features and neurologic defects in early pregnancyleading to malformations in some cases [] Multiple congenitalabnormalities have been observed after weekly MTX treatmentsat a mg dose during the first months of pregnancy [] evenfetal death [] Verberne had reviewed cases of congenitalanomalies after in utero exposure to MTX and proved that somecongenital anomalies such as microcephaly craniosynostosistetralogy of Fallot pulmonary valve atresia limb reductiondefects and syndactyly were truly part of the œfetal methotrexatesyndrome [] Administration of MTX in childhood mightalso cause manifestations including visual defect [] andSmith“Magenis syndrome [] among patients with specificmutations us special care should be taken with pregnantpatients and children in particular e Risk Factors of MTX Associated Side Eï¬ects e mostcommon risk facts of MTX induced adverse eï¬ects areadvanced age age years and underlying disease incaused by MTX administration with100 g alcohol concluding renal andor hepatic insufficiency and lung diseaseespecially patients with chronic hepatitis B and diabetesmellitus [“] Patients with a history of alcohol intakemight have a greater risk of liver fibrosis and hepatotoxicitysumption per week [] Also preexisting hypoalbuminemiaand past use of any of the DMARDs and protonpumpinhibitors have been described in studies to increase theincidence of MTX induced side eï¬ects [ ] Moreovertaking drugs that may interact with MTX at the same timemight also be dangerous these drugs include salicylatescotrimoxazole chloramphenicol sulfonamides cyclosporine and pyrimethamine [] Although no significantprotective eï¬ect of folate supplementation on MTX relatedtoxicity has been found [] the folate deficiency is anotherreason for the side eï¬ects based on clinical cases []Heidari found that MTX administration elevatedkidney ROS levels decreased tissue antioxidant capacityincreased lipid peroxidation and depleted renal glutathionestores eir research data indicate that MTX caused tissuedamage and organ dysfunction through oxidative stresserefore they proposed that patients with preexistingmitochondrial defects might be vulnerable to MTX inducedrenal injury []e use of highdose MTX HDMTX is also the riskfactor of adverse eï¬ects MTX induced liver fibrosis is morelikely to become morphologically evident with high cumulative doses possibly largely exceeding to mg[ ] and the side eï¬ects caused by omeprazole use inthe past were found in cancer patients receiving HDMTXtreatment []e distribution of MTX in vivo also plays a role in MTXrelated side eï¬ects As MTX tends to accumulate in theextravascular compartment patients with pleural eï¬usionascites and massive edema should get extra caution due tothe risk of toxicity from reabsorption of extravascular fluid[]Another noteworthy risk factor is UV UV recall phenomenon also known as MTX associated UV reactivationhas been reported [ ] It is reactivity of sunburn areaswithin to days of the treatment with MTX [ ]According to Adams and associates this phenomenon mightbe due to the immune response by uncontrolled sunburninduced ‚ammation released by MTX [] Patients whopreviously suï¬ered sunburns deserve more detailed monitoring when methotrexate is needed Is Folate Supplementation Necessary for OphthalmicPatients To prevent MTX associated side eï¬ectsit iscommon to take folate [as either folic acid FA or folinicacid FLA] in clinic [ ] However there is noconsistent and evidencebased guideline for folate supplementation in ophthalmic patientsFolate and folic acid play significant roles in the de novosynthesis of purines and thymidylate which are required forDNA replication and repair [] Funk and associates founda significant reduction of circulating folate concentrations in of patients receiving MTX treatment [] Patientstreated with highdose MTX HDMTX got routine folatesupplementation to reduce HDMTX associated side eï¬ects[“] After a systematic literature review of HDMTXtherapy and folate supplementation Van der Beek et al[] found lower incidence of MTX associated adverseeï¬ects in regimens with higher cumulative doses and earlieradministration of folate supplementation in similar HDMTX dosage studies Folate supplementation in patientswith lowdose methotrexate is also being studied Ortiz et al[] had proved the protectivefolateeï¬ectof 0cJournal of Ophthalmologysupplementation by conducting a Cochrane review including more than patients taking lowdose MTX Untilnow folate supplementation had been proved to prevent andimprove MTX associated eï¬ects including gastrointestinalrespiratory and neurologic side eï¬ects [ ] Mori et alsupported the protective eï¬ect by demonstrating that patients treated with lowdose MTX without folate supplements were significantly associated with the development ofmyelosuppression and pancyt ia []However Arabelovic and associates™ preliminary studyshowed a significant increase of MTX dose needed []since folic acid fortification enriched cereal grain productswere fully implemented in the USA and Canada [] isconveyed a message to us that high dose of folate supplementation might have ‚uence on the efficacy of MTXAlDabagh found that the reduction in efficacy ofMTX cannot be ignored while folate supplementation didmake a significant reduction in associated adverse eï¬ects[] Salim declared the decreasing ‚uence betweenthe anti‚ammatory eï¬ect of MTX and folate supplementation by carrying out a doubleblind clinical trial []Chladek had conducted an labeltwowaycrossover study supporting the opinion above [] Additionally because of the unequal distribution of folic acidand MTX in organs and tissues [] MTX discontinuationis more common for some MTX associated side eï¬ects inophthalmic clinic [] rather than higher dosage of folatesupplementationere are no ophthalmic studies to demonstrate theprotective eï¬ects of folic acid supplementation us although the folate supplementation is widely used amongpatients treated with lowdose MTX [ ] the necessityand standardized dosage of folate supplementation in specific patients [] as well as the MTXfolate interactionstill warrant further studies DiscussionMethotrexate as one of the alternative pharmacologicalsteroidsparing immunosuppressive agentsis becomingmore and more popular as the preferred treatment in severalautoimmune conditions requiring longterm immunosuppression [] Lowdose MTX has anti‚ammatory andimmunomodulatory properties by increasing levels of intracellular and extracellular adenosine [] which is thefoundation of ophthalmic MTX treatment e standardizedand recommended administration of ophthalmic MTXtreatment is once a week starting with a dose of mg andescalating every to weeks up to “ mgweek whennecessary [ ] In patients with insufficient response toMTX alone cyclosporin with or without azathioprine wasadded []To avoid side eï¬ects split doses of MTX administrationand folate supplementation are gradually being used inophthalmic clinic Prescription of to mg of folatesupplementation has a significant role in MTX safety []but the higher dosage is less applied even with higher dose ofMTX [] Prophylactic folate supplementation is notnecessary in most patients [] ere is also research toconvey that ml100 g or above dosage of fish oil is aseï¬ective as folinic acid in therapeutic potential in preventingbone loss during MTX chemotherapy [] For some resistant andor mortal adverse eï¬ects the discontinuation ofMTX will work instantlyWith the increasing longterm use of MTX it is importantto monitor patients™ blood examination results including bloodroutine and liver and renal functions As pancyt ia can be alate manifestation [] elevation of urea creatinine aminotransferases and albumin as well as electrolytes disturbancesmay result in MTX associated liver and renal side eï¬ects []Plasma MTX level is not a reliable predictor for adverse eventsin MTX therapy [] On the contrary circulating folate levelsand folate polyglutamate distribution change sensitively withMTX exposure and exogenous folate supply [] and could beused as a biomarker of MTX efficacy [] It should be notedthat as erythrocytes have a halflife of approximately daysthe results of blood examinations might reflect both pretreatment and posttreatment status which need to be analyzedcarefully []Numerous studies had been conducted to prove thatMTX could be used as a welltolerated safe and eï¬ectivefirstline treatment Hence the MTX administration shouldnot continue to be stigmatized as a œcancer drug or to bediscouraged because of associated adverse eï¬ects Contrarily the indication and the routes of administration areabout to gradually widenConflicts of Intereste authors declare that they have no conflicts of interestReferences[] R Q H Kloos R Pieters C van den Bos œe eï¬ect ofasparaginase therapy on methotrexate toxicity and efficacy inchildren with acute lymphoblastic leukemia Leukemia Lymphoma vol no pp “ [] R K Bath N K Brar F A Forouhar and G Y Wu œAreview of methotrexateassociated hepatotoxicity Journal ofDigestive Diseases vol no pp “ [] W Wang H Zhou and L Liu œSide eï¬ects of methotrexatetherapy for rheumatoid arthritis a systematic review European Journal of Medicinal Chemistry vol pp “[] J Smolen and R Landew´e œEULAR recommendations forthe management of rheumatoid arthritis with synthetic andbiological diseasemodifying antirheumatic drugs update Annals of the Rheumatic Diseases vol no pp “ [] J A Singh K G Saag S L Bridges œ Americancollege of rheumatology guideline for the treatment 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chemotherapy for the treatment ofrecurrent intraocular lymphoma British Journal of Ophthalmology vol no pp “ [] E Kim C Kim J Lee and Y Cho œA case of primaryintraocular lymphoma treated by intravitreal methotrexateKorean Journal of Ophthalmology vol no pp “[] J Smith J T Rosenbaum D J Wilson œRole ofintravitreal methotrexate in the management of primarycentral nervous system lymphoma with ocular involvementhistorical image Ophthalmology vol no pp “ [] CC Chan and D J Wallace œIntraocular lymphomaupdate on diagnosis and management Cancer Controlvol no pp “ [] K L Larkin U S Saboo G M Comer œUse ofintravitreal rituximab for treatment of vitreoretinallymphoma British Journal of Ophthalmology vol no pp “ [] C P Fox E H Phillips J Smith œGuidelines for thediagnosis and management of primary central nervoussystem diï¬use large Bcell lymphoma British Journal ofHaematology vol no pp “ [] A Sadaka R Sisk J Osher O Toygar M Duncan andinfusion forœIntravitreal methotrexateC Riemannproliferative vitreoretinopathy Clinical Ophthalmologyvol pp “ [] N G Lambert DJ Wilson D M Albert andW D Chamberlain œIntravitreal methotrexate for recurrentepithelial downgrowth JAMA Ophthalmology vol no p [] A M Joussen F E Kruse HE V¨olcker and B KirchhofœTopical application of methotrexate for inhibition of cornealangiogenesis Graefe™s Archive for Clinical and ExperimentalOphthalmology vol no pp “ [] S K Kurup C Gee and C M Greven œIntravitrealmethotrexate in therapeutically resistant exudative agerelated macular degeneration Acta Ophthalmologica vol no pp e145“e146 [] H E M Khalil H A Raafat N A Azab H E Haroun andH A Elgendi œe role of intraocular methotrexate in themanagement of uveitis and posterior segment involvementin Behçet™s disease patients e Egyptian Rheumatologistvol no pp “ [] S R J Taylor A Banker A Schlaen œIntraocularmethotrexate can induce extended remission in some patients in noninfectious uveitis Re

neprilysin nep is a neutral endopeptidase it is also known by different functional names such as common acute lymphoblastic leukemia antigen calla the cluster of differentiation cd10 endoprotease endopeptidase and membrane metalloen dopeptidase nep is a member of m13 family of zinc peptidase in the body nep cleaves many peptides such as atrial natri uretic peptides btype natriuretic peptides angiotensins i ii ii en ix bradykinin substance p endothelin i ii amyloid dorphin neurotensin vasopressin etc [“] the progression of various pathological conditions such as kidney and heart disease obesity diabetes [ ] few malignancies such as colon can a ˆ—corresponding author email address anoopkishoremanipaledu a kishore 101016jmolstruc2020129073 elsevier bv all rights reserved cer lung cancer and melanomas [“] etc is associated with the peptidase activity of nep in the us food and drug ad ministration fda approved sacubitrilvalsartan the combination of a neprilysin inhibitor and an angiotensin receptor blocker arb respectively commonly known as angiotensin receptor neprilysin inhibitor arni for heart failure with reduced ejection fraction further in clinical trials involving sacubitrilvalsartan treatment groups performed well in the renal failure population as compared to treatment with an arb valsartan alone there fore nep has gained considerable attention in the last decade for its peptide degrading property and its inhibition has therapeutic potential in multiple diseases but the known and available nep inhibitors are limited hence drug repurposing using different in silico tools can aid in speeding up the process of drug discovery for the development of new nep inhibitors the role of nep has been extensively studied in various dis eases the study report of the paradigm trial highlighted the role 0c r sankhe e rathi and s manandhar of molecular structure of nep inhibitors in the population of heart failure with reduced ejection fraction in an invivo study of subtotal nephrec tomy the renoprotective effect of sacubitrilvalsartan was found to be stronger as compared to valsartan alone according to the result of the uk harpiii trial the combination of sacubi trilvalsartan is effective and is welltolerated in the chronic kidney disease population similarly various studies are focussed on the importance of nep on chronic kidney and cardiovascular dis eases nep inhibition in streptozotocininduced diabetic mice im proved outcomes of cardiac function for heart failure with reduced ejection fraction in diabetic nephropathy the combination of the nep inhibitor thiorphan with an angiotensin receptor blocker and an angiotensinconverting enzyme ii activator showed significant improvement in the condition by modulating components of the reninangiotensin system and natriuretic peptide system the activation of the leptinaldosteroneneprilysin axis contributes to the pathogenesis of cardiac complications in obese patients in obesity and type diabetes nep inhibition showed improve ment in insulin sensitivity and glycaemic control the inhibition re sults in modulation of several peptides with glucoregulatory prop erties such as bradykinin cholecystokinin glycogen like peptide glucosedependent insulinotropic peptide secretin and vasoactive intestinal polypeptide leading to improved glucose homeostasis and weight loss a study conducted to evaluate the effect of nep on nociception concluded that nep inhibition can be a good strategy for pain management in cancers such as colon cancer [ ] lung cancer [ ] and melanomas the increased levels of nep is correlated with neoplastic progression the peptidase ac tivity of nep and its interaction with akt focal adhesion kinase is assumed to contribute to the pathogenesis of colon cancer in aggressive melanomas cd10 nep is the biomarker for detec tion a recent report has highlighted the role of arni in enhanc ing anti‚ammatory and natriuretic peptide systems in covid patients [ ] additionally the use of arni is also recom mended for patients suffering from covid19 all these find ings highlighted the need for designing novel nep inhibitors but de novo drug development is resource intensive and time consum ing hence drug discovery by repurposing the existing drugs can be an attractive strategy with the benefit of reduced developmen tal risk especially in the case of nep inhibitors the computation repurposing is known as ˜ insilico drug re purposing™ in in the us approximately of drugs ap proved was through the drug repurposing approach the con cept of drug repurposing has been already practiced in cardio vascular disorders cancer obesity erectile dysfunction smoking cessation stress psychosis etc drug repurposing using al ready approved drugs reduces the time and money on preliminary screening toxicity studies clinical trials bulk manufacturing and formulation development on the other hand the establishment of new drug candidates requires lots of time and resources a good example is the case of allopurinol which was originally approved for cancer and is now available for the treatment of gout in this context we decided to identify a series of inhibitors for nep using insilico drug repurposing the protein structure of the extracellular domain of nep with sacubitralat the active metabo lite of sacubitril was used in the current study the inhibitor bind ing pocket in the protein structure of the extracellular domain of human nep pdb id 5jmy has already been revealed by schier ing nikolaus the inhibitor binding pocket contains the catalytically essential triad of his583 his587 and glu646 for our drug repurposing study the structures of fda approved drugs were downloaded from the zinc database based on the binding pocket of the nep inhibitor the high throughput virtual screening of existing fda approved drugs was done to find out a new se ries of nep inhibitors to the best of our knowledge this is the first study based on drug repurposing approach that is being re ported and employed for the development of nep inhibitors using receptorinhibitor complex materials and methods in the current study the maestro molecular platform version by schrodinger llc was used to perform molecular dock ing and simulation studies on an hp desktop system with linux ubuntu lts platform intel haswell graphics card 8gb ram and intel core i34160 processor protein preparation and grid generation xray crystallographic structure of the extracellular domain of human nep pdb id 5jmy was downloaded from the rcsb pro tein data bank the pdb id 5jmy has a resolution of ˚a prior to docking and simulation studies the biological unit of protein was prepared using ˜protein preparation wizard™ in schrodinger suite during the process of protein preparation the protein was subjected to import and refine review and modify and minimize processes in protein preparation wizard missing side chains and residues were filled using the prime tool the active site and cat alytically important residues were retained in the protein structure the water molecules beyond ˚a were deleted and stages were generated for hetero atoms to generate low energy state protein energy minimization was done using opls3e optimized potential for liquid stimulation force field and the prepared protein was used for molecular modelling to generate a grid around the lig and the receptor grid generation workflow was used by keeping all functional residues in the grid ligand preparation the structures of fda approved drugs from zinc database were downloaded for ligand preparation the lig prep tool was employed the lowest energy 3d structures with cor ± under the opls3e related chiralities were generated at ph force field in this process all the ligands were preprocessed which includes generation of tautomers ionization state at ph ± using epik addition of hydrogen bond charged group neu tralization and ligand geometry were optimized ligand docking all the molecular docking studies were carried out using the ligand docking tool glide gridbased ligand docking with ener getics module the glide module was used for predicting ligand protein binding modes and ranking different scoring functions are involved in glide such as highthroughput virtual screening htvs standard precision sp and extra precision xp first all the drugs were docked with htvs mode but computationally htvs docking does not use descriptor and explicit water technol ogy as used in the xp mode hence to avoid falsepositive results few drugs were reanalyzed using sp and xp modes [ ] free ligand binding energy calculation the prime module was used to determine absolute ligand binding affinities to nep using mmgbsa molecular mechanics energies generalized born and surface area continuum solvation method the mmgbsa assay of top eight xp docked drugs was performed using pose viewer file of glide xp mode the prime mmgbsa method is dependent on the vsgb solvation model that uses a variabledielectric generalized born model and water as a solvent under the opls3e force field to calculate binding energy 0cr sankhe e rathi and s manandhar of molecular structure adme analysis for the assessment of the adme profile the qikprop tool from the maestro modeling platform was used the qikprop tool helps in the prediction of the druggable property of best four hits based on adme analysis during this process various descriptors such as molecular weight cardiotoxicity qplogherg predicted octanolwater partition coefficient qplogpow permeability qp pcaco polar surface area psa human oral absorption oral absorption and lipinski rule of five were calculated induced fit docking ifdsp table docking score and prime mmgbsa score of top eight drugs sr no drug dock score xp kcalmol mmgbsa 01g bind sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 zinc000000402909 zinc000000601283 zinc000000000797 zinc000003831594 zinc000028973441 ifdsp was carried out using the inducedfit docking module from maestro molecular modelling platform based on the xp glide docking score binding energy crucial residues involved and adme analysis four zinc0 zinc0 zinc0 and zinc0 drugs were selected for ifd“sp docking in ifd based on the bfactor side chains were trimmed with receptor and van der waals scaling of and respectively and a maximum of poses were set for each ligand further prime sidechain prediction and minimization were performed in which refinement of all residues within ˚a of the ligands™ pose and side chains were performed this pro cess allows the ligand structure and conformation to accommodate nearby reorienting side chains the ligands and residues were min imized in inducedfit protein structure all the ligands were rigor ously docked and ifd score for each was calculated using the for mula ˆ— prime_energy ˆ— glide score ˆ—ifd score glide_ecoul molecular dynamics md simulation the flexibility of the receptor is restricted in gridbased dock ing systems like xp and ifd these do not mimic the actual bio logical systems where the protein and drug are solvated in wa ter hence to tackle this problem md simulation was performed based on the glide docking score free binding energy and ifd score four drugs were selected for md simulation for 20ns for md simulation three steps were performed viz system builder mini mization and md simulation the docked complex of protein and ligand were selected and the system model was made by prede fined spc solvent under orthorhombic boundary conditions next the system model was subjected to energy minimization until a gradient threshold reached kcalmol ˚a balanced at k tem perature and bar pressure via npt ensemble in the final step minimized ligandprotein complex were subjected to md simula tion bioisostere replacement for optimization of adme and biological properties of top two selected compounds zinc0 and zinc0 the bioisostere replacement of functional group was performed the bioisosteric replacement tool from maestro molecular modelling platform was employed to create bioisosteric structures of better potency and adme profile further the results of the generated bioisosteres were analysed through interaction of ligands with crucial amino acid residues xp glide docking score free binding energy and adme analysis results nep was prepared at a neutral ph of αhelical α subdomains were present in the extracellular domain both helical subdomains of nep are connected with the linker region ± two and essential catalytic triad are present in the central cavity of both subdomains in the central cavity the catalytically impor tant zinc atom is coordinated with the side chains of amino acid residues his583 his587 and glu646 [ ] in the protein the cocrystallized ligand sacubitrilat is bound to the active site of nep and showed crucial interactions with his583 his587 and glu646 residues a fourth interaction was provided by the car boxylate oxygen adjacent to the p1 methyl group of sacubitri lat to generate a receptor grid receptor grid generation workflow was used and the cubic box of specific dimensions was generated around sacubitrilat to perform molecular docking studies ligand docking around ligands from zinc database were screened with htvs docking mode of glide panel htvs docking mode utilizes a small period to a large set of drugs by reducing the final torsional refinement and comprehensive sampling but during htvs dock ing mode the number of intermediate conformational sampling is limited hence a total of drugs with dock scores less than kcalmole were filtered and reanalyzed in sp docking mode after performing sp docking around drugs were subjected to an extensive xp docking mode of glide panel xp docking mode is more accurate avoids the possibility of falsepositive results and gives an appropriate correlation between a good pose of drugs and a good dock score finally based on xp dock score and pivotal interactions eight active drugs zinc0 zinc0 zinc0 zinc0 zinc0 zinc0 were identified for further screening the docking score of cocrystalized ligand sacubitralat was found to be all the eight selected drugs showed docking scores between to given in table zinc0 zinc0 all the eight drugs showed similar interaction as sacubitri lat schiering nikolaus et al had reported that the hydropho bic interaction of sacubitrilat with phe544 was towards the shal low s1 pocket of nep protein the charge positive interac tion with arg717 and polar interaction with asn542 were found to be common in sacubitrilat and selected eight drugs even in this study all the eight drugs showed hydrophobic interactions with phe544 sacubitrilat also showed interactions with asn542 arg717 arg110 and arg102 our eight selected drugs showed in teractions with atleast two of the aforementioned residues insilico docking studies also showed that all the eight drugs showed in teraction with his711 which then formed a hydrogen bond with zinc causing the stabilization of zinc transition state this in teraction with zinc and its stabilization might result in decreased catalytic activity of nep as it is a zinc dependent endopeptidase nep degrades various peptide substrates at the amino sides of hydrophobic amino acids according to the reports the pro tein structure of nep consists of a large hydrophobic pocket con taining the side chains ala543 ile558 phe563 met579 val580 0c r sankhe e rathi and s manandhar of molecular structure his583 val692 and trp693 the cocrystalized ligand sacu bitrilat showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 the eight se lected drugs also showed hydrophobic interaction with ala543 ile558 phe563 met579 val580 val692 and trp693 but the hydrophobic interaction with ile558 met579 and trp693 were missing in interactions of zinc0 zinc0 and zinc0 respectively sacubitrilat and the selected eight drugs showed polar pipi stacking and cation interaction with his583 the interactions with side chains of ala543 ile558 phe563 met579 val580 his583 val692 and trp693 may con tribute to inhibition of the peptidase activity of nep according to previous reports amino acid residue glu584 is important for peptidase activity and residues such as ala543 and asn542 are important for nep inhibition in the current study all eight selected drugs possess interaction with glu584 asn542 and ala543 the 2d interaction diagrams with a summary of all non bonding interactions are given in table free ligand binding energy calculation the primemmgbsa was employed to calculate the binding en ergy of the top eight drugs with selected docked poses all the 01g bind eight drugs showed stability in the docked pose with 01g bind ing energy kcalmol described in table the ing energy of cocrystallized drug sacubitrilat was found to be 9651kcalmol the cocrystalized ligand and the eight drugs were found to be stable with docked pose this finding indicates that the selected drugs may act as nep inhibitors induced fit docking ifdsp after the virtual docking studies based on the ligand interac tion and binding energy of the eight drugs four ligands showing good values were taken forward for induced fit docking ifd in virtual docking protocol the interactions occur between the bind ing site of the rigid protein and the flexible ligand but this is not the case with the actual ligandprotein interactions in the body where the target protein undergoes backbone or sidechain move ments after binding with ligands this induces alteration in binding sites of the protein also in the body the ligand binding site on the proteins conforms to the ligand shape and binding mode ifd was conducted to resolve the shortcomings of rigid docking pro tocols ifd has two main applications first it generates the most accurate active complex structure of ligand which is not possi ble in virtual molecular docking with rigid protein structure sec ond ifd avoids falsenegative results of virtual docking in virtual docking screening of the ligands was done with the single confor mation of ligands however in ifd confirmers were generated for each ligand hence ifdsp was carried for zinc0 zinc0 zinc0 and zinc0 and a maximum of conformers were generated for each ligand based on molecular docking and binding energy further the ifd score and ligand interaction were analyzed for selected drugs the ifd score and 3d ligand interactions are given in fig zinc0 showed similar nonbonding interactions as predicted in xp docking the zinc0 exhibits a new hbond interaction with his711 with similar nonbonding interactions as observed in xp docking in ligand interactions of zinc0 the new hbond interaction was observed with his711 and lost with glu584 the hydrophobic interaction with ala543 val580 met579 phe689 val692 trp693 phe563 and phe106 was also lost similarly new hydrophobic interaction was observed with ile718 and lost with ile558 and phe544 the new pipi stacking interactions were observed with trp693 and phe106 and missing with amino acid residue his583 the pipi cation interaction with arg717 was retained and lost with arg110 as predicted in xp docking zinc0 retained hbond interaction with his711 and glu584 showed new hbond inter action with trp693 and lost hbond interaction with arg717 the new pipi stacking interaction was observed with phe106 zinc0 also showed new hydrophobic interaction with phe689 and met579 and hydrophobic interaction missing with tyr545 it also showed similar hydrophobic interaction patterns with other amino acid residues as predicted in xp docking adme analysis adme properties of the four drugs were analyzed using the quikprop module the adme profile was assessed using vari ous descriptor calculations such as molecular weight qplogherg qplogpow qppcaco human oral absorption psa and lipinski rule of five given in table all the selected drugs obey the lip inski rule of five molecular dynamics md simulation molecular dynamics is used to simulate ligandprotein com plexes in presence of systems with biological relevance it includes the explicit solvent representation with the entire protein the main advantage of md stimulation is that it represents the actual conditions of the biological system it provides a highly dynamic protein structure and the ligandprotein complex is solvated with water as happens in the biological system ifd however pro vides limited flexibility which is insufficient to mimic the actual conditions of a biological system hence md simulation studies were carried out to get insights into the top four drugs in terms of binding stability and nonbonding interactions with crucial amino acid within the drugbinding pocket of nep protein in a dynamic state in md simulation the frame was captured for 20ps which results in the generation of frames for 20ns stimulation time and saved in a trajectory further rmsd root mean square devi ation for nep protein and ˜lig fit prot™ for the ligands were com puted to estimate the stability of ligandprotein complex based on molecular docking score binding energy and ifd score the md simulation was carried out for four ligand protein complexes viz zinc0 01427nep docked complex complex zinc0 01533877nep docked complex complex zinc0 0601283nep docked complex complex and zinc0 03831594nep docked complex complex for com plex rmsd values for protein and ligand were found to be ˚a and ˚a respectively the rmsd values were found to be in the acceptable range ˚a but the drift in the ligandprotein complex was observed for a period of 05ns20ns in case of complex the ligandprotein stabilization was observed from 022ns and 59ns respectively and drift was observed for 720ns in complex the rmsd values are ˚a and ˚a for protein and ligand respectively for complex the rmsd values were found to be ˚a for both the complex was initially stable but there was drift for 313ns and eventually stabilization was observed for 1320ns according to the results obtained from md simulation complex is possibly more stable than complex and similarly complex showed rmsd value of ˚a for both the protein and the ligand the com plex showed initial drift from to 13ns but eventually stabi lized from 1320ns overall better stability in protein and ligand was observed in complex and compared to complexes and the rmsd plot of selected ligandprotein complexes are given in fig further the binding pattern and nonbonding interactions were analyzed for all four complexes the binding pattern was found to be different for all four complexes in complex the signifi 0cr sankhe e rathi and s manandhar of molecular structure table 2d interaction diagrams of top eight drugs with a summary of all nonbonding interactions sr no drug 2d ligand interaction diagrams nonbonding interaction sacubitrilat zinc000001533877 zinc000000001427 zinc000001851195 hbond glu584 his711 arg717 arg102 asn542 hydrophobic met579 val580 ile558 phe689 val692 trp693 phe563 phe106 ile718 ala543 phe544 polar his583 his587 asn542 salt bridge zn806 arg102 pipi stacking trp693 his583 charged positive arg102 his711 arg717 arg110 charged negative asp650 glu646 glu584 hbond arg717 glu584 ala543 asn542 hydrophobic ile718 phe689 val692 trp693 ala543 phe544 met579 val580 phe106 ile558 phe563 polar thr721 his587 his583 asn542 salt bridge zn806 his711 arg110 pipi cation his583 charged positive his711 arg717 arg110 charged negative glu646 asp650 glu584 hbond ala543 his711 glu584 hydrophobic ile558 phe544 ala543 val580 met579 ile718 phe689 val680 trp693 phe563 phe106 polar asn542 his583 his587 salt bridge zn806 pipi stacking his583 trp693 charged positive arg717 his711 charged negative asp650 glu646 hbond glu584 his711 ala543 trp693 hydrophobic ile718 ile558 ala543 phe544 phe689 ala690 val692 trp693 met579 val580 phe563 phe106 polar thr721 his587 his583 asn542 salt bridge zn806 pipi stacking trp693 charged positive arg717 his711 arg110 charged negative asp650 glu646 glu584 zinc000000402909 hbond his711 glu584 hydrophobic ile718 ala543 phe544 phe689 val692 trp693 met579 val580 phe106 phe563 polar his587 his583 asn542 pipi stacking phe106 his583 salt bridge zn806 charged positive arg717 his711 charged negative asp650 glu646 glu584 continued on next page 0c r sankhe e rathi and s manandhar of molecular structure table continued sr no drug 2d ligand interaction diagrams nonbonding interaction zinc000000601283 zinc000000000797 hbond his711 glu584 hydrophobic phe544 ala543 trp693 val692 phe689 val580 met579 phe106 ile558 phe563 polar his587 his583 asn542 salt bridge zn806 pipi stacking his583 pipi cation arg717 arg110 charged positive arg102 arg110 his711 arg717 charged negative asp709 glu646 glu584 asp650 hbond asn542 hydrophobic ile718 val580 met579 phe689 val692 trp693 ile558 ala543 phe544 phe563 phe106 polar his587 his583 asn542 salt bridge zn806 pipi stacking his711 phe544 his583 pipi cation his711 charged positive arg717 his711 charged negative glu646 glu584 asp650 zinc000003831594 hbond glu584 his711 arg717 hydrophobic val580 ala543 phe544 tyr545 phe106 phe563 ile558 trp693 val692 polar his587 his583 asn542 salt bridge zn806 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 zinc000028973441 hbond glu584 his711 hydrophobic met579 val580 phe544 ala543 phe106 trp693 val692 phe563 ile558 polar his587 his583 asn542 salt bridge zn806 pipi stacking phe106 pipi cation arg110 his711 charged positive arg717 his711 arg110 arg102 charged negative glu646 glu584 asp650 0cr sankhe e rathi and s manandhar of molecular structure fig 3d ifd ligand interactions and scores of the top four selected drugs ligand interaction of a zinc0 b zinc0 czinc0 0601283d zinc0 with different amino acid residues of nep fig rmsd plot of ligandprotein complexes rmsd plot of a zinc0 b zinc0 c zinc0 d zinc0 with the active site of nep 0c r sankhe e rathi and s manandhar of molecular structure table adme analysis of top four selected drugs using qikprop compound id molecular weight qplogp ow qplogherg qplogs qppcaco oral absorption psa rule of five sacubitrilat zinc000001533877 zinc000000001427 zinc000000601283 zinc000003831594 fig ligandprotein interaction diagram obtained after md stimulation ligand interaction of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep cant hbond interactions were observed with amino acid residues glu584 ala543 and his711 and pipi interaction with his583 and trp693 as predicted in xp docking the hydrophobic interac tions with ala543 trp693 met579 and phe689 were retained in md simulation on the other hand hydrophobic interactions with ile558 phe544 and phe563 were missing in md simulation the hydrophobic interaction with ala543 val580 ile718 val692 and phe106 was weaker affecting the stability of the ligand protein complex similarly the water bridgetype interaction with glu584 was observed in complex strong hbond interaction was shown by asn542 arg717 glu584 and ala543 additional hbond interactions were also observed with his711 and glu646 the hydrophobic interaction with ala543 ile718 phe689 trp693 met579 val580 ile558 phe106and phe563 were weakly con tributing to the stability of ligandprotein complex and the inter action was lost with the amino acid residue phe544 additional water bridge type of interaction was shown by asn542 glu646 and ala543 the pipi cation interactions were retained with his583 as predicted in xp docking in complex hbond interac tion was retained with glu584 and his711 and new hbond inter action was observed with asp709 and arg110 in md simulation complex showed weak hydrophobic interaction with ala543 phe544 val580 trp693 phe563 ile558 and phe106the hy drophobic interaction was lost with amino acid residues met579 phe689 and val692 the new pipi stacking interaction was ob served with his711 however pipi stacking interaction was missing with his583 the new pipi cation interaction was observed with arg717 and pipi cation interaction was missing with arg110 as compared to xp docking the additional water bridge type of inter action was shown by asp709 and glu584 in complex hbond interaction was retained with his711 and arg717 new hbond in teractions were found with trp693 and ala543 whereas hbond interaction was lost with glu584 complex showed strong hy drophobic interaction with trp693 and ala543 whereas weak hy drophobic interaction with val680 phe106 phe563 ile558 and val692 in contrast to xp docking similarly hydrophobic interac tion was missing with amino acid residues phe544 and tyr545 the additional water bridge type of interaction was observed with ala543 among all four complexes complexes and were found to more stable the additional hbond interactions in complexes and may contribute to the stability of the ligandprotein com plexes the ligandprotein md interaction diagrams and histograms of selected complexes are given figs and bioisostere replacement the zinc0 indomethacin a nonsteroidal anti ‚ammatory drug and zinc0 tyropanoic acid a ra diocontrast agent were found to be more stable in md simulation for 20ns the zinc0 is anti‚ammatory antipyretic 0cr sankhe e rathi and s manandhar of molecular structure fig histogram of ligandprotein complexes histogram of a zinc0 b zinc0 c zinc0 d zinc0 with different amino acid residues of nep and analgesic in nature it is commonly used in rheuma toid arthritis acute shoulder pains osteoarthritis spondylitis and acute gouty arthritis zinc0 is known as sodium tyropanoate which is employed in xray diagnosis and imaging of gallstones though they exhibit good binding affinity for nep one of the major disadvantages of zinc0 is its rapid elimination from the body [ ] therefore bioisostere re placement of zinc0 and zinc0 was per formed to enhance biological activity and surpass rapid excretion bioisosteres are the molecules which are generated by replace ment an atom or a group of atoms from the parent drug with other functional groups two main advantages associated with bioisostere replacement are first it will result in generation of new bioisostere molecules with similar biological characteristics of the parent drug second bioisosteres can overcome various prob lems associated with the parent drug™s activity pharmacokinetics and toxicity during the bioisosteric replacement and bioisosteric structures of zinc0 and zinc0 respec tively were generated out of these the top two bioisosteres were identified based on the ligand interactions with the crucial amino acid residues of nep docking score the binding energy calculated employing mmgbsa and adme parameters the top two selected bioisosteres of zinc0 and zinc0 are il lustrated by fig the docking scores of the bioisosteres of zinc0 structure structure are and with binding en ergies and kcalmol respectively similarly the dock ing scores of structure and of zinc0 were found to be and with binding energies and Ï Ïkcalmol respectively table further assessment was done based on the ligand interactions with crucial amino acid residues of the protein compared to the parent drugs table structure of zinc0

"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a

"SARSCoV2 infection associated respiratory disease COVID19 has evolved into a pandemic but being anew form of virus pathogenesis of disease causation is not fully understood and drugs and vaccinesagainst this virus are still being tested so that no effective drugs or vaccines have been advised byregulatory authority In this context the Ministry of AYUSH Government of India has recommended˜Ayush Kwath™ to improve the immunity and combat the infection Our objective of this literature reviewis to review the role of immunity in pathogenesis of COVID19 and role of Ayush Kwath against the virusand regulation of immunity Current review was conducted using a search of available literature onCOVID19 and immunity Vyadhikshamatwa Ayurveda and COVID19 Rasayana Coronavirus SARSCoV immunomodulatory effects of medicinal plants TulsiHoly BasilOcimum sanctum DalchiniCinnamonCinnamomum zeylanicum SunthiGingerZingiber officinale and MarichBlack PepperPiper nigrum Ayurveda being an ancient science have both medicinal and cultural values and had stimulated our kitchenand ‚uenced what we ate in different seasons and the remedies we used for common ailments Herbssuch as Tulsi Marich Sunthi Dalchini are the most commonly used and easily available drugs in homeThus Ayush Kwath due to its immunemodulatory antiviral antioxidant anti‚ammatory antiplatelet antiatherosclerotic hepatoprotective renoprotective properties seems to be effective inimmunoregulation for controlling viral infections like COVID19 Further preclinical and clinical trialsneed to be done for the evaluation of safety and efficacy of this polyherbal formulation The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciencesand Technology and World Ayurveda Foundation This is an access under the CC BYNCNDlicense httpcreativecommonslicensesbyncnd40 IntroductionCOVID19 also known as severe acute respiratory syndromecorona virus SARSCoV2 is an infectious disease believed to beoriginated from bats and transmitted to human beings [] Being anew form of virus pathogenesis of disease causation is not fullyunderstood and drugs and vaccines against this virus are still beingtested so that no effective drugs or vaccines have been advised byregulatory authority Not only for Coronavirus have many otherviruses also lack preventive vaccines and effective antiviral medications Studies have explored that these viruses can form drugresistant mutants which decrease the existing drug™s efficacy Sothese viruses can be a threat to the mankind for long time [] Corresponding authorEmail shankargautammohpgovnpPeer review under responsibility of Transdisciplinary University BangaloreHigh mortality among immunecompromised and those withsome underlying pathology implies that the factors that improveimmunity can prevent serious manifestations due to COVID19infection [] Many herbal products are found to have immunemodulatory and antiviral property so their discovery can be amilestone in the prevention and control of COVID19 [] In thiscontext the Government of India has recommended to take ˜AyushKwath™ in order to boost the immunity As this is a new formulationthis needs to be validated scientifically We have made an attemptto review the immunepathogenesis of COVID19 and the role ofeach herb over it Immunopathogenesis of COVID19The ˜S™ protein of coronavirus can bind to host cells through theACE2 receptor found in the oral and nasal mucosa [] Other siteswhere ACE2 receptors are found are lungs stomach intestinebladder heart and kidney [] Variable presentation of disease in101016jjaim202008003 The Authors Published by Elsevier BV on behalf of Institute of Transdisciplinary Health Sciences and Technology and World Ayurveda Foundation This isan access under the CC BYNCND license httpcreativecommonslicensesbyncnd40Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxdifferent age groups serious manifestations that are seen morecommonly in immunecompromised old aged and in those withunderlying pathology many asymptomatic cases in pediatric agegroup and presence of lymph ia in the majority of the casesthese factors implies that immunity has a vital role in the pathogenesis of COVID19 [16e8] It is assumed that our immune systemhas lack of memory against such a virus that gave it an edge overhumans []Viruses cause cell destruction mainly in two ways direct cytopathic effects ofthe virus and immune response mediateddestruction [] COVID19 cannot lyse the cells directly as the majorpathway of cell destruction is due to immunemediated destruction[] It has been mentioned that unlike adults less vigorous cellmediated immune response in alveoli of children results in beingasymptomatic in the majority of cases []The pathogenesis can be split into two stages Nonsevere andSevere [] Nonsevere stageThe virus fuses with the host cell membrane and enters insidethe host cell through airway epithelium [] The virus propagates and multiplies inside the host cell and can reach lower airwayand alveoli In adults with good innate cellular and humoral immunity propagation of virus can be limited and viral load reachingalveoli can be reduced thus recovery can take place within 2e3weeks with mild symptoms []Humoral immunity prevents the viruses to enter new cells whilecellmediated immunity targets on eradicating virusinfected cells[] In this stage a strong immune system can be helpful inpreventing the propagation of the virus thus reducing the severityof the disease [] Severe stageOnce the immune system is breached the virus propagates andreaches the lower respiratory tract and alveoli Then the virus canpenetrate alveoli and reaches systemic circulation causing viremia[] The virus binds to multiple ans having ACE2 receptor protein During this stage cellmediated immunity becomes robustand starts releasing various pro‚ammatory cytokines IFNaIFNg IL1B IL6 IL12 IL18 IL33 TNFa etc and chemokinesCCL2 CCL3 CCL5 CXCL8 CXCL9 CXCL10 etc causing damage tomultiple ans known as Cytokine storm [] We may need tosuppress the ‚ammation for improvement during this severestage []Tocilizumab and anti‚ammatory interleukin IL10 are proposed to have a therapeutic role in the reduction of severity and mortality of COVID19[] As increased risk of thromboembolic phenomena is alsofound to be associated with COVID19 prophylactic antithromboticmedications are advised during this stage []IL6 receptor antagonist Ayurveda purview Disease conceptIt seems that most early cases had a history of contact with theoriginal market for seafood but the disease has now advanced to betransmitted through human to human contact [] Thus this disease can be considered as Communicableboth contagious and infectious diseases In Ayurveda epidemics are discussed under theterm of Janapadodhwamsa [ CSVi ] by Charaka and Marakaby Sushruta [ SSSoo ] The symptoms like fever coughbreathing difficulty headache and vomiting resemble with clinicalfeatures of SARS [ SSSoo ] Dalhana in his commentary hasmentioned that symptoms like anosmia cough catarrh will occurafter the entry of contaminated air through the nasal ingwhich is similar to typical clinical features of COVID19 [ SSSoo] Furthermore this disease can be classified as Adidaivika BalaPravritta Vyadhi ABPV Sansargaja Upsargaja and Aupasargic RogaABPV are those diseases arising due to causes that cannot becontrolled by human intelligence Upasargaja Vyadhi are thosefeverlike diseases that manifest due to close contact with diseasedpersons [ SSSoo ] whereas Sansargaja Vyadhi resides withpeople who are cursed by almighty god ie due to ‚uence ofinvisible forcesforces behind human control [ SSSoo ]Aupasargic Vyadhi is defined in two different ways by Sushruta oneas a disease which spreads from one person to another person [SSNi ] and another as ˜¦Upadravasangyah™ ie complications or associated diseases that manifest after primary disease [SSSoo ] Susruta mentions the diseases like Jwara Kusthaskin diseases Shosha tuberculosis Netrabhisyandi conjunctivitis and other Aupasargika roga alike communicable diseasescan be spread through Prasanga intimate relationship GatraSansparsha direct contact Nishwasa breathing or airborneSahabhojana eating together Sahashayana sleeping togethersharing and using of others™ clothes ornaments ointments etc [SSNi ]Agantuja Vyadhi Ì´ diseases of exogenous origin occurs due tophysicalexternal factors like Bhuta Visha Vayu Agni and Praharatrauma etc without any involvement of Vataadi Dosha initiallyhowever in later stage dosha are involved in the disease process[ CSSoo1145] Cakrapaṇidatta clarifies that Bhuta meansVisakṛimi or a virulent anism [ CSSa1121] Krimi may beSahaja natural or Vaikarika pathogenic anisms that may bevisible macroscopic or invisible to the naked eye microscopic[ CSVi ]Thus it is difficult to correlate this disease with specific Ayurveda terminology but while interpreting the disease on the basis ofSamprapti by considering the causative agent and the clinical features like fever Jwara cough Kasa anorexia Aruchi fatigueTandra generalized body ache or myalgia Angamarda andTiredness it can be contemplated as an Agantuka Vyadhi whichlater on due to the involvement of dosha develops to Nija Vyadhi asKaphaVatolvana Hina Pitta Sannipataja Jwara Severe Vata andKapha with mild Pitta [ CSSoo CSChi ] Whiletalking about the pathogenesis of fever in Ayurveda Charakamentioned that when Vataadi dosha either singly or in Sansristatwo dosha or in Sannipataja all three dosha got aggravated thenit enters Amashaya and mixed with Rasa Dhatu causing obstructionof Rasavaha and Swedavaha Srotas resulting in the destruction ofAgni Agni then spreads out from its Sthana to whole over the bodycausing the febrile condition [ CSNi120 CSChi3129]Immunity concept in AyurvedaStrength health lifespan and vital breath are dependent on thecondition of Agni [ CSSoo ] Charaka has mentioned theterm Vyadhikshamatwa and states that during certain conditions ordue to certain factors even unwholesome unhealthy food doesnot produce disease immediately all unwholesome diet are notequally harmful all dosha are not equally powerful all persons arenot capable of resisting diseases [ CSSoo ] This suggeststhat the body™s immune system plays a crucial role in diseasedevelopment The equilibrium state of Dhatu is called Swasthya [CSSoo ] The person who is desirous to be healthy should adopthealthy practices related to diet conduct and activities [ CSSooImmunity can be considered in Ayurveda as] ThusPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxVyadhikshamatwa and Oja which depends on the condition of AgniDosha and DhatuThere are three factors Aahara Swapna and Brahmacharya dietsound sleep and celibacy that support the life with which the bodywill be endowed with strength complexion and development tilllife span [ CSSoo ] Bala Ì´ StrengthImmunity is of threetypescongenital time affected and acquired Congenital is thatwhich is developed naturally in the body and mind time affected isdue to seasonal variation and age factor and acquired one is produced by the proper application of diet and exercise [ CSSoo ] Thus not only diet but also performing yoga or exercises withproper methods by giving rest in between exercises as Rasayanatherapy will increase acquired strength [ CSSoo1136] Oja isalso called Bala is the essence of all Sapta Dhatu being located inHridaya combines with Rasa and circulates through the Dhamaniand performs Tarpana or Prinanam of the whole body [ SSSoo CS1774] The equilibrium state of Kapha promotesstrength that™s why normal Kapha is called Oja [ CSSoo ]Normal pure blood promotes strength complexion health andlifespan [ CSSoo ] While dealing with Sannipataja JwaraSusruta in Uttarsthana mentioned Abhinyasa Jwara also called asHataujasa Jwara indicating the loss or deranged condition of Oja[ SSUtt ]The word ˜Rasayana Rasa þ ayana™ refers to nutrition and itstransportation in the body for attaining excellent Rasadi Dhatuswhich leads to gain longevity freedom from disorders optimumstrength of physique and sense ans [ CSChi ]Rasayana promotes nutrition by explicitly enriching the nutritionalvalue of Rasa by enhancing Agni ie digestion metabolism andabsorption by Srotashodhana Consequently any medication thatimproves Rasa™s consistency would enhance the health of all bodytissues Role of Ayurveda and traditional medicineEvery society has its own medical system which is deeplyrooted in its culture and guided by its philosophy of life Beingculturally and linguistically diverse countries there developedseveral types of traditional medicines TM based on practices skilltraditional knowledge based on beliefs theories and experiencesindigenous to different cultures Ayurveda Traditional Chinesemedicine TCM Ancient Egyptian medicine Sowa Rigpa etc system of medicine remain the most ancient yet living traditions inSouth East Asia Western Pacific Eastern Mediterranean Africaregion Up to percent of the population in some Asian and African countries depend on TM for primary health care PHC needs[] Still there is a high trend of using many herbs in religious andcultural works therapeutically for common ailments and as spicesfor foods according to occasion specific and seasonal regimes Ayurveda and TM have made a significant contribution to the prevention and alleviation of various communicable and noncommunicable diseases for thousands of years A long history ofusing many herbal remedies and experiences passed from generation to generation has resulted in people relying on herbal remedies and some simple home remedies for common diseases can beused even by illiterate citizens The selfcare an integral part ofPHC with home remedies using various herbs is the most commontreatment for India Nepal Bhutan and China for different flucommon cold fever GI disorders etc Prevention of smallpox inChina has been an epochmaking effort in the period of mankind™spreventive care One observational study found that the prevalencebetween the total number of COVID19 cases per million populationand the grams of spice supply per capita per day is clearly interrelated Most nations with lower spice intake per capita reportedmore COVID19 cases per million population and vice versa []Nevertheless with the invention of drugs many herbal remediesused historically have become modern medicines Few notableexamples include morphine digoxin artemisinin and colchicine Asmany herbs are found to have immunomodulatory role and possessantiviral activity many people are being optimistic over the traditional system of Medicine Ayurveda and TCM have descriptions ofimmunomodulation along with antiviral treatments even targeted to the coronavirus family []The key factor for COVID19 to occur and evolve is the interaction between the virus and an individual™s immune system [] Asmedicinal plants enhance NK cell activity inhibit activated transcription factor ATF2 downregulate Th17related cytokinesincluding transcription factor RORc IL17A and Th2related cytokines including IL5 IL13 and IL6 inhibit GATA3 IL4 IL6 IL1bRt IL17A TNFa expression and increase the secretions of ILit shows that natural products have potentimmunemodulatory and immuneboosting effects that may behelpful during the infection course by increasing innate immuneresponse to infections []INFg etc Ayush KwathConsidering the importance of immunity boosting measures inthe wake of COVID19 outbreak the Ministry of AYUSH Government of India with the interest of health promotion of the massesrecommends ˜Ayush Kwath™ or ˜Ayush Kudineer™ or ˜Ayush Joshanda™which comprises of four medicinal herbs Table [] Theherbs like holy basil cinnamon ginger black pepper are highlyavailable accessible and widely used in the kitchen and areconvenient to educate and train about its use to community healthworkers community and even to all public that they can have costeffective treatment with herbal home remedies This will help topromote immunity and to lower the gatherings at hospitals andpharmacies in this pandemic This type of public health measurewould eventually promote ˜health for all™ with the theme ˜ourhealth in our own hands™ making responsible to each and everypeople by active involvement in their own health instead of relyingon mass distribution of some medicine As people leave theirhomes to earn a living this herbal decoction will ensure broadaccess to health care The WHO SEARO adopted a resolution torevitalize PHC through health systems strengthening to achievehealth for all with the emphasis on health promotion and diseaseprevention [] This Kwath is not just a mechanical mixtureinvented for the COVID19 pandemic but it is a revival of healthtraditionMethod of preparation and useTake all the ingredients in dry form as per standards laid downin Ayurvedic Pharmacopoeia and make coarse powder Make sachets or tea bags each of g of powder or mg tablet of aqueousextract to be consumed like tea or hot drink by dissolving in mlof boiled water once or twice daily Gud JaggeryDraksha Resinsandor Lemon Juice can be added while consuming the formulation TulsiMany invitro animal and human experimental scientificstudies showed that due to presence of eugenol phenolic compoundslinoleic acid etc compounds Tulsi has antimicrobialincluding antibacterial antiviral antimalarial antidiarrhealantioxidantcardioprotectiveimmunemodulatory properties and is thus recommended as a treatmentfor a range of diseases including features like cough fever asthmaanxiety diarrhea gastric cardiac and genitourinary disorders[32e36] Due to its anti‚ammatory and antioxidant properties itanti‚ammatoryhepatoprotectiverenoprotectiveanalgesicantipyreticPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxTable Contents and properties of Ayush KwathSN NameScientific name Parts used Main chemicalRasaVirya AyurvedicSansthanika KarmaProportion RemarksTulsiOcimumsanctum LinnLeavesconstituentsVolatile oil PhenolAldehyde EugenolAscorbic acid Linoleicacid CaroteneDosha karmaKatu Tikta Ushna KaphavatashamakaPittabardhakaDalchini CinnamomumStem Bark CinnamaldehydezeylanicumBreyncuminaldehydeEugenolKatu TiktaMadhuraUshna KaphavatashamakaPitta vardhakaSunthiZingiberofficinale RoscRhizomeZingiberene Zingiberol KatuUshna KaphavatashamakaVedanahara DeepanaPachana AnulomanaKrimighna HridhyaRaktashodhakaKasahara SwasaharaKshayanashakaMutrala VishaghnaJwaraghna esp useful inVatashlaishmikaVishama and Jirna JwaraDeepana PachanaVajikaranaVataanulomanaYakridutejaka GrahiHriyottejakaOjovardhakaRaktashodhakaShelshmaharaYakshmanashakaMutrajananaDeepana PachanaVrishyaShoolaprashamanaRaktashodhakaHridhyottejakaShothahara KaphaghnaSwasahara JwaraghnaAampachana partsPrabhava Specialaction Krimighna parts partsPrabhava KrimighnaContraindicationsPandu KushthaMutrakriccharaktapitta Grishma andSharada Ritu MarichPiper nigrumLinnFruitPiperine PiperidinePiperettine andChavicineKatuUshna Kaphashamaka Deepana Pachana partYakriduttejakaVatanulomanaKrimighnaHriddhyottejakaKaphaghnaKaphamissarakaJwaraghna espVishamjwarapratibandhakaprotects against toxic chemicalinduced injury enhance the antioxidant enzymes and protect cellular anelles and membranes byclearing damaged free radicals []The compounds such as ursolic acid carnosol rosmarinic acidcirsilineol apigenin eugenol and cirsimaritin present in O sanctumincrease haemoglobin concentration enhance SRBC agglutinin titers decrease cyclooxygenase CoX2 and lipoxygenase LOX5enzymes activity suppress NFkB classical pathway up regulationof IL2 IFNg and TNFa down regulation of IL1b and produce ofSRBC antigenspecific antibodies which represent a major defensemechanism to assess Tcelldependent antibody responses ie Tulsiby enhancing immune response boost the defense mechanismagainst the infection [38e40] Several studies have shown that Tulsiaqueous and methanol extract of leaf and seed oil besidesimproving vital capacity also is an immunemodulator and regulator as it enhances immune response by increasing Thelper andNK cells phagocytic activity and index with the rise in lymphocytecount neutrophil count and antibody titer []In an acute toxicity study it did not produce any hazardoussymptoms or CNS and ANS toxicities or death and did not show anychange in water and food consumption body weight and hematological and biochemical profiles [] DalchiniIt is a potent immune system booster and is used in variousailments like flu indigestion edema cough etc [] Cinnamonbark contains cinnamaldehyde benzaldehyde cuminaldehyde andterpenes [] In one study cinnamon at high dose mgkgshowed immunestimulant activity as it significantly increased thephagocytic index serum immunoglobulin levels and antibody titerand decreased the percentage reductions in neutrophil countCinnamon low dose mgkg increased serum immunoglobulinlevels only This showed that high dose increases both cell mediated and humoral immunity whereas low dose showed effect onlyon humoral immunity [] The studies also suggest that cinnamaldehyde can act as a strong regulator of monocytemacrophagemediated immune responses by inhibition of PI3K PDK1 and NFkBactivation of signaling components In addition to this by theactivation of CD29 and CD43 it blocked cell migration cellecelladhesion induced but not cellfibronectin adhesion and it wasable to suppress both the production of nitric oxide NO and upregulation of surface levels of costimulatory molecules CD69 andCD80 and pattern recognition receptors TLR2 and CR3 []Cinnamon bark decrease systemic levels ofIFNg withoutaltering the levels of IL4 or IL2 inhibit antiCD3 AbstimulatedIFNg and IL4 at the mRNA and secreted protein levels enhance IL protein secretion at the cellular level which helps to decrease celldeath inhibit IL2mRNA expression inhibit antiCD3induced p38JNK ERK12 and STAT4 activation but not IkBa degradation orSTAT6 and ultimately alter the ‚ammatory responses in T cellsThis shows the immunemodulatory effect of cinnamon on cytokine secretion and the involvement of intracellular signaling molecules in activated T cells It also causes a reduction in the subG1Please cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxphase accompanied by an increased ratio of apoptotic cells tonecrotic cells [] The constituents like cinnamaldehyde cinnamophilin etc are found to be a thromboxane A2 receptor antagonistanticoagulative antiatherosclerotic and thus prevents unnecessary clumping of platelets and atherosclerotic CVD []In a systematic review of its adverse events relatively few selflimiting adverse effects were reported like allergic reactions andgastrointestinal disorders on clinical trials case reports and caseseries The evidence available show that cinnamon is safe for use asspice in daily diets or as a medication [] However its use fortherapeutic reasons in high doses or for prolonged periods cancause some adverse effects and should be observed clinically SunthiAn alcohol extract increases the immunological status of micewith increased phagocytosis by macrophages whereas crudeextract was also shown to increase humoral and cellmediatedimmune responses [] The bioactive compounds of ginger suchas nevirapine bsitosterol 6gingediol germacrene methyl6shogaol 6gingerol alinalool 6shogaol gingerdion zingibereneetc are known to inhibit viral replication among these the mostpotent inhibitors of reverse transcriptase RT enzyme is bsitosterol which is predicted to be used as nonnucleoside reversetranscriptase NNRTIs HIV1 inhibitors [] It is reported thatGinger contains TNFa which is also known as an anti‚uenzacytokine [] The rhizome of Ginger and its main componentslike gingerols shogaols etc inhibit prostaglandin and leukotrienebiosynthesis inhibit cyclooxygenase and lipoxygenase activitiesinhibits the synthesis of pro‚ammatory cytokines such as IL1TNFa and IL8 without any significant effect in IL6 levelsinhibit the excessive production of NO PGE TNFa and IL1beta reduce the elevated expression of NFkB and TNFa downregulate ‚ammatory iNOS and COX2 gene expression inhibitthromboxane synthetase raise levels of prostacyclin without aconcomitant rise in PGE or PGE alpha inhibit platelet aggregation decrease agerelated oxidative stress markers and enhancefibrinolysis [53e58]The concentration of IgM and eosinophil count in nonsmokerswas significantly increased in a comparative study of the effect ofginger extract among male smokers and nonsmokers whereas theconcentration of hemoglobin and lymphocyte count in smokerswas strongly increased This indicates that in nonsmokers gingerresults in a stronger antibody response or humoral immunity thanin smokers []According to Ayurveda it is contraindicated to be used in a fewdiseases Kushtha Pandu Mutrakriccha Raktapitta and in Grishmasummer and Sharada autumn Ritu There are few minor adverseeffects recorded that did not need care such as mild gastrointestinal symptoms sleepiness mild diarrhea during prior few days oftreatment It is also explained that ginger has the ability to induceheartburn and as a gastric irritant with doses above g [] Duringpregnancy ginger did not pose a major risk for side effects oradverse events [] MarichIt has been also found to increase bioavailability thus enhancethe therapeutic efficacy of many drugs vaccines and nutrients andhave immunemodulatory antioxidant antiplatelets antihypertensive antiasthmatic antipyretic analgesic anticarcinogenicanti‚ammatory antidiarrheal antispasmodic anxiolytic antidepressants hepatoprotective antiulcer antithyroids antiapoptotic antimetastatic antimutagenic antibacterial antifungal[62e65] The extract and itsand antiamoebic propertiesconstituents like piperine regulate the balance of the cytokinesproduction of Th1 Th2 Th17 and Treg cells reduce the accumulation of ‚ammatory cells inhibit the expressions of GATA3 IL4IL6 IL1b Rt IL17A and TNFa increase INFg and IL10 secretions in BALF Bronchoalveolar lavage fluid and increasemacrophage activation and T and B cell proliferation []Beside this Marich possess cytotoxic activity suppresses thelevels of total IgE antiOVA IgE antiOVA IgG1 and histaminerelease in serum ameliorates fibrosis and ltration of ‚ammatory cells inhibits the allergic responses inhibitsTh2Th17 responses and mast cells activation inhibits NFkB cFos cAMPresponse elementbinding CREB and activated transcription factor ATF2 suppresses PMAinduced MMP9 expression inhibitsPKCaextracellular signalregulated kinase ERK and reducesNFkBAP1 activation In addition piperine also inhibits the Pglycoprotein Pgp and CYP3A4 functions [67e69] Piper nigrum isfound to have dose dependent antifertility effects on mice [] DiscussionAccording to Ayurveda therapeutics is of two types Swasthasyorjaskarawhich promotes strength immunity in the healthyand Roganutwhich alleviates disorders Both of these groupsperform both of these functions but Rasayana and Vajikarana aremostly used for promotive treatment CSChi [] AyushKwath has both immune promoting and disease alleviating properties which can be achieved by various treatment modalities likeRasayana Satwawajaya Yuktivyapashraya Vyadhi Viparitarthakarichikitsa etcThe Katu and Tikta Rasa Usna Virya and Deepana PachanaYakriduttejaka properties of Ayush Kwath help to improve Agni andSrotosodhana improves microcirculation and tissue perforationthus promotes proper digestion metabolism and absorption andacts as Rasayana for the development of preceding Dhatu andfinally form Oja Oja itself acts as immunity to prevent diseaseImmunity is dependent on the condition of Agni Ayush Kwath withits Agni promoting and Kaphashamaka properties balance Kaphaand with Raktashodhaka Hridhya Krimighna properties purify theblood It is already mentioned that natural Kapha and pure bloodpromote Oja and Bala respectively Krimighna is the Prabhavaspecial action of Tulsi and Sunthi which directly acts againstpathogens The properties like Jwaraghna esp VatashlaishmikaVishama Kasahara Swasahara Kshayanashaka ShoolaprashamanaSwothahara Kaphaghna Hridayaottejaka Yakridutejaka have directrole to alleviate various clinical signs symptoms and complicationsAs this disease is considered as KaphaVatolvana Hina PittaSannipataja Jwara the Kapha Vata Shamaka properties of AyushKwath can play a significant role in balancing the vitiated doshasAfter six days of Jwara Charaka suggests the decoction of Pachanadrugs in the case of Amdosha and Shamaniya drugs in Niramadosha[ CSChi ] This shows that Yuktivyapashraya and Vyadhiviparita chikitsa can be done even after the involvement of Dosha inlater stages Ayush Kwath has potential psychoneuroimmunemechanisms via evidence of a reduction in depression anxietyand stress in controlled trials and shows meaning response as it is aspecific remedy for cough and respiratory problems this shows therole of Satvawajaya Chikitsa in its management []Immunity plays a key role in the pathogenesis of COVID19 bothduring the early nonsevere stage and during the severe stage ofthe disease The earlystage strong immune response may preventthe propagation and spread of viruses inside the body thusreducing the severity of cases and early termination of infectionWhile during later stage strong cellmediated immunity of thebody against the virus itself can be a factor responsible for graveconsequences due to cytokine storm The target during the earlyPlease cite this as Gautam S Immunity against COVID19 Potential role of Ayush Kwath J Ayurveda Integr Med 101016jjaim202008003 0cS Gautam Journal of Ayurveda and Integrative Medicine xxx xxxx xxxlikesteroidsand IL6 receptorstage should be to reduce viral propagation while at a later stageshould be to reduce the ‚ammatory response of the immunesystem Medicinal herbs with immune booster property can be anoption during the early nonsevere stage while herbs with anti‚ammatory and antithrombotic properties can be an optionduring a later or severe stage Cytokine storm that is believed as amajor factor responsible for complications and death of COVID19patients has been found to be reduced with anti‚ammatorydrugsantagonists Anti‚ammatory interleukins IL10 in modern medicine [] Therole of medicinal herbs with anti‚ammatory property on thecytokine storm is still lacking in research Like anti‚ammatoryinterleukins and IL6 receptor antagonist Tocilizumab IL thatare proposed in modern medicine to have a therapeutic role in thereduction of severity and mortality of COVID19 Cinnamon barkthat is found to decrease INFg and IL4 Its antiatheroscleroticanticoagulative and antiplate

" postmortem studies can provide important information for understanding new diseases and smallautopsy case series have already reported different findings in covid19 patientsmethods we evaluated whether some specific postmortem features are observed in these patients and if thesechanges are related to the presence of the virus in different ans complete macroscopic and microscopicautopsies were performed on different ans in covid19 nonsurvivors presence of sarscov2 was evaluatedwith immunohistochemistry ihc in lung samples and with realtime reversetranscription polymerase chainreaction rtpcr test in the lung and other ansresults pulmonary findings revealed earlystage diffuse alveolar damage dad in out of patients andmicrothrombi in small lung arteries in patients latestage dad atypical pneumocytes andor acute pneumoniawere also observed four lung infarcts two acute myocardial infarctions and one ischemic enteritis were observedthere was no evidence of myocarditis hepatitis or encephalitis kidney evaluation revealed the presence ofhemosiderin in tubules or pigmented casts in most patients spongiosis and vascular congestion were the mostfrequently encountered brain lesions no specific sarscov2 lesions were observed in any an ihc revealedpositive cells with a heterogeneous distribution in the lungs of of the patients rtpcr yielded a widedistribution of sarscov2 in different tissues with patients showing viral presence in all tested ans ie lungheart spleen liver colon kidney and brains in autopsies revealed a great heterogeneity of covid19associated an injury and theremarkable absence of any specific viral lesions even when rtpcr identified the presence of the virus in many anskeywords covid19 sarscov2 autopsy rtpcr immunohistochemistry correspondence isabellesalmonerasmeulbacbe1department of pathology erasme hospital universit libre de bruxellesulb route de lennik brussels belgium2centre universitaire inter rgional d™expertise en anatomie pathologiquehospitali¨re curepath chirec chu tivoli ulb rue de borfilet 12a jumet belgiumfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cremmelink critical care page of severeacuteincluding coronavirusesrespiratorysyndrome coronavirus sarscov and middle eastrespiratory syndrome coronavirus merscov causesevere acute respiratory failure which is associated withhigh mortality rates the novel sarscov2 strainexhibits phylogenetic similaritiesto sarscov andcauses coronavirus disease covid19 which hasresulted in more than deaths worldwide so faras the pandemic has progressed the pathophysiology ofthis viral infection has become clearer in particular ithas been shown that sarscov2 can directly alter cellfunction by a link to the angiotensin converting enzyme ace2 receptor which is almost ubiquitous in thehuman body nevertheless the mechanisms behind the high mortalityand severe an dysfunction associated with covid19remain poorly understood controversies exist regardingthe occurrence of fatal complications such as pulmonaryembolism or diffuse endothelial injury [ ] as well as onthe roles of direct viral cellular injury or concomitantcomorbidities in the fatality of this disease in this setting autopsy is of great importance to helpphysicians understand the biological characteristics andthe pathogenesis of covid19 most of the previously reported postmortem findings focused on lung morphologyand few data are available on complete postmortemanalyses of other ans [ ] the aim of this study wastherefore to investigate the presence of specific features ofviral injury as well as the distribution of the virus in different ans of patients who died from covid19methodsstudy designin this postmortem study we included the first adultpatients years who died in our hospital either in acovid19 unit or an intensive care unit from march with confirmed sarscov2 infection ie positivertpcr assay on nasopharyngeal swab andor bronchoalveolar lavage specimen exclusion criteria were lack offamily consent and a delay of more than days after deathbefore postmortem examination the study protocol wasapproved by the local ethics committee p2020218data collectionrelevantwe collected demographics comorbiditiesclinical dataincluding duration between symptomonset or hospitalization and death the results of chestcomputed tomography scan andif available microbiological tests and medical treatments eg hydroxychloroquine antivirals or antibiotics and use of ansupport acute respiratory distress syndrome ardsand acute kidney injury aki were defined accordingto standard definitions [ ]postmortem procedurethe belgian public health institute sciensano guidelines were integrated into our postmortem procedure the cadavers were kept in the refrigerator at °cand autopsies were performed to h after death toensure the safety of the autopsy team personal protective equipment consisted of two superposed disposablelatex gloves plastic sleeves ffp3 mask scrub hat clearface visor surgical gown plus plastic apron and rubberboots in the postmortem room dirty and clean circulations were used in the airlocks to allow decontaminationall analyses were performed at normal pressurespleen bone marrow kidney bladderusing standard surgical pathology processing completesets of tissue samples were collected for diagnosis andbiobanking the material was biobanked by biobanqueh´pital erasmeulb be_bera1 cub h´pital erasmebbmrieric the banked material consists of samplesper an including the trachea thyroid lymph nodesheartliverstomach colon and brain for the lungs we collected sixsamples per lobe ie a total of samples except fortwo patients who had undergone lobectomy for cancerand from whom only samples were taken for safetyreasons complete brain removal was not allowed butwith the help of a neurosurgeon in cases we used anew safe procedure with drills and protective devicesto avoid air dispersion to obtain between and samples from different brain regions as detailed inthe additional file additional material formalinfixed paraffinembedded ffpetissues underwentstandard processing to provide hematoxylin and eosinhestained sections special stains and immunohistochemistry ihc were used for lung masson™s trichromeperiodic acidschiff [pas] gomorigrocott anticmvihc antihsv ihc antipneumocystis j ihc and kidneypas masson™s trichromejones methenamine silversamplesmorphological analysismorphological analysis was performed on he stainedglass slides using the secundos digital platform tribvnhealth care chatillon francefor digital diagnosisafter the acquisition of whole slide digital scans — magnification using a nanozoomer ht slide scanner hamamatsu hamamatsu city japansarscov2 detection by immunohistochemistrysince no antibody against sarscov2 has been validatedfor ihc on ffpe tissues we selected an antisarsnucleocapsid protein antibody standard ihc was appliedas previously described to 4μmthick postmortem lungsections one sample for each lung lobe per patient to display sarsnucleocapsid protein invitrogen pa141098dilution on dako omnis agilent technologies 0cremmelink critical care page of santa clara ca usa using the envision flexdetection system according to the manufacturer™sprotocol the sections were counterstained withhematoxylin negative tissue controls were obtainedfrom patients who had an autopsy before the covid pandemic semiquantitative ihc evaluation wasperformed by two senior pathologists nd mr as follows negative ˆ’ between one and five positive cellsper whole slide scattered cells more than five cellsper whole slide but no foci isolated cells andwith foci more than cells in one — field sarscov2 detection by rrtpcrtotal nucleic acid was extracted from ffpe tissues usingthe maxwell rsc dna ffpe kit reference as1450promega corporation madison wi usa and thepromega maxwell extractor following the protocol described by the manufacturer onestep rtpcr assaysspecific for the amplification of sarscov2 e envelopeprotein gene were adapted from a published protocol briefly μl of rna ng was amplified in μl reaction mixture containing μl of taqman fastvirus 1step master mix life technologies μm ofeach forward acaggtacgttaatagttaatagcgt and reverse atattgcagcagtacgcacacaprimers and μm of probe famacactagccatccttactgcgcttcgbbq the amplification condition was °c for min for reverse transcriptionfollowed by °c for s and then cycles of °c for s and °c for s a clinical sample highly positivefor sarscov2 was diluted and used as a positive control in each analysis a clinical sample obtainedfrom a patient who was autopsied before the covid19pandemic was used as a negative control the quality ofthe rna from the samples showing negative results wasassessed by amplification of the human met rna according to a validated iso15189 accredited methodused as a routine diagnostic method in our laboratorystatistical analysisdata are reported as counts percentage or medians[interquartile ranges iqrs] all data were analyzedusing graphpad prism version graphpad software san diego ca usaresultsstudy cohortthe main characteristics of the study cohort malesout of median age [“] years are given intable the time period between the onset of symptoms and death ranged from to days median days and between admission and death from to days median days all except two patients had atleast one comorbidity including hypertension n diabetes n cerebrovascular disease n coronaryartery disease n and solid cancer n none ofthe patients had tested positive on admission for therespiratory syncytial virus or influenza a and b viruseseleven of the patients were treated with mechanicalventilation eleven patients died in the icu and on themedical ward the main causes of death were respiratoryfailure n and multiple an failure n laboratory data are reported in additional file table s1macroscopic findingsone patient had had a left pneumonectomy and onepatient a right bilobectomy the lungs were typicallyheavy and the lung parenchyma had a diffuse firmconsistency with redtan and patchy darkred areas ofhemorrhage thrombi were found in the large pulmonary arteries in patients and lung infarction in patientspleural adhesions associated with pleural effusions were observed in cases we observed cardiomegaly in and hepatomegaly in patients the kidneys were often enlargedwith a pale cortex and petechial aspect but no hemorrhageor infarct the gut had advanced postmortem autolysiswith no evidence of specific lesions except for one patientwho had ischemic enteritis in the patients for whombrain samples were available one had had a recently drainedsubdural hematoma and another a cerebral hemorrhagemicroscopic findingsfile as shown in figs and and additionaltable s2the main pulmonary findings includedearlystage diffuse alveolar damage dad which consisted ofinterstitial and intraalveolar edema withvariable amounts of hemorrhage and fibrin depositioninterstitial mononuclearhyaline membranes minimalinflammatoryii pneumocytehyperplasia microthrombi were noted in the smallpulmonary arteries in patients ten of the patientsalso had advanced dad lesions ie fibroblastic proliferation within the interstitium and in the alveolar spaces patients had evidence of acute pneumonia or bronchopneumonia had atypical pneumocytes and three hadsyncytial multinucleated giant cells we observed no viralinclusions or squamous metaplasiaand typeinfiltrateall the patients who survived more than weeksn had late dad lesions there was no relationship between the delay from onset of symptoms todeath orfrom hospitalization to death and thepresence of other histological lesions including bronchopneumonia pneumonia microthrombiischemiclesions pulmonary emboli or pulmonary infarct in of the patients who had not received mechanicalventilationthe delay between hospitalization anddeath was less than days in this group only casehad microthrombi the other patients had longer 0cremmelink critical care page of table characteristics of the study populationidct scancomorbiditiesagesexrrtpcrpostime todeathantemorteman failureardsakiposnegnegmfmfmcadcvddiabeteshypertensioncadcrfliver cirrhosiscopdcancerhypertensioncancercvdcopdcancerggoposmaggopospostreatmentscause of deathmechanicalventilationantibioticshydroxychloroquineantibioticscorticosteroidsmechanical ventilationhydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationhydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmohydroxychloroquineremdesivircorticosteroidsantiobioticshydroxychloroquineantibioticscardiogenicshockmofrespiratory failurerespiratory failurerespiratory failuremesentericischemiamofrespiratory failurerespiratory failureantibioticsseptic shockmofmechanical ventilationrrthydroxychloroquinelopinavirritonavirantibioticsmechanical ventilationecmorrthydroxychloroquineoseltamivirantibioticshydroxychloroquineantibioticsrespiratory failurerespiratory failuresudden deathmechanical ventilationhydroxychloroquineantibioticsmofhydroxychloroquinerespiratory failurehydroxychloroquineantibioticsrespiratory failureardsakihypoxichepatitisardsakiardsardsakihypoxichepatitisardsakiardsakihypoxichepatitisakiardsakiardsakiardsardsakihypoxichepatitisardsardsakihypoxichepatitismhypertensioncrfbcposmnonebcposmfhypertensioncadcvdcrfdiabeteshypertensiondiabetesemphysemaposggoposmhypertensiondiabetesggobcposmmmfdiabetesliver cirrhosiscancerdiabeteshypertensioncaddiabetesdiabeteshypertensiondiabetesbcposggoposggobcggobcpospos 0cremmelink critical care page of table characteristics of the study population continuedidcomorbiditiesct scanagesexmggolpposrrtpcrtime todeathfmhypertensiondiabetesliver transplanthypertensioncvdggobcposggobclpposantemorteman failureardsakipulmonaryembolismardsakipulmonaryembolismardsakipulmonaryembolismtreatmentscause of deathmechanical ventilationrrthydroxychloroquineremdesivirantibioticsmechanical ventilationrrthydroxychloroquineantibioticsmechanical ventilationecmorrthydroxychloroquineantibioticsseptic shockmofseptic shockmofseptic shockmoftime to death time from admission to death days cause of death was reported by the attending physician m male f female rrtpcr reverse transcription realtime polymerase chain reaction used as diagnostic laboratory test neg negative pos positive cad coronary artery disease cvd cerebrovascular disease lp lobarpneumonia ggo groundglass opacity ma minor abnormalities bc bilateral consolidation copd chronic obstructive pulmonary disease crf chronic renal failureards acute respiratory distress syndrome aki acute kidney injury ecmo extracorporeal membrane oxygenation rrt renal replacement therapy mof multiplean failuredelays between hospitalization and death daysthey had no microthrombififteen patients had signs of chronic ischemic cardiomyopathy of different severities and patients had signsof acute myocardial infarction there was no evidence ofcontraction bands or myocarditis histological evaluationof the kidneys was limited because of moderate to severepostmortem autolysis occasional hemosiderin granuleswere observed in the tubular epithelium in patientsand pigmented casts in in the medulla edematousexpansion of the interstitial space without significant inflammation was observed in patients chronic renal lesions ie nodular mesangial expansion and arteriolarhyalinosis glomerulosclerosis or chronic pyelonephritisfig main histological findings green finding present gray finding absent black unavailable 0cremmelink critical care page of fig pulmonary histological findings a earlystage diffuse alveolar damage dad hyaline membrane he — magnification with a zoom ona giant cell — magnification b fibrin thrombi in a pulmonary artery he — magnification c latestage dad fibroblastic proliferationhe — magnification d latestage dad fibroblastic proliferation trichrome staining — magnification e acute pneumonia he — magnification f antisarscov immunohistochemistry ihcpositive cells — magnificationwere also observed no microthrombi were identifiedbut one patient had a thrombus in an interlobar arteryliver examination revealed congestive hepatopathyand steatosis but no patchy necrosis hepatitis or lobular lymphocytic infiltrate the histological changes in theabdominal ans including the esophagus stomachand colon are reported in additional file table s2most of the findings were related to chronic underlyingdiseases except for one case of ischemic enteritisbrain samplesshowed cerebral hemorrhage orhemorrhagic suffusion n ischemic necrosisn edema andor vascular congestion n anddiffuse or focal spongiosis n we found no evidence of viral encephalitis or vasculitis isolated neuronalnecrosis or perivascular lymphocytic infiltrationfocalsarscov2 detection in the lungs by ihcsarscov2 was identified by ihc in the lungs of ofthe patients fig howeverthere was largevariability in the distribution of sarscov2positivecells in the lung parenchymasarscov2 detection by rtpcrsarscov2 rna was detected in at least one anfrom every patient fig in the lung rtpcr waspositive in patients with threshold cycle ct valuesvarying from to viral rna was alsodetected in the heart n the liver n thebowel n the spleen n and the kidney n as well as in of the cerebral samples ct valuesfor nonpulmonary ans ranged from to eight patients had positive rtpcr in all tested ansabnormalitiesdiscussionthis postmortem study showed several histopathologicalin covid19 nonsurvivorshowever none of the findings was specific for direct viralinjury even though sarscov2 was detected in all examined ans using rtpcr we decided to performcomplete autopsies rather than other techniques such aspostmortem core biopsies so as to obtain a better overview of all ans especially the lungs we collected samples from each lobe this approach enabled us to 0cremmelink critical care page of fig detection of sarscov2 by immunohistochemistry ihc in ffpe post mortem lung samples of patients semiquantitative evaluationœˆ’ negative result œ scattered positive cells between and positive cellswhole slide œ positive isolated cells cellswhole slide butno foci œ foci of positive cells more than positive cells in one — field na not availabledocument the considerable heterogeneity of histologicallesions and of sarscov2 spread through the bodythe diagnosis of sarscov2related an injury ischallenging postmortem histologicalfindings wereheterogeneous and often associated with chronic underlying diseases in a previous autopsy study in covid19patients the authors reported that dad associatedwith viral pneumonia was almost impossible to distinguishfrom that caused by bacterial pneumonia no obviousintranuclear or intracytoplasmic viralinclusions wereidentified in another report desquamation of pneumocytes and hyaline membrane formation are frequentlydescribed in ards of many different causes especially inearlyphase ards the presence of multinucleatedcells with nuclear atypia is used to diagnose herpes virusinfection in daily practice as in previous reports [ ]we also observed the presence of multinucleated cellswithin lung alveoli in three patients however the significance of multinucleated cells is unclear and may not bespecific of sarscov2 infection finally some ofthe microscopic features of these patients are compatiblewith an changes secondary to shock or systemicinflammation and no histological finding could be specifically ascribed to sarscov2in the absence of typical postmortem viral featuresour results show that rtpcr is feasible on ffpe blocksand could be used in postmortem analyses to identifythe presence of sarscov2 in multiple ans and tounderstand the spread of the virus within the humanbody the discordant rtpcr and ihc results fordetection of sarscov2 in the lungs may be explainedby the different sensitivity of these assays which washigher for the rtpcr whereas lowlevel viral replication might not be detected by ihc moreover ihc wasbased on the only available antibodies which aretargeted against sarscov new antibodies againstsarscov2 need to be developed to improve theaccuracy of ihc in the analysis of tissue samples fromsuspected or confirmed covid19 patientsmost of the previous postmortem studies in covid19patients were conducted using needle biopsies and weretherefore rather limited in terms of sampling our completeautopsy analysis identified considerable heterogeneity ofsarscov2 spread through the human body and providesa more accurate description of macroscopic and microscopic an alterations as for previous coronavirusdiseases [ ] the lungs are the most affected ans incovid19 however dad findings werehighly 0cremmelink critical care page of fig molecular detection of sarscov2 rna in postmortem samples detection of sarscov2 by reverse transcription realtime polymerasechain reaction rtpcr in ffpe postmortem tissues of patients œ positive result œˆ’ negative result œna tissue not available nc noninformative test result due to lowquality rnaheterogeneous including both earlyonset and additionallate lesions this finding could be explained by the heterogeneity of the pulmonary injury including compliant lungsin the early phase and a more dense and nonrecruitablelung in the late phase as some patients died outsidethe icu without receiving mechanical ventilation we couldnot estimate lung compliance before death the heterogeneity could also reflect different treatments eg fluid administration or corticosteroids or different complications asan example half of the patients had concomitant acutepneumonia and it is difficult to conclude whether the dadreflected the natural timecourse of the viral disease or wassecondary to superimposed complications such as nosocomial infections in a recent report needle postmortem biopsies suggested that covid19 is not associated withdad but rather with an acute fibrinous and anizingpneumonia afop consequently requiring corticoid treatment a diagnosis of afop is based on the absence ofhyaline membranes and the presence of alveolar fibrin ballshowever hyaline membranes are heterogeneously distributed in the lung parenchyma with dad and complete lunganalysis not just biopsies are necessary to exclude theirpresence moreover afop may be a fibrinous variant ofdad the limitation of lung biopsy was also shown inanother study in which only of lung samples werepositive for sarscov2 using rtpcr when compared to almost in our series in addition we did notfind specific œendothelitis as previously reported in a smallcase series considering the heterogeneity of postmortem covid19 associated lesions molecular and ihcassessments are mandatory in the histological analysis ofcovid19 tissue samplespatients with covid19 often have altered coagulation and a prothrombotic status with the possible development of acute pulmonary embolism pe in ourstudy three patients had pe already diagnosed beforedeath four patients had pulmonary infarction in a previous study acute pe was considered as the main causeof death in four patients however the inclusion ofpatients who died before hospital admission and the lackof specific thromboprophylaxis during the hospital staymay account for the differences in the severity of pewhen compared to our study although we frequentlyobserved the presence of microthrombi in the lung parenchyma this feature is also reported in other forms ofards regardless of etiology [ ] as such whetherdiffuse pulmonary thrombosis is a main contributor ofthe fatal course of severe hypoxemia in covid19 0cremmelink critical care page of patients remains to be further studied in a systematicreview of pathologicalfindings in covid19 polak identified a timeline in the histopathologicalfindings in the lung with epithelial dad denudationand reactive pneumocytes atypia and vascular microvascular damage thrombi intraalveolar fibrin depositschanges present at all stages of the disease but fibroticchanges interstitialfibrous changes only appearingabout weeks after the onset of symptoms few patientshad fibrosis at early stages and in these cases it waslikely because of preexisting lung disease our resultsare consistent with those of polak except forthe lack of late fibrotic changes which may be related tothe use of antiinflammatory drugs at high doses fornearly all our patients we did not observe specific viral an injury such asmyocarditis hepatitis or encephalitis the cases ofœacute cardiac injury reported in covid19 clinicalstudies do not necessarily translate into myocarditisor acute myocardialischemia only two had acutemyocardial ischemia similar to data reported in septicpatients ie elevated troponin without overt cardiacischemia however using rtpcr we found thevirus in almost all the examined ans this suggeststhat the virus can bind to most cells probably via theace2 receptor which is ubiquitous but may notdirectly cause an injury as extrapulmonary directviral injury eg encephalitis hepatitis or myocarditishas only been reported in very few cases we suggest thatsarscov2 infection may be just the trigger for anoverwhelming host response which could secondarilyresult in covid19associated an dysfunction asrtpcr mightitremains unclear whether this represents active viral replication into the tissues or previous cellular infectionwithout clinically relevant significance just detect residual viral genomethis study has several limitations i we only includedpatients who had had a positive rtpcr on nasopharyngeal swab andor bronchoalveolar lavage to ensurethat only true positive cases were enrolled we decidednot to include three patients who had had thoracic ctscan findings suggestive of covid19 but had negativertpcr results this limitation in our study reflects thedifficulty of diagnosing covid19 on a clinical basis iithe sample size was relatively small and autopsies wereonly carried out from to h after death this delaydid not allow us to properly analyze the gastrointestinaltract and kidneys which showed signs of autolysis inparticular acute tubular injury in the proximal tubuleswas indistinguishable from autolysis iii we could notdetermine the timecourse andor sequence of anspread of the virus and no specific hypothesis regardinghow sarscov2 spreads eg hematogenously couldbe identified and iv the time to death differed frompatient to patient as did the course of the disease andtreatments received which limits a precise clinicalpathological correlation of histological findings related tocovid19 finally we did not evaluate specific mechanisms involved in the pathogenesis of an injurythese results underline the heterogeneity of an injuriesduring covid19 disease and the absence of specificsarscov2 lesions using rtpcr sarscov2 couldbe detected in all ans even those without evidentmicroscopic lesionssupplementary informationsupplementary information accompanies this paper at httpsdoi101186s13054020032185additional file critical careautopsycovid additional materialprocedure to obtain brain samplesadditional file critical careautopsycovid additional table s1laboratory findings on the day of admissionadditional file critical careautopsycovid additional table s2detailed histological findings in all patientsabbreviationsace2 angiotensin converting enzyme afop acute fibrinous andanizing pneumonia aki acute kidney injury ards acute respiratorydistress syndrome covid19 coronavirus disease ct threshold cycledad diffuse alveolar damage ffpe formalinfixed paraffinembeddedhe hematoxylin and eosin ihc immunohistochemistry iqrs interquartileranges merscov middle east respiratory syndrome coronaviruspas periodic acidschiff pe pulmonary embolism rtpcr realtime reversetranscription polymerase chain reaction sarscov severe acute respiratorysyndrome coronavirusacknowledgmentsthe authors thank nathalie lijsen christophe valleys gees lacroixbarbara alexiou dominique penninck nicole haye and audrey verrellen fortechnical and logistic supports prof frdric schuind and dr djameleddineyahiacherif for neurosurgical procedure egor zindy diapath ulb forproofreading the paper and dr mariepaule van craynest for trainees™supervisionauthors™ contributionsis had the idea for and designed the study and had full access to all thedata in the study and takes responsibility for the integrity of the data andthe accuracy of the data analysis is ft jlv and cd drafted the paper mrcv ll pl mlr cm alt jcg lp rdm sd sr nd lp and od collected thedata mr nd and rdm did the analysis and all authors critically revised themanuscript for important intellectual content and gave final approval for theversion to be published all authors agree to be accountable for all aspectsof the work in ensuring that questions related to the accuracy or integrity ofany part of the work are appropriately investigated and resolvedfundingthis study received financial support from fonds y bo«l brussels belgiumfonds erasme pour la recherche mdicale brussels belgium and œappel  projet spcial covid19 ulb brussels belgium the cmmi is supported bythe european regional development fund and the walloon region ofbelgium walloniabiomed grant no project œcmmiulbsupport the center for microscopy and molecular imaging and its diapathdepartment cd is a senior research associate with the fnrs belgiannational fund for scientific research 0cremmelink critical care page of availability of data and materialsthe data that support the findings of this study are available from thecorresponding author on reasonable request participant data without namesand identifiers will be made available after approval from the correspondingauthor and local ethics committee the research team will provide an emailaddress for communication once the data are approved to be shared withothers the proposal with a detailed description of study objectives andstatistical analysis plan will be needed for the evaluation of the reasonabilityto request for our data additional materials may also be required during theprocess d'haene n melndez b blanchard o de n¨ve n lebrun l vancampenhout c design and validation of a genetargeted nextgeneration sequencing panel for routine diagnosis in gliomas cancersbasel corman vm landt o kaiser m molenkamp r meijer a chu dk detection of novel coronavirus 2019ncov by realtime rtpcr eurosurveill de hemptinne q remmelink m brimioulle s salmon i vincent jl ards aclinicopathological confrontation chest “ menter t haslbauer jd nienhold r savic s hopfer h deigendesch n ethics approval and consent to participatethe study protocol was approved by the local ethics committee erasmehospital p2020218 the ethical committee has waived the need for writteninformed consentpostmortem examination of covid19 patients reveals diffuse alveolardamage with severe capillary congestion and variegated findings of lungsand other ans suggesting vascular dysfunction histopathology epub ahead of print httpsdoi101111his14134franks tj chong py chui p galvin jr lourens rm reid ah lungpathology of severe acute respiratory syndrome sars a study of autopsy cases from singapore hum pathol “ nicholls jm poon ll lee kc ng wf lai st leung cy lungpathology of fatal severe acute respiratory syndrome lancet “ hwang d chamberlain d poutanen s low de asa sl butany j pulmonarypathology of severe acute re

chrysoperla nipponensis okamoto which has the unique diapause phenotype distinguishable from nondiapause adult is an ideal model anism for studying the mechanism of reproductive diapause however there is no reliable and effective reference genes used for the reproductive diapause study of c a0nipponensis therefore in this study we evaluated the expression stability of candidate reference genes tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin αtub in adults under diapause and nondiapause induction conditions using four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method results showed that arp3 and tub1 were the most stable reference genes in all samples and in the adult tissues group arp3 and rps5 were the most stable reference genes in the development degree group αtub and ef1a were unstable reference genes under the conditions of this study meanwhile to verify the reliability of the reference genes we evaluated the relative expression levels of vg and vgr in different treatments significant upregulation and downregulation in expression level of two genes in response to diapause termination and diapause fat body tissue was respectively observed when using arp3 as the reference gene but not when using an unstable reference gene the reference genes identified in this work provided not only the basis for future functional genomics research in diapause of c a0nipponensis and will also identify reliable normalization factors for realtime quantitative realtime polymerase chain reaction data for other related insectskey words chrysoperla nipponensis okamoto reference genes qrtpcr reproduction diapausedue to the advantages of high sensitivity rapidity specificity and accuracy bustin et a0al valasek et a0al vanguilder et a0al shakeel et a0al quantitative realtime polymerase chain reaction qrtpcr has been widely used in the study of animals plants and microanisms roy et a0al jia et a0al zhang et a0 al ding et a0 al sun et a0 al qrtpcr is the most commonly used method for the expression analysis of target genes however the reliability of qrtpcr results in different samples is determined by a variety of factors among which the use of stably expressed reference genes is an important link for accurate detection of gene expression changes by qrtpcr bustin et a0 al at present several commonly used reference genes for data normalization include tubulin actin ribosomal protein elongation factor 1α glyceraldehyde3phosphate dehydrogenase 18s ribosomal rna and other genes bustin vanguilder et a0al however more and more studies have found that these reference genes do not show consistent expression patterns under different experimental conditions and even affect the reliability of experimental results therefore in order to obtain stable and reliable normalization factors reference genes with stable expression are usually used for correction and standardization to reduce errors between samples selection and evaluation of reference genes have become a necessary step before quantifying the expression of target genes accuratelynowadays there have been many studies on the selection of reference genes for insects such as sesamia inferens helicoverpa armigera aphis gossypii myzus persicae etc lu et a0 al shakeel et a0al zhang et a0al ma et a0al kang et a0al in these studies four statistical algorithms including genorm normfinder bestkeeper and ˆ†ct method were mainly used to analyze the ct values obtained by qrtpcr of reference genes under various experimental conditions shakeel et a0 al finally the stability of the candidate reference genes was determined according to the geometric mean value of each gene ranked using different the authors published by oxford university press on behalf of entomological society of americathis is an open access distributed under the terms of the creative commons attribution noncommercial license httpcreativecommonslicensesbync40 which permits noncommercial reuse distribution and reproduction in any medium provided the original work is properly cited for commercial reuse please contact spermissionsoupcom 0c of insect science vol no algorithms and the most suitable reference gene was selected for the target gene expression analysis xiao et a0al kang et a0al chrysoperla nipponensis okamoto as one of the important predatory natural enemies of agricultural and forestry pests prefers to eat aphids thrips and other pests okamoto nie et a0al because of its characteristically wide geographical distribution and broad range of host prey niijima syed et a0al it has good prospects for widespread application in biological control mcewen et a0 al memon et a0 al reproductive diapause is an important way for c a0nipponensis adults to escape from adverse environments xu et a0al at present there have been many reports on the diapause of c a0nipponensis xu et a0al found that the body color of c a0nipponensis was green during the reproductive period but turned brown and yellow during the diapause period chrysoperla nipponensis belongs to the photoperiodic sensitive insect the adult diapause was induced by short photoperiods xu et a0al chen et a0al found that different photoperiods affected the material content eg protein and glycogen of c a0nipponensis diapause induced by the short photoperiod was beneficial to the storage of c a0nipponensis chen et a0al we expect an exponential increase of diapause research on c a0nipponensis at the molecular level in the near future thus stable and reliable reference genes are important for accurately quantifying gene expression of c a0nipponensisribosomal proteins and ribosomal rna have been used as reference genes in previous diapause studies for example williams et a0al used ribosomal protein rp49 as a reference gene to study the natural variation of drosophila melanogaster diapause williams et a0 al and sim and denlinger used ribosomal protein large subunit rpl19 as a reference gene in a study of ovarian development of culex pipiens during overwintering diapause sim and denlinger this indicates that under the same experimental conditions the selected reference genes in different species research are also differentin this research candidate reference genes were selected including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub whose expression profiles were measured by the qrtpcr the stability was analyzed by four statistical algorithms genorm normfinder bestkeeper and ˆ†ct method in different developmental stages reproductive and diapause of adults and among different tissues the optimal reference genes under different conditions were determined which contributed to the accurate expression of target genes for future researchmaterials and a0methodsinsecta stable population of c a0 nipponensis was maintained in an artificial climate chamber rsz intelligent artificial climate chamber changzhou guohua jiangsu province under the following conditions a0± °c temperature a0± relative humidity rh and long photoperiod of l d h in our laboratory the eggs were collected by cutting the stalk and incubated in fingertip tubes a0cm in diameter and a0cm in height the primary hatching larvae were fed megoura japonica matsumura whose host plant was vicia faba l a0the adults were paired immediately after emergence in a bottle a0cm in diameter and a0cm in height fed a dry brewer™s yeast feed mixed with sucrose in a ratio of and then minced in a mortar and sifted through mesh and honey water the diapause adults used in this study were kept under the conditions of short photoperiod of ld h in all processes from eggs larvae pupae to adults whereas the nondiapause adults were kept under the conditions of long photoperiod of ld a0hsample collection development degree samples from individuals from varying developmental stages included female adults in the diapause induction period “ d under the short photoperiod the diapause maintenance period “ d under the short photoperiod the diapause termination period “ d under the short photoperiod and the reproduction period “ d under the long photoperiod each sample which included three to four females was independently replicated three times as three biological replicates adult tissues reproduction seven different tissues were collected from reproductive adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of reproductive adults under long photoperiod were collected on the 10th day after emergence each tissue required about “ a0mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues diapause seven different tissues were collected from diapause adults including ovarian fat body head wings antennae front thorax and epidermis the tissues of diapause adults under short photoperiod were collected on the 20th day after emergence each tissue required about “ a0 mg of material and each tissue sample collection was independently replicated three times as three biological replicates adult tissues samples from reproductive and diapause adult tissues all samples samples from group and all treatments were immediately frozen with liquid nitrogen and stored in an ultralow temperature refrigerator at ˆ’°c prior to rna extractiontotal rna extraction and cdna synthesisin this study total rna was extracted using minibest universal rna extraction kit takara japan and dnase i a0 was used for digestion of the membrane rna integrity was estimated by agarose gel electrophoresis rna concentration and purity were measured with a nanodrop one spectrophotometer thermo scientific then 1μg rna was reversetranscribed into the firststrand complementary dna cdna according to the hiscript ii q rt supermix for qpcr gdna wipers vazyme nanjing china instructions and stored at ˆ’°c all cdna was diluted 10fold with dnasernasefree sterile water before usecandidate reference gene selection and primer a0designaccording to several commonly used reference genes candidate reference gene sequences were obtained by screening from the existing transcriptome of c a0 nipponensis in the laboratory namely tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub in order to ensure the predictive accuracy of the selected sequences we conducted blast alignment all primers were designed using primer premier based on the following criteria gc content “ annealing temperature “°c and primers length “ a0 bp and the specificity of each pair of amplicons was determined by qrtpcr followed by agarose gel electrophoresis and melting curve analysis the amplification efficiency of the pcr was calculated by using the formula e a0 a0 ˆ’slopeˆ’ the slope was obtained by the standard curve which was generated by qrtpcr of a series of continuously diluted cdna samples 0c of insect science vol no realtime qrtpcr analysisthe 20µl total reaction volume were configured according to the protocol of chamq sybr qpcr master mix vazyme contained 10µl a0× chamq sybr qpcr master mix a0µl a0μm of each gene specific primer a0µl of cdna and a0µl of ddh2o the amplification reaction program was set as follows predenaturation at °c for a0s followed by cycles of denaturation at °c for a0s annealing at °c for a0 s the melting curves were analyzed in the “°c temperature range after amplification step the reaction was performed on a roche lightcycler96 instrument to obtain ct values amplification curves melting curves and standard curves all samples were carried out in four technical and three biological replicates and the negative control no template was performed in paralleldata analysisct values for all samples were exported into an excel spreadsheet and were used to analyze the stability of candidate reference gene expression by genorm normfinder bestkeeper and ˆ†ct method the comprehensive ranking were performed following methods adapted from xiao et a0 al the optimal number of genes was determined by the pairwise variation vnn1 between the normalization factors calculated by genorm among the four algorithms genorm and normfinder need to convert the original ct value according to the corresponding requirements before analysis in genorm the stability ranking of genes was determined by the expression stability value m value in bestkeeper the stability ranking of genes was determined by the coefficient of variation cv and sd in normfinder the stability ranking of genes was determined by the gene expression stability value sv in ˆ†ct method the stability of genes was ranked according to the sd of genes ˆ†ct values in four statistical algorithms genes with the lowest value were the most stably expressedvalidation of reference a0genesin most insects vitellogenin vg and vitellogenin receptor vgr play important roles in the reproductive process of female insects vg is taken up by developing oocytes through receptormediated endocytosis rme thereby promoting the development of oocytes and the formation of eggs in this process vgr is the main receptor mediating endocytosis previous studies have shown that reproductive diapause arrests development of oogenesis and vitellogenesis tatar and yin and the expression levels of the vg and the vgr in nondiapause female were significantly higher than those in reproductive diapause female jiang et a0al in order to evaluate the effectiveness of the selected reference genes the expression levels of the target genes vg and vgr were respectively detected by qrtpcr in the different development degree and tissues of adults and the most unstable reference gene was used for comparison in parallel the reaction system and program were the same as for qrtpcr of reference genes and four technical and three biological replicates were performed for each treatment the relative expression levels of vg and vgr were respectively calculated in excel using the ˆ†ˆ†ct method the differences of target genes™ expression levels were analyzed by tukey™s test using spss software spss inc under different experimental conditionsresultsselection and primer performance of candidate reference a0genesthe candidate reference genes including tub1 arpc5 ef1a 128up rps5 rps26e gapdh arp3 actin and αtub were selected to identify the normalization factors for qrtpcr analysis and the sequence information has been submitted to genbank and the accession numbers are shown in supp table online only to determine the amplification specificity of the primers agarose gel electrophoresis and melting curve analysis were performed all primer pairs showed a single band and a single peak fig a0 to obtain correlation coefficient r2 and amplification efficiency e of pcr a standard curve was generated with the 10fold dilution series of cdna the results showed that the amplification efficiency ranged from to and the correlation coefficient varied from to supp table [online a0only]expression profiling of candidate reference a0genesto evaluate expression levels of the candidate reference genes the cycle threshold ct values under different groups were obtained by qrtpcr and represented by boxplot fig a0 the results indicated that ct values of all candidate reference genes were different under different conditions and also varied under the same condition overall ct values ranged from to among them the genes with higher expression abundance were tub1 “ rps26e “ and actin “ followed by 128up “ arp3 “ arpc5 “ rps5 “ and ef1a “ the genes with lower expression abundance were αtub “ and gapdh “ according to the ct value range of each candidate reference gene the genes with relatively stable expression were tub1 and arp3 whereas the most unstable genes were ef1a actin and αtubexpression stability of candidate reference genes under different conditionsin this study four statistical algorithms were used to analyze the expression stability of the candidate reference genes under different conditions including genorm normfinder bestkeeper and ˆ†ct method as different statistical algorithms would generate different ranking patterns the comprehensive ranking of genes was finally determined through the geometric mean of sequencing bestkeeper the original ct values were used for analysis evaluated the stabilities of the candidate reference genes according to the cv and sd of the ct values ˆ†ct method the difference values of original ct values were used for analysis performed stability ordering according to the mean sd of ˆ†ct value genorm and normfinder the original ct values were converted for analysis sequenced the candidate reference genes according to the stability values the lower the stability value the more stable the gene expressiondevelopment degree of a0adultsthe expression stability of the candidate reference genes at different periods of reproductive and diapause female showed that the top four ranked genes identified by the genorm bestkeeper normfinder and δct method were similar but the rank order was slightly different arp3 and rps5 were the first and second stably expressed genes in the four statistical algorithms supp table [online only] and comprehensive ranking analysis fig a0 as for the third and fourth ranked gene tub1 and actin identified by comprehensive ranking analysis were the same as those generated by genorm and normfinder while bestkeeper selected arpc5 and tub1 ˆ†ct method selected actin and rps26e supp table [online only] αtub was ranked by genorm bestkeeper normfinder and δct method as the least stable gene among the candidate reference 0c of insect science vol no fig specificity a and product length b of qrtpcr amplification for ten candidate reference genesgenes during different development degrees of adults supp table [online a0only]adult a0tissuesthe expression stability ranking of candidate reference genes in different tissues of reproductive and diapause females was varied according to the four statistical algorithms in different tissues of reproductive females the top four genes were rps5 arp3 arpc5 and tub1 respectively fig a0 but the rank order of the four genes was significantly different among different statistical algorithms rps5 was ranked first by genorm and ˆ†ct method and was ranked third and fourth by normfinder and bestkeeper respectively arp3 was ranked first by bestkeeper and was ranked second third and fifth by normfinder δct method and genorm respectively arpc5 was ranked first and second by genorm and δct method and was ranked fourth and fifth by normfinder and bestkeeper respectively tub1 was ranked first and second by normfinder and bestkeeper and was ranked fifth and sixth by δct method and genorm respectively supp table [online only] however the four statistical algorithms found that ef1a was ranked as the least stable gene in the tissues from reproductive females supp table [online only]in different tissues of diapause females the top four genes were different from those of different tissues of reproductive females tub1 was ranked first by the comprehensive ranking followed by rps26e arp3 and 128up fig a0 through analysis it was found that ˆ†ct method and normfinder displayed the same rankings for expression stability of candidate reference genes under this condition supp table [online only] tub1 was identified as the most stably expressed gene by genorm ˆ†ct method and normfinder although it was ranked fifth by bestkeeper rps26e ranked steadily among the four statistical algorithms was ranked second by ˆ†ct method and normfinder whereas it was ranked first and third by genorm and bestkeeper respectively arp3 was ranked first by bestkeeper with the smallest coefficient of variation whereas it was ranked third by ˆ†ct method and normfinder in addition to being ranked third by genorm 128up was ranked fourth by bestkeeper ˆ†ct method and normfinder supp table [online only] however in tissue from diapause females actin was consistently identified as the gene with the most unstable expression by the four statistical algorithms supp table [online only]in the reproductive and diapause female tissues the expression stability of the candidate reference genes was different in order to accurately determine the expression of the target genes the expression stability of candidate reference genes under the two conditions was analyzed according to the comprehensive ranking 0c of insect science vol no fig expression profiles of candidate reference genes in c a0nipponensis expression data are displayed as ct values for each reference gene using a box and whisker plot in different experimental conditions the line across the box is the median the box indicates the 25th and 75th percentiles the whiskers represent the 10th and 90th percentilesfig expression stability and comprehensive ranking of reference gene measured by the geomean method a a0lower geomean value indicates more stable expressiontub1 and ef1a were the most stable and unstable genes respectively whereas arp3 128up and arpc5 were ranked second third and fourth respectively fig a0 tub1 was the best candidate reference gene identified by normfinder and ˆ†ct method and was ranked third and sixth by bestkeeper and genorm respectively arp3 was the most suitable candidate reference gene selected by bestkeeper and was ranked second by normfinder and ˆ†ct method and fifth by genorm 128up and arpc5 were the most stable candidate reference genes identified by genorm whereas they were ranked separately fifth and sixth by normfinder and ˆ†ct method and seventh and fifth by bestkeeper respectively supp table [online only] ef1a was identified as the least stable gene by genorm normfinder and δct method although bestkeeper selected actin as the least stable gene supp table [online only]all a0samplesin order to determine the best reference gene suitable for the different conditions of adults the stability of the ten candidate reference genes was ranked for all samples arp3 was identified as the most stable gene by the comprehensive ranking followed by tub1 arpc5 and rps5 whereas ef1a was identified as the least stable gene fig a0 0c of insect science vol no but the most and least stable genes identified by different statistical algorithms were slightly different arp3 was selected as the most stable reference gene by bestkeeper and ˆ†ct method although tub1 and arpc5 were selected as the most stable reference gene by normfinder and genorm respectively ef1a was selected as the least stable reference gene by genorm and ˆ†ct method despite it being ranked ninth by bestkeeper and normfinder supp table [online only]the best combination of candidate reference genes under different conditionsaccording to the pairwise variation vnn1 between the normalization factors and cutoff value calculated by genorm the number of reference genes required for optimum normalization in each experimental condition was determined the cutoff value of vnn1 a0 suggested that n reference genes were enough to make gene expression normalization otherwise n reference genes were needed the analysis results showed that all v23 were indicated that the optimal number of reference genes under each condition was two fig a0 more specifically arp3 and rps5 were the most stable gene combinations under adult developmental stage and reproductive adult tissues conditions tub1 and rps26e were the most stable gene combinations under adult diapause tissues conditions and arp3 and tub1 were the most stable gene combinations under adult tissues and all samples groups table a0relative expression levels of target genes vg and vgr genbank mt308983 mt522179 in the whole adult and from tissues of reproductive and diapause adults respectively when the most stable reference genes arp3 andor rps5 were used as normalization factors at different periods of reproduction and diapause the expression patterns of the target genes vg and vgr were consistent with low expression in the diapause period and rich expression in the late diapause and reproduction period however when the most unstable reference gene αtub was used as a normalization factor neither the target gene vg nor vgr showed a consistent expression pattern fig a0 under different tissue conditions when the most stable reference genes tub1 andor arp3 were used as the normalization factors the expression level of the target gene vg in the fat body of the reproductive female was significantly higher than that in the ovary of the reproductive female the expression level of the target gene vgr in the fat body of the reproductive female was lower than the ovary of the diapause female while when the most unstable reference gene ef1a was used as the normalization factor the expression pattern differed with normalization by tub1 and tub1arp3 fig a0 in general when the most stable reference genes were used as the normalization factors the accurate expression pattern of the target gene could be obtainedvalidation of reference a0genesthe stability of reference gene is very important for the analysis of expression level of target gene vg and vgr which are important for insect reproduction were selected to verify the applicability of the selected reference gene we examined the discussionqrtpcr has become an important means to explore gene expression level due to its high sensitivity rapidity specificity and accuracy and was widely used in physiology studies that investigated insect diapause such as drosophila melanogaster williams fig pairwise variations vnn1 was calculated by genorm to determine the optimal number of reference genes for accurate normalization in different conditions the cut off values under indicate that no additional genes are required for the normalization 0c of insect science vol no et a0al culex pipiens sim and denlinger leptinotarsa decemlineata lehmann et a0 al chrysopa septempunctata liu et a0al and pieris melete wu et a0al however the selection of appropriate reference genes was the key to accurately analyze the gene expression level for example under conditions of injury heatstressed and experimentally varied diets table recommendation for the best combination of reference genes based on the genorm and comprehensive rankings under various experimental conditionsgroupreference genemostdevelopment degreeadult tissues reproductionadult tissues diapauseadult tissuesall samplesarp3rps5tub1tub1 arp3 rps5arp3rps26earp3tub1leastαtubef1aactinef1aef1athe best reference gene was different in drosophila melanogaster ponton et a0al under biotic factors and abiotic stress inappropriate selection of the reference genes in locusta migratoria resulted in significant differences in the expression level of the target gene chitin synthase chs1 yang et a0al diapause of most insects was mainly affected by photoperiod and temperature in past studies the screening of reference genes of drosophila melanogaster ponton et a0al leptinotarsa decemlineata shi et a0al helicoverpa armigera zhang et a0al bombyx mori guo et a0al and harmonia axyridis qu et a0al under main environmental factors was completed by genorm normfinder bestkeeper and ˆ†ct method in this study the expression profiles of candidate reference genes of c a0nipponensis were analyzed under different conditions by the same four statistical algorithms genorm vandesompele et a0 al normfinder andersen et a0al bestkeeper pfaffl and ˆ†ct method silver et a0al different algorithms produced different stability rankings in order to obtain statistically consistent and accurate results we finally ranked the gene based on their stabilities determined by fig validation of selected reference genes under different periods a and tissues b of reproductive and diapause female in c a0 nipponensis relative expression levels of the vg and vgr in different samples using different normalization factors the most and least stable genes asterisks indicate significant differences in the expression levels of the vg and vgr r reproduction period d1 the diapause induction period d2 the diapause maintenance period d3 the diapause termination period 0c of insect science vol no comprehensive analysis method xiao et a0 al and selected the most stable reference genes under each condition as far as we know actin which played an important role in cell contraction and cytoskeletal maintenance was found in virtually all eukaryotic cells and was considered as an ideal reference gene for many anisms sürencastillo et a0al shakeel et a0al for example actin was used as a reference gene for normalization in the determination of genes related to reproductive and nutritional signaling such as vitellogenin of chrysopa septempunctata liu et a0al however in this study three genes related to actin were selected for analysis among which actin was similar to actin of c a0septempunctata which was also a member of the neuroptera in our study actin was the most unstable gene in the diapause female tissues but the actinrelated protein arp3 which was structurally homologous with actin showed better stability arp3 was selected as the most stable reference gene in adults of different developmental levels and all samples while it was the second most stable gene in the reproductive adult tissues and all adult tissues although arp3 was ranked as the third most stable gene in the diapause adult tissues it showed relatively stable expression in the expression profile tubulin which played an essential role in maintaining cell shape movement and intracellular material transport was also often used as a reference gene but different types of tubulin have different stability for example in the study of helicoverpa armigera the expression of βtub was relatively stable compared with that of αtub under almost all conditions zhang et a0 al similarly in this study αtub showed unstable expression and was the least stably expressed gene in adults of different developmental stages whereas tub1 was considered to be the most stably expressed reference gene in diapausing adult tissues and all adult tissues and was the second most stable gene in all samples ribosomal protein rp widely distributed in various tissues played an important role in protein biosynthesis and was widely used as a reference gene in many insects lu et a0al koyama et a0 al sun et a0 al in this study rps5 was considered to be stable in the tissues of reproductive females and ranked second among different developmental stages of adults elongation factors ef was a protein factor which promoted polypeptide chain to extension during the translation of mrna and was recommended as the ideal reference gene under different conditions of a variety of insects chapuis et a0 al ponton et a0 al however some studies showed that ef1α was one of the most unstable genes under certain conditions fu et a0 al in our study ef1a was found to be the most unstable gene in the reproductive adult tissues all tissues and all samples and the second least stable gene in the adults of different developmental stages and diapause adult tissues therefore it was not suitable for the study of c a0nipponensisrecently an increasing number of studies have demonstrated the importance of using multiple stably expressed reference genes for the accuracy of qrtpcr analysis ling and salvaterra yuan et a0 al kang et a0 al however this does not mean that the more reference genes increase the reliability of the results the study has indicated that either too few o

"The occurrence of PLC is extremely rare in liver carcinoma. Herein we report the case of a patient with PLC after liver transplantation due to liver carcinoma. PLC was confirmed by clinical manifestations imaging studies and cytologic examination of exfoliated cells in the pleural effusion. Liver carcinoma Liver transplantation Metastasis Pulmonary lymphangitic carcinomatosis Background Primary liver carcinoma is a malignancy originating from hepatocytes and/or intrahepatic biliary epithelial cells. In China there are more than 90 million carriers of the hepatitis B virus (HBV) accounting for 40% to 45% of HBV carriers worldwide. The high prevalence of HBV in China is the underlying reason why liver carcinoma is the malignancy with the highest morbidity and mortality rates in China. Currently resection and liver transplantation are major strategies for the treatment of liver carcinoma. For patients with hepatic cirrhosis liver transplantation can cure both the cancer and liver cirrhosis. However liver carcinoma may recur or metastasize after resection or liver transplantation mainly via the hematogenous route. Although lymphatic metastasis can occur metastasis is usually found in the hepatic hilus upper abdomen and retroperitoneal lymph nodes [1]. Pulmonary lymphangitic carcinomatosis (PLC) is a special manifestation of metastatic cancer in the lymphatic vessels of the lung that is characterized by diffuse or focal growth. Most PLC cases originate from adenocarcinomas. PLC is rare in liver carcinoma patients. To the best of our knowledge no studies reported to date have described PLC after liver transplantation. Case presentation A 45-year-old man was admitted to our hospital with a complaint of repeated episodes of abdominal distension. He was diagnosed with HBV-induced hepatic cirrhosis and liver carcinoma (T3N0M0). He underwent liver transplantation without any metastasis before the operation. Pathological analysis identified a tumor (12 cm?—?8 cm?—?10 cm) in the right lobe of the liver within which the cancer cells were arranged in nests and pleomorphism was seen. These findings together with the results of immunohistochemistry demonstrated features of mixed liver carcinoma: ?-fetoprotein (+) hepatocytes (+) CD34 (+) CD19 (+) CD10 (focal +) synaptophysin (-) chromogranin A (-) and cytokeratin (pan +) (). The function of the graft liver was favorable. FK506 was used alone for antirejection therapy. Immunohistochemical staining of mixed liver carcinoma tissue specimens. (A) Cancer cells were arranged in nests and showed atypia. The interstitium was rich in sinusoids and invasive growth was noted. (B) Image showing cytokeratin 19 (CK19) (+). (C) Image showing CK7 (+). All three images are stained with hematoxylin and eosin and were scanned at 100— original magnification. Two months later the patient developed a dry cough of unknown etiology and his condition deteriorated 1 week later. Expectoration was occasionally present accompanied by chest tightness shortness of breath and hypoxemia (75 mmHg partial pressure of oxygen). Fever and chills were absent and the patient™s white blood cell count neutrophil count and inflammatory factors were normal. His sputum culture was negative. Lung computed tomography (CT) suggested infectious lesions in the lung which were characterized by interstitial changes. Right-sided pleural effusion and segmental atelectasis in the lower lobe of the right lung were noted. Several enlarged lymph nodes were identified in the mediastinum (A). Thoracentesis was immediately performed and approximately 2000 ml of light yellow fluid was collected. The patient™s chest tightness and shortness of breath improved significantly. Posttransplantation interstitial pneumonia was considered at first. FK506 was discontinued and methylprednisolone (40 mg every 12 hours) caspofungin sulfamethoxazole (SMZ) and aminophylline were administered. Computed tomography scans of the lungs. (A) Soon after the appearance of the patient™s respiratory symptoms a computed tomography (CT) scan revealed septal thickening of the peribronchovascular interstitium pleural effusion segmental atelectasis in the right lower lobe of the lung and several enlarged lymph nodes in the mediastinum. (B) Discontinuation of anti-infection therapy and 5 days after thoracentesis extensive involvement of the parenchyma with septal thickening was evident with reticulonodular densities in all lung fields. Five days later a lung CT scan showed reexpansion of the right lung and diffuse exudate in the interstitium. Multiple nodules were found in both lungs (B). Pulmonary function tests showed severe obstructive ventilatory dysfunction and moderate reduction in carbon monoxide diffusion capacity. Examination of exfoliated cells in the pleural effusion showed cancer cells (). Positron emission tomography (PET)-CT indicated multiple nodules and patchy or cloudy shadows with high density in both lungs (maximal standardized uptake value (SUV) approximately 6.27). Several enlarged lymph nodes were found in the mediastinum hepatic hilus and retroperitoneum (maximal SUV approximately 8.39). Moreover lesions with increased density were found in the left third rib the right upper femur and the left acetabulum which were accompanied by an increase in fluorodeoxyglucose. The patient was diagnosed with PLC after liver transplantation due to liver carcinoma. Cancer cells among the exfoliated cells in the pleural effusion are shown. All slides are stained with hematoxylin and eosin and were photographed under light microscope at 400 — original magnification. The treatment with steroid and aminophylline continued to improve the status of the patient™s interstitial lesions. Although antirejection therapy was stopped rejection did not occur and the function of the graft liver was stable. Oral capecitabine was administered but was not effective. The patient experienced increasing chest tightness and shortness of breath and he died as a result of respiratory failure 1 month later. Discussion PLC was first described by Troisier in 1873. About 30% to 40% of malignancies may present with metastasis to the lung and PLC accounts for approximately 6% to 8% of metastatic cancer in the lung. Most PLCs originate from adenocarcinomas and they are most often due to lung cancer followed by breast cancer and gastric cancer [23]. Patients with renal cancer cervical cancer thyroid cancer and melanoma rarely develop PLC [4-6]. The pathologic features of PLC include infiltration of cancer cells and interstitial edema in and around lymphatic vessels as well as infiltration of inflammatory cells caused by lymph node metastasis in the lung. The metastatic cancer in the mediastinal and pulmonary hilar lymph nodes may obstruct lymphatic drainage resulting in retrograde migration of cancer cells into terminal lung tissues via lymphatic vessels or anterograde migration of cancer cells in the pleura into the pulmonary hilar lymph nodes through intrapulmonary lymph vessels. In addition a cancer embolus may form in the terminal vessels of the lung due to hematogenous metastasis which can invade the surrounding lymphatic vessels. Thus hilar and mediastinal lymph node metastasis may be present or absent in PLC depending on the route of metastasis of the primary cancer. Extrahepatic metastasis of liver carcinoma is mostly found in the lung adrenal gland bone and central nervous system. Hematogenous spread is thought to be the most common extrahepatic metastatic route [78]. "

glioma initiates from glial cells and contains several types such as astrocytoma and oligodendroglioma1 over a quarter of brain tumors are glioma which causes a large number of cancerrelated deaths every year around the world1 the current clinically therapeutic strategies are surgery combined with chemotherapy and radiotherapy2 however the prognosis of glioma patients remains not well post therapy3 hence it is urgently required to discover new molecular mechanism for glioma therapyboth long noncoding rna lncrna and microrna mirna belong to noncoding rnas which have no proteincoding ability lncrna is characterized with more than nucleotides while mirna is about nucleotides in length4 lncrna and mirna are involved in various cellular processes including cell division invasion and survival5 dysregulation of lncrna or mirna usually causes tumor initiation and progression67 for example lncrna linc00152 upregulation promotes gastric cancer growth and metastasis8 lncrna snhg6 overexpression facilitates lung cancer cell proliferation and metastasis9 mir3405p dysregulation promotes tumorigenesis of esophageal squamous cell carcinoma10 in addition mir126 cancer management and research “ du this work is published and licensed by dove medical press limited the full terms of this license are available at wwwdovepresscomtermsphp and incorporate the creative commons attribution “ non commercial unported v30 license httpcreativecommonslicensesbync30 by accessing the work you hereby accept the terms noncommercial uses of the work are permitted without any further permission from dove medical press limited provided the work is properly attributed for permission for commercial use of this work please see paragraphs and of our terms wwwdovepresscomtermsphpcorrespondence jun wu weiwen qiu email wwwwjjjj924163com weiwenqhotmailcomsubmit your manuscript wwwdovepresscomdovepresshttp102147cmars262279 0cdu dovepresssuppresses colon cancer cell survival and induces apoptosis11 besides lncrna has been identified as potential competing endogenous rna cerna for mirna to function in cancer12 the potential roles underlying lncrna and mirna still require much investigation and the relationship between lncrna and mirna also needs to be definedlinc00173 is an oncogene in lung cancer and breast cancer1314 the function of linc00173 in glioma is unclear in the current study we found that linc00173 was upregulated in glioma tissues linc00173 high expression was associated with a low survival rate linc00173 depletion suppressed proliferation migration and invasion of glioma cells linc00173 was discovered to sponge mir765 to elevate nutf2 expression taken together our findings supported that linc00173 plays essential oncogenic roles in glioma through activating mir765nutf2 pathwaymaterials and methodsclinical samplesthirtyseven glioma tissues and normal tissues were collected from lishui city people™s hospital patients received no radiotherapy or chemotherapy before surgery all tissues were stored in liquid nitrogen association between linc00173 expression and clinical characteristics in glioma tissues was analyzed in table written informed consent was obtained from every patient this study was approved by the ethics committee of lishui city people™s hospital no and the table association between linc00173 expression and clinical characteristics in glioma tissuesfeaturesage years‰gendermalefemalegradei“iiiii“ivtumor size cm‰low n19high n18pvalueexperiments were conducted in accordance with the declaration of helsinkicell culture and treatmentthe normal human astrocyte nha and glioma cell lines were purchased from institute of biochemistry and cell biology of the chinese academy of sciences shanghai china cells were cultured using pmi1640 medium invitrogen carlsbad ca usa supplemented with fetal bovine serum fbs invitrogen shrnas against linc00160 mir6293p mimics mir6293p inhibitors and negative controls were obtained from genepharma and transfected into glioma cells using lipofectamine invitrogen according to the manufacturer™s instructions efficiency was validated using qrtpcr after hqrtpcrtotal rna was extracted from tissues and cell lines using trizol invitrogen carlsbad ca primescript rt reagent kit rr047a takara holdings inc tokyo japan was used to generate cdna from rna template qpcr was completed through sybr premix ex taq„¢ ii takara japan gapdh was the normalized control relative expression was calculated through the ˆ’δδct methodluciferase reporter assaythe fragment of linc00173 or nutf2 containing indicated mir765 binding site was constructed into pmir report vector for luciferase reporter assay glioma cells were transfected with report vector and mir765 mimics after h the luciferase reporter activity was measured through the dualluciferase reporter assay system promega madison wiwestern blot assaycells were lyzed using radioimmunoprecipitation buffer beyotime shanghai china protein concentration was determined by a bca protein assay kit thermo fisher scientific ma then proteins were separated using sdspage and transferred onto pvdf membranes after blockage using bsa for h the membrane was incubated the primary antibodies at °c overnight after washed times using pbst the membranes were incubated with horseradish peroxidaselabeled second antibody followed by detection the enhanced chemiluminescence reagent emd millipore usathrough submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du cck8 and colony forming assaysproliferation was measured using cck8 and colony formation assay cck8 assay was performed using the cck reagent dojindo kumamoto japan according to the manufacturer™s instructions and absorbance was determined at nm using a microplate reader biotek winooski vt for colony formation assay cells were seeded into 6well plates and cultured for days then the cells were fixed with methanol and stained with crystal violet for minutesedu assaycells were plated into 96well plates and incubated with edu μl at °c for h followed by detection using facstranswell migration and invasion assaystranswell plates corning ny were used to measure migration and invasion according to the manufacturer™s instructions in brief cells were suspended into μl serumfree medium and seeded into the upper chamber while the lower chamber was filled with µl of complete medium after incubation for cells in the lower chamber were fixed with methanol and stained with crystal violet for minutes migrated and invaded cells were counted through a light microscopestatistical analysisgraphpad prism graphpad ca usa was used to analyze results data were presented as means±standard deviation sd significant differences were analyzed using student™s ttest or oneway anova survival rate was analyzed by the kaplan“meier method and log rank test p005 was considered to be significantresultslinc00173 expression is elevated in gliomathe expression of linc00173 was firstly analyzed through qrtpcr we found that linc00173 level was elevated in glioma tissues compared with normal tissues figure 1a besides we found that linc00173 was also upregulated in glioma cell lines compared to nha cells figure 1b then according to the median value of linc00173 glioma tissues were classified into two groups after analysis we found that linc00173 high expression correlated with poor prognosis figure 1ctransfection of linc00173 enhanced glioma cell proliferation migration and invasionto explore the function of linc00173 u87 and u251 cells were chosen after shlinc00173 linc00173 expression was significantly downregulated figure 2a through cck8 assay we observed that linc00173 knockdown suppressed the proliferation capacity of glioma cells figure 2b and c which was validated by edu and colony formation assays figure 2d and e afterwards transwell assay was performed results indicated that linc00173 loss inhibited migration and invasion of glioma cells figure 2f and g thus linc00173 exerted oncogenic roles by affecting proliferation migration and invasionlinc00173 worked as the sponge for mir765linc00173 has been found to serve as cerna for several mirnas such as mir490 and mir2181314 to determine the mechanism of linc00173 in glioma we also figure linc00173 expression is elevated in glioma a the level of linc00173 in glioma tissues was measured b the expression of linc00173 in glioma cell lines and nhas c association between overall survival and linc00173 expression in glioma patients p005cancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 enhanced glioma cell proliferation migration and invasion a qrtpcr analysis of linc00173 expression in u87 and u251 cells b“e proliferation ability was measured using cck8 edu and colony formation assays f and g migration and invasion capacity was evaluated after linc00173 knockdown in glioma cells p005suppressed the supporting their direct performed bioinformatics analysis using mirdb we identified that mir765 was the most potential candidate because it scored the highest to validate it we constructed luciferase reporters figure 3a followed by luciferase reporter assay results showed that mir765 activity of linc00173wt only figure 3b interaction pulldown assay further demonstrated their interaction figure 3c qrtpcr found that linc00173 overexpression suppressed the level of mir765 figure 3d next bioinformatics analysis using mirdb and targetsan implied that nutf2 is the most potential target of mir765 the corresponding luciferase reporters were further constructed figure 3e luciferase reporter assay also demonstrated the interaction between nutf2 and mir765 figure 3f besides nutf2 expression was suppressed by mir765 mimics figure 3g moreover nutf2 level was decreased after linc00173 knockdown while mir765 inhibitors reversed it figure 3h finally we found that mir765 level was negatively correlated with linc00173 or nutf2 in glioma tissues figure 3i and jlinc00173 promoted glioma progression through mir765nutf2 pathwaywe noticed that nutf2 expression was upregulated in glioma tissues figure 4a and b suggesting an oncogenic role to investigate whether linc00173 regulates glioma progression through mir765nutf2 we restored the expression of nutf2 in shlinc00173 transfected cells cck8 and transwell assays demonstrated that nutf2 restoration successfully rescued the capacities of proliferation migration and invasion in glioma cells transfected with shlinc00173 figure 4c“f therefore linc00173 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du figure linc00173 worked as the sponge for mir765 a bioinformatics analysis indicated the binding sites between linc00173 and mir765 b u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter linc00173wt or linc00173mut then relative luciferase activity was determined c rna pulldown assay using biotinlabeled mirnas d relative expression of mir765 after linc00173 knockdown e bioinformatics analysis indicated the binding sites between mir765 and nutf2 f u87 cells were transfected with mir765 mimics or negative controls mirnc and luciferase reporter nutf2wt or nutf2mut then relative luciferase activity was determined g qrtpcr analysis for nutf2 expression h western blotting analysis for nutf2 protein level i and j correlation analyses of linc00173 mir765 and nutf2 in glioma tissues using pearson™s correlation coefficient p005contributes to glioma progression through mir765nutf2 pathwaydiscussionas the most malignant brain tumor glioma leads to a huge number of deaths patients with glioma display a poor prognosis therefore it is of great significance to reveal the mechanism underlying glioma progression in this study we found that linc00173 was upregulated in glioma tissues and cells linc00173 overexpression predicted a poor prognosis moreover linc00173 knockdown the proliferation migration and invasion of glioma cells linc00173 was also found to inhibit mir765 and promote nutf2 expression summarily our research discovered that linc00173 is an important oncogenic lncrna in gliomasuppressed the potential roles of lncrna in glioma have been researched for a long time many lncrnas have been identified to participate in glioma development for example lncrna nck1as1 enhances growth and metastasis of glioma through targeting mir13823p to activate β catenin signaling2 lncrna ccat2 contributes to glioma progression by activating vegfa pathway15 lncrna linc00467 upregulation promotes glioma development through repressing p53 level16 previous study showed that linc00173 downregulation promotes nonsmall cell lung cancer cell growth and survival17 however another study showed that linc00173 enhances chemoresistance and facilitates tumor progression in small cell lung cancer13 besides linc00173 contributes to breast cancer development14 yet how linc00173 works in glioma remains undermined in our study we found that linc00173 was upregulated in glioma tissues linc00173 knockdown inhibited the proliferation migration and invasion of glioma cells therefore our data discovered that linc00173 is a new oncogene in glioma for the first timecancer management and research submit your manuscript wwwdovepresscom dovepress 0cdu dovepressfigure linc00173 promoted glioma progression through mir765nutf2 pathway a and b nutf2 expression in glioma tissues and normal tissues according to tcga data using gepia tool and qrtpcr c and d proliferation was measured by cck8 assay e and f migration and invasion potential was determined by transwell assay p005lncrna has been found to serve as mirna sponge in tumor cells for instance lncrna ttnas1 sponges to promote breast cancer metastasis18 mir1405p lncrna cdkn2bas1 sponges mir3245p to regulate cellcycle progression in laryngeal squamous cell cancer19 previous studies also revealed linc00173 was a sponge for some mirnas such as mir4903p and mir2181314 in our study we did not observe linc00173 sponges above mirnas however through bioinformatics we identified linc00173 targeted mir765 in glioma we demonstrated their direct interaction and found that linc00173 overexpression inhibited mir765 expression mir765 has important roles in cancers mir765 was found to suppress tongue squamous cell carcinoma development20 mir765 also promotes myeloma and osteosarcoma progression2122 besides mir765 plays oncogenic or anticancer roles in gastric cancer and breast cancer2324 its role in glioma remains unclear our results suggested that mir765 was a tumor suppressor in gliomalncrnamirnamrna regulatory axis is widely observed in cancer for example linc00703mir181a klf6 axis suppresses the development of gastric cancer25 linc00312mir9cdh1 axis was found to promote breast cancer progression26 through bioinformatics we found that mir765 targeted nutf2 in glioma moreover we showed that nutf2 expression was regulated by linc00173mir axis the function of nutf2 in cancer is nearly unknown in our work we found that nutf2 expression was upregulated in glioma tissues compared to normal tissues moreover we found that nutf2 overexpression promoted the proliferation migration and invasion of glioma cells indicating nutf2 was an oncogene in gliomain conclusion our study showed that linc00173 acted as a sponge for mir765 to promote nutf2 expression and linc00173mir765nutf2 axis plays a critical function in promoting glioma progressionfunding this work was supported by zhejiang province analytical testing and experimental animal program lgd19h and zhejiang province welfare technology applied research project 2017c37111 disclosureall authors declare no conflicts of interest in this workreferences ostrom qt cioffi g gittleman h cbtrus statistical report primary brain and other central nervous system tumors diagnosed in the united states in neuro oncol 201921suppl 5v1“ v100 101093neuoncnoz150the of glioma huang l li x ye h et al long noncoding rna nck1as1 promotes sponging microrna13823p and activating the trim24wntbetacatenin axis j exp clin cancer res 101186s13046 tumorigenesis through chen w lei c liu p et al progress and prospects of recurrent glioma a recent scientometric analysis of the web of science in world neurosurg 2020134e387“e399 101016jwneu20 submit your manuscript wwwdovepresscom dovepress cancer management and research 0cdovepress du sun b meng m wei j wang s long noncoding rna pvt1 contributes to vascular endothelial cell proliferation via inhibition of mir190a5p in diagnostic biomarker evaluation of chronic heart failure exp ther med “ 103892etm20208599 feng s yao j chen y functional role of reprogrammingrelated long noncoding rna lincrnaror in glioma j mol neurosci “ 101007s120310140488z zhang d zhou h liu j mao j long noncoding rna asb16as1 promotes proliferation migration and invasion in glioma cells biomed res int sun l zhao m wang y neuroprotective effects of mir27a against traumatic brain injury via suppressing foxo3amediated neuronal autophagy biochem biophys res commun “ 101016jbbrc201612001 shi y sun h downregulation of lncrna linc00152 suppresses gastric cancer cell migration and invasion through inhibition of the erkmapk signaling pathway onco targets ther “ 102147otts217452 li k jiang y xiang x et al long noncoding rna snhg6 promotes the growth and invasion of nonsmall cell lung cancer by downregulating mir1013p thorac cancer wang x gu m ju y zhou j pik3c3 acts as a tumor suppressor in esophageal squamous cell carcinoma and was regulated by mir340 5p med sci monit 202026e920642 1012659msm923909 wei l chen z cheng n microrna126 inhibit viability of colorectal cancer cell by repressing mtor induced apoptosis and autophagy onco targets ther “ 102147 otts238348 chen y shen z zhi y long noncoding rna ror promotes radioresistance in hepatocellular carcinoma cells by acting as a cerna for microrna145 to regulate rad18 expression arch biochem biophys “ 101016jabb201803018 zeng f wang q wang s et al linc00173 promotes chemoresistance and progression of small cell lung cancer by sponging mir218 regulate etk expression oncogene “ to 101038s4138801909842 fan h yuan j li x et al lncrna linc00173 enhances triplenegative breast cancer progression by suppressing mir490 3p expression biomed pharmacother 1010 16jbiopha2020109987 sun sl shu yg tao my lncrna ccat2 promotes angiogenesis in glioma through activation of vegfa signalling by sponging mir424 mol cell biochem ““ 101007 s1101002003712y zhang y jiang x wu z et al long noncoding rna linc00467 promotes glioma progression through inhibiting p53 expression via binding to dnmt1 j cancer “ 107150 jca41942 yang q tang y tang c diminished linc00173 expression induced mir1825p accumulation promotes cell proliferation migration and apoptosis inhibition via agernfkappab pathway lung cancer am j transl res in nonsmallcell “ xue j zhang z li x ren q wang q long noncoding rna ttnas1 promotes breast cancer cell migration and invasion via sponging mir1405p oncol lett “ 1038 92ol201911222 liu f xiao y ma l wang j regulating of cell cycle progression by the lncrna cdkn2bas1mir3245prock1 axis in laryngeal squamous cell cancer int j biol markers “ 1011771724600819898489 ding j yang c yang s linc00511 interacts with mir765 and modulates tongue squamous cell carcinoma progression by targeting lamc2 j oral pathol med “ 101111 jop12677 long s long s he h chen g microrna765 is preregulated in multiple myeloma and serves an oncogenic role by directly targeting sox6 exp ther med “ 103892 etm20197473 lv db zhang jy gao k microrna765 targets mtus1 to promote the progression of osteosarcoma via mediating erkemt pathway eur rev med pharmacol sci “ 1026355eurrev_201906_18040 jiao y yuan c wu h li x yu j oncogenic microrna765 promotes the growth and metastasis of breast carcinoma by directly targeting ing4 j cell biochem yuan l ma t liu w et al linc00994 promoted invasion and proliferation of gastric cancer cell via regulating mir7653p am j transl res “ yang h peng m li y zhu r li x qian z linc00703 acts as a tumor suppressor via regulating mir181aklf6 axis in gastric cancer j gastric cancer “ 105230jgc2019 19e43 chen y qiu f huang l et al long noncoding rna linc00312 regulates breast cancer progression through the mir9cdh1 axis mol med rep “ 103892mmr201910895cancer management and research publish your work in this journal cancer management and research is an international peerreviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes enhanced survival and quality of life for the cancer patient the manuscript management system is completely online and includes a very quick and fair peerreview system which is all easy to use visit httpwwwdovepresscomtestimonialsphp to read real quotes from published authors dovepress submit your manuscript here 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"Ethnopharmacological relevance Tetrastigma hemsleyanum Diels et Gilg Themsleyanum a rare herbal plant distributed in subtropical areas of mainland China has become a focus of scientific attention in recent years because of its high traditional value including uses for treatment of children with fever pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Aim This systematic review aims to provide an insightful understanding of traditional uses chemical composition pharmacological effect and clinical application of T hemsleyanum and lay a foundation for the further study and for the utilization of T hemsleyanum resource Materials and methods A domestic and overseas literature search in known databases was conducted for published s using the relevant keywords Results One hundred and fortytwo chemical constituents identified from T hemsleyanum have been reported including flavonoids phenolic acids polysaccharide anic acids fatty acids terpenoids steroids amino acid and others Among these components flavonoids and polysaccharides were the representative active ingredients of T hemsleyanum which have been widely investigated Modern pharmacological studies have shown that these components exhibited various pharmacological activities such as antiinflammatory antioxidant antivirus antitumor antipyretic antihepatic injury immunomodulatory antibacterial etc Moreover different toxicological studies indicated that the clinical dosage of T hemsleyanum was safe and reliable Conclusions Modern pharmacological studies have well supported and clarified some traditional uses and T hemsleyanum has a good prospect for the development of new drugs due to these outstanding properties However the present findings did not provide an indepth evaluation of bioactivity of the extracts the composition of its active extracts was not clear Moreover they were insufficient to satisfactorily explain some mechanisms of action Data regarding many aspects of T hemsleyanum such as links between the traditional uses and bioactivities pharmacokinetics quality control standard and the clinical value of active compositions is still limited which need more attention Introduction Tetrastigma hemsleyanum Diels et Gilg T hemsleyanum mostly known as œSan ye qing is a kind of folk plant Because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource It mainly grows in the eastern central southern and south western provinces of China such as Zhejiang Jiangsu Guangxi Fujian and Yunnan provinces Peng and Wang T hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models Xu As an edible plant the leaves of T hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body Sun while the aerial parts of T hemsleyanum developed as potential new traditional chinese medicine TCM preparations Guo Corresponding author Ningbo Research Institute of Zhejiang University Ningbo Zhejiang People™s Republic of China Email address px4142163com X Peng 101016jjep2020113247 Received May Received in revised form July Accepted August JournalofEthnopharmacology2642021113247Availableonline12August2020037887412020ElsevierBVAllrightsreserved 0cT Ji Abbreviations T hemsleyanum Tetrastigma hemsleyanum Diels et Gilg TCM UPLCESIQTOFMSMS Ultra high performance liquid Traditional Chinese Medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκB 5hydroxytryptamine norepinephrine dopamine prostaglandin E2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin MIC GSH MDA NFκB 5HT NE DA PGE2 MAPK mitogenactivated protein kinase LPS Celegans Caenorhabditis elegans TNFα IL1 IL6 IL12p40 interleukin subunit p40 sTNFR1 soluble TNF receptors IL10 IL1 IL4 iNOS TLR4 MD2 MyD88 myeloid differentiation protein JNK GPT GOT ALP SOD interleukin interleukin interleukin inducible NO synthase Tolllike receptor myeloid differentiation factor2 cJun Nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory The root tubers of T hemsleyanum are extensively used either alone or in combination with other herbal medicines in TCM clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain Sun Chen and Guo Therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal In February T hemsleyanum was awarded as the new œeight famous kinds of TCM in Zhejiang province meant that it has become a key object of industrialization development of Zhejiang™s dominant large varieties of medicinal materials In COVID19 broke out and has caused more than deaths in China and infection cases have been reported in more than countries Hua Shi Xuan Fei mixture Approval number of Zhejiang medicine Z20200026000 which composed of T hemsleyanum has been approved by Zhejiang Provincial Drug Administration for clinical treatment of COVID19 Futhermore the modern pharmacological studies had shown that T hemsleyanum also had effects of antiinflammatory Ji antioxidant Hossain antivirus Ding antitumor Lin antipyretic Yang and Wang antihepatic injury Ma et al immunomodulatory Xu antibacterial Chen hypoglycemic Ru 2018ab etc Numerous reports have demonstrated that the biological activities of T hemsleyanum are attributed to its many chemical components Fu Wang has reported isolated alkaloids from the aerial parts of T hemsleyanum Wang Ru extracted a novel polysaccharide TDGP3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin A secretory immunoglobulin A Epithelialmesenchymal transition ALT AST HA LN TBiLi TP IFNÎ IgA SIgA EMT MMPs matrix metalloproteinase TIMPs matrixmetallo proteinase Cytc CAT GSHPx glutathione peroxidase Tregs TGF COX2 Foxp3 PDL TAOC CCl4 CEF HVJ VSV A F S1 S2 PEF CFF EAF BAF Cytochrome c catalase regulatory T cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast Hemagglutinating virus of Japan vesicular stomatitis virus alkalicontaining extract of T hemsleyanum ketonecontaining extract of T hemsleyanum crude extract of T hemsleyanum crude extract of T hemsleyanum Petroleum ether extractions of T hemsleyanum ethanol extract Chloroform extractions of T hemsleyanum ethanol extract ethyl acetate extractions of T hemsleyanum ethanol extract nbutanol extractions of T hemsleyanum ethanol extract T hemsleyanum with a molecular weight of — Da by enzymolysisultrasonic assisted extraction method Ru 2019ab Large amounts of flavonoids were found in leaves aerial parts and root tubers of T hemsleyanum Xu 2014ab Deng Yu In addition T hemsleyanum also contains a variety of functional components such as anic acids Hu phenolic acids Liu minerals Fan amino acids Fu etc In recent years wild resources of T hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments In it was listed in the preferentially protected crop germplasm resources of Zhejiang province Based on our team™s preliminary research Peng Peng 2016ab Li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of T hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization Materials and methods The available information about the traditional uses phytochemicals and pharmacological properties of T hemsleyanum was searched via Web of Science Google Scholar PubMed Science Direct China National Knowledge Infrastructure CNKI and Springer search using Chinese or English as the retrieval languages The keywords used include T hemsleyanum root tubers of T hemsleyanum Radix Tetrastigma JournalofEthnopharmacology26420211132472 0cT Ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words All references were from experimental studies and published prior to April were reviewed All chemical structures were drawn using ChemDraw Pro software heatclearing were Botanical characteristics T hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose It is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus The optimum pH is between and The root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter Fig The epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section The stem of T hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node Palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate The leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base The flowers of T hemsleyanum are small yellow green and ovate The flowering stage of T hemsleyanum ranges from April to June and the fruit phase is normally from August to November When the flower withered it will form a small green round fruit with the size of millet When it is mature the fruit will turn from green to red the berries are spherical and soft spherical Traditional uses T hemsleyanum belonging to the family Vitaceae was firstly recorded in Ben Cao Gang Mu Ming Dynasty AD The aliases of Sanyeqing include Shi Hou Zi Shi Bao Zi Shi Lao Shu Lan Shan Hu Lei Dan Zi Po Shi Zhu Tu Jing Wan Sou Jia Feng San Ye Dui golden wire hanging gourd golden bell golden wire hanging potato etc The root tubers or whole grass of T hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of TCM such as Zhi Wu Ming Shi Tu Kao Qing Dynasty Wu Jiangxi herbal medicine Common folk herbal medicine in Zhejiang All of these ancient works described the effects of toxicityremoving T dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women National compilation team of Chinese herbal medicine In the TCM culture the properties of T hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional Chinese medicine and Zhong Hua Ben Cao Shanghai Science and Technology Press The channel tropism was lung heart liver and kidney meridians Decocting with water or mashing for external application are the traditional possess methods of T hemsleyanum Considering its extensive traditional effects many prescriptions containing T hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies Excitingly it has reported that Jinlian disinfection drink containing san ye qing combined with interferon can treat Covid19 He Jinqi Tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was Wei Moreover Zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage III primary liver cancer Jiang and Gong In addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea Gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites Ji Chemical compounds of Themsleyanum The chemical constituents of T hemsleyanum have been widely investigated Sun Sun Zeng Xu 2014ab Fu Fan Chen Ding 2015a Fig The aerial part A root tuber B and raw herb C of T hemsleyanum JournalofEthnopharmacology26420211132473 0cT Ji b Ding a total of one hundred and fortytwo compounds have been isolated and identified from T hemsleyanum until now The information about compound name molecular weight compound formula detection method analysis sample is summarized in Table Flavonoids and their glycosides Modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from T hemsleyanum Lin Zhang Table To date fiftyone flavonoids and their glycosides have been extracted and identified from T hemsleyanum In this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on C3² and C4™ on the B ring of flavonoid aglycone At present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids Among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry UPLCESIQTOFMS has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution Our team used UPLCESIQTOFMS to identify chemical constituents from the aerial part of T hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc Sun According to the report Liu total flavonoids of T hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase MAPK and nuclear factorκB NFκB in lung tissue Moreover the flavonoids of T hemsleyanum had the activity of antilung cancer Wei Luteolin a flavonoid found in T hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers Muhammad It is certain that T hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs Polysaccharide Saccharide is another important active ingredient extracted from T hemsleyanum Shao Polysaccharide has great potential in clinical application because of its unique pharmacological activity However due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures Guo Table The prescriptions and traditional uses of T hemsleyanum in China Prescriptions name Qingteng Fengshi Qufengshi Yaojiu Main composition Jiu Traditional use T hemsleyanum Parabarium chunianum Tsiang Zanthoxylum nitidum Roxb DC T hemsleyanum Deeringia amaranthoides Lam Merr Blumea aromatica Wall DC T hemsleyanum Deeringia amaranthoides Lam Merr Zanthoxylum nitidum Roxb DC Panax notoginseng Burk FH Chen T hemsleyanum Gypsum Lonicera japonica Thunb Houttuynia cordata Thunb Ophiopogon japonicus Linn f KerGawl T hemsleyanum T hemsleyanum Lysimachia christinae Hance Imperata cylindrica Citrus reticulata Blanco T hemsleyanum ginsenoside Astragalus propinquus Schischkin T hemsleyanum Nepeta cataria L Lonicera japonica Thunb Saposhnikovia divaricata Trucz Schischk Huatuo Fengtongbao capsule Sanyeqing Gypsum Decoction Sanyeqing Power Zhonggan mixture Jinqi Tablet Hua Shi Xuan Fei mixture extracted the polysaccharides from roots of T hemsleyanum RTP1 RTP2 and RTP3 were successively found by protein precipitation and purification Moreover further study indicated RTP31 was high purity polysaccharide with a molecular weight of kDa and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively Ru 2018ab extracted a polysaccharide THP from T hemsleyanum with the average molecular weight estimated as kDa The results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of In Ru 2019ab successfully extracted polysaccharide THDP3 from T hemsleyanum with molecular weight of kDa which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of Moreover TDGP3 mainly consists of †’4αDGalAp1†’ †’4DGalp1†’ and †’4αDGlcp1†’ residues as backbones and DManp1†’ †’36DManp1†’ and αDAraf1†’residues as branches Phenolic acids Phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring As a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects Twenty three phenolic acids No52“ Table have been reported in the aerial parts of T hemsleyanum such as caffeic acid chlorogenic acid 1OgalloylDglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid There were twentyone phenolic acids in the root tuber of T hemsleyanum some of which were the same as aerial parts Alkaloids Alkaloids are a group of basic anic compounds containing nitrogen that exist in nature Alkaloids are stored in small quantities in T hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare Wang Fu extracted the aerial parts of T hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and Treatment of joint pain wind cold dampness arthralgia Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis Treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture Treatment of infantile hyperpyretic convulsion Treatment of blood avalanche leucorrhea Treatment of liver cancer Treatment of malignant tumor Treatment of Covid19 Usage Oral administration “ mL once times a day Oral administration mL once times a day Oral administration capsules once times a day References Ministerial standard Ministerial standard Ministerial standard One dose a day decoct twice in water and take it “ times after mixing Oral administration Oral administration mL once times a day Oral administration capsules once times a day Oral administration mL once times a day Xu Gao Jiang and Gong Wei Zhejiang Provincial Drug Administration JournalofEthnopharmacology26420211132474 0cT Ji Detection Mode Analysis parts of sample Reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber Sun Sun Zeng Sun Sun Sun Zeng Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Zeng Sun Zeng Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Xu 2014b Sun Zeng Sun Zeng Sun Zeng Zeng Sun Sun Sun Sun Xu 2014b Sun Xu 2014b Sun Zeng Sun Xu 2014b Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Fu Sun Sun Xu 2014b Fan Xu 2014b Fan Sun continued on next page UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Table Chemical constituents isolated from the different parts of T hemsleyanum Name Flavonoids and their glycosides quercetin quercitrin quercetin3Oglucoside quercetin3Orutinoside quercetin3galactoside quercetin3Oxylosylglucoside quercetin3Oxylosylglucose7Orhamnoside orientin orientin2²²Orhamnoside Isoorientin isoorientin2²²Orhamnoside isoorientin cid0 ²²Oxyloside vitexin vitexin2²²Orhamnoside vitexin2²²Oglucoside vitexin2²²Oarabinoside isovitexin isovitexin2²²Orhamnoside isovitexin2²²Oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8Cxylosyl6Cglucoside apigenin6CαLarabinose8CDglucose eriodictyol eriodictyolOhexoside I eriodictyolOhexoside II luteolin luteolin6 8diChexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3Oneohesperidin kaempferol3Orhamnoside kaempferol7Orhamnose3Oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3Ocarfuran7Orhamnosyl glucoside daidzein biochanin A procyanidin dimmer procyanidin B1 procyanidin B2 procyanidin trimer Phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR13CNMR MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS JournalofEthnopharmacology26420211132475 0cT Ji Table continued Name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1OgalloylDglucose protocatechol glucoside epigallocatechin vanillic acid1Ofuran celery glucosyl ester protocatechuic acid1Ofuran celery glucosyl ester methoxyphenol1Ofuran glycosylOglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid Alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid Scid0 trolline Fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid Dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether Trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid diMeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid Terpenoids and steroids sitosterol daucosterol campesterol Stigmasterol 6Obenzoyl daucosterol ergosterol taraxerone Taraxerol αamyrine pteroside Z ganoderic acid H 3epipapyriferic acid oleanic acid Saponins Ginsenoside Rh1 Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS 1HNMR LCMS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS NMR UV MS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS GCMS TCL HNMR CNMR MS GCMS GCMS IR HNMR EIMS IR HNMR MS IR HNMR MS IR HNMR MS IR EIMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS HNMR CNMR MS Analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber Reference Sun Sun Sun Sun Sun Sun Zeng Sun Xu 2014b Zeng Zeng Zeng Zeng Xu 2014b Xu 2014b Chen Fu Fu Fu Fu Fu Fu Fu Fu Fu Fu Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Sun Chen Ding Sun Sun Guo Ru Ru Ru Ru Sun Sun Sun Ding UPLCESIQTOFMSMS root tuber Sun continued on next page JournalofEthnopharmacology26420211132476 0cT Ji Table continued Name Ginsenoside Rh2 Vinaginsenoside R1 Amino acid and derivatives Phenylalanine pyroglutamic acid glutimic acid hexose Tryptophan Lglutamic acid Detection Mode UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS UPLCESIQTOFMSMS Analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part Reference Sun Sun Sun Sun Sun Sun Sun respectively a carboline By comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and Scid0 trolline The chemical structures were shown in Fig identified as indole a

" the widely recognized anticancer potential of aspirin has created a broad interest to explore theclinical benefits of aspirin in cancer therapy however the current understanding of the molecular mechanismsinvolved in the anticancer potential of aspirin remains limitedmethods cancer stemness assays which contained aldh side population chemoresistance sphere formationand tumorigenesis were performed to validate aspirin function in vitro and in vivo histone modification assay wasperformed to check the effect of aspirin on histone methylation as well as the activity of hdac and kdm6abinhibitor in vivo assay was performed to evaluate therapeutic effects of various inhibitor combination mannersresults in regards to in vitro studies aspirin diminishes cancer cell stemness properties which include reducing thealdh subpopulation side population chemoresistance and sphere formation in three cancer types in regards toin vivo studies aspirin decreases tumor growth and metastasis and prolongs survival in addition our resultsshowed that aspirin inhibits inflammationrelated stemness gene expression especially icam3 identified by a highthroughput sirna platform in regards to the underlying molecular mechanism of action aspirin reduces histonedemethylase kdm6ab expression that mediates histone methylation and suppresses gene expression via a coxindependent manner in regards to therapeutic strategies aspirin combined hdm inhibitors icam3 downstreamsignaling srcpi3k inhibitors or icam3 inhibitor lifitigrast prevents cancer progression in vivos the aforementioned findings suggest a molecular model that explains how aspirin diminishes cancercell stemness properties these findings may provide novel targets for therapeutic strategies involving aspirin in theprevention of cancer progressionkeywords aspirin histone methylation cancer stemness icam3 cox therapeutic strategies correspondence shenwenzhi2011126com1department of pathology and institute of precision medicine jining medicaluniversity hehua road jining chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang stem cell research therapy page of cancer stem cell csc was found as the chief culprit toinitiate tumor occurrence to enhance tumor malignancyand to cause tumor recurrence whereby the maintenanceof cancer cell stemness mainly depends on the tumormicroenvironment or also called the œniche [“] currently the specific properties of csc were identified likehigh aldh1 activity aldehyde dehydrogenase sidepopulation chemoresistance and other cd moleculescd44 cd133 or markers sox2 oct4 nanoglgr5 positive in cancer clinical therapy which targetedhighly tumorigenic cscs may provide new targets orinsight for cancer therapy however unfortunately cscshad demonstrated a relative resistance to conventionalchemotherapy and radiotherapy moreover the cancer cellstemness and the resultant tumor initiationmalignancycould be maintained by the tumorassociated inflammation factors within the tumor microenvironment niche[ ] our previous work presented a mediumthroughput sirna screen platform to identify inflammation genes that regulate cancer cell stemness and obtainedseveral novel candidates agents that target these genesmay inhibit both inflammation and cancer cell stemnessat the same timeaspirin a nonsteroidal antiinflammatory drugiscommonly used as an antipyretic analgesic antiinflammatory and antithrombotic agent [ ] recentobservational and epidemiological studies have shownthat regular prolonged use of aspirin reduces the riskfor several cancers eg colorectal esophageal breastlung prostate liver and skin cancers [“] althoughthe benefits of aspirin for cancer patients have beenwidely appreciated the mechanism remains unclear previous studies attribute the anticancer potential of aspirin to the inhibition of cyclooxygenase2 cox2which is upregulated in various cancer cells [ ] ofnote an increasing body of evidence suggests that aspirin exhibits anticancer effects in a coxindependentmanner histone modification is a reversible process mediated bythe epigenetic enzymes [ ] histone methylation andacetylation are two important chemical modifications thatact in transcriptional activation or inactivation chromosome packaging and dna damagerepair [ ] histone demethylases hdms and histone deacetylaseshdacs are the key enzymes that remove methyl andacetyl groups respectively to regulate gene transcriptionin this regard aspirin was reported to affect hdacs expression and suppress progression of some cancers like aspirin mediates h3k27 acetylation to prevent coloncarcinogenesis and aspirin cooperates with p300 to activate h3k9 acetylation further to promote colorectal cancer cell apoptosis [ ] however the specific rolesand mechanisms of aspirinmediated histone methylationin cancer stemness remains insufficient thus we studiedthe role of aspirin on histone methylation and the attendantand cancerprogressioneffects on cancercellstemnessthe aldh subpopulationour results indicated that aspirin diminishes various cancer cell stemness properties which include reducingside populationchemoresistance and sphere formation in three cancertypes in vitro aspirin inhibits tumor growth and metastasis as well as prolongs survival in vivo aspirininhibits inflammationrelated stemness genes especiallyicam3aspirin reduces histone demethylasekdm6abexpression which mediates histone methylation respectively with a coxindependent manner and aspirin hdm inhibitors aspirin icam3downstream signaling srcpi3k inhibitors and aspirin icam3 inhibitor lifitigrast all reduce cancer progressionin vivo the abovementioned findings demonstrate apromising mechanism of action and potential therapeutic strategy of aspirin in the prevention of cancerprogressionmethodscell culturemdamb231 breast cancer cell and a549 lung cancercell were purchased from atcc hepg2 was obtainedfrom the chinese academy of sciences mdamb231breast cancer cell and hepg2 liver cancer cell were cultured in dmem medium a549 lung cancer cell was cultured in medium allculture media weresupplemented with fbs gibco and were grown at °c in co2 incubators all cells were passaged forless than months before renewal from frozen earlypassage stocks and tested to ensure thatthey weremycoplasma negativecytotoxicity assayaspirin was purchased in sigma cat a2093 and dissolved in dmso mdamb231 a549 and hepg2 cellswere cultured in 96well plate and treated with variousconcentrations and mm for a549 cells and mm for mdamb231 cells and and mm forhepg2 cells of aspirin for h cell activity was testedby applying cck8 kit dojindo china following themanufacturer™s instructionsaldefluor assaythe aldeflour assay kit stemcell technologies vancouver canada was used to measure aldh enzymaticactivity in three cancer cell lines mdamb231 a549and hepg2 in brief cells were treated with aspirin for h and — cells were suspended in aldeflour 0czhang stem cell research therapy page of assay buffer containing aldh1 substrate and incubated for min at °c cells treated with the specific aldh inhibitor deab served as the negative control stained cellswere analyzed on bd facs calibur flow cytometer bdbiosciences san jose ca data analysis was performedusing flowjo software tree star inc ashland orside population assaymdamb231 a549 and hepg2 cells treated with aspirin for h were harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml hoechst dye wasadded at a final concentration of μgml μgmla549 and μgml hepg2 in the presence or absence of μm fumitremin c ftc the followingsteps were described previously [ ]cell apoptosis assaythe mdamb231 a549 and hepg2 cells were treatedwith aspirin and cisplatin ddp μgml for h simultaneously then harvested and resuspended in prewarmed staining buffer pbs buffer added fbs at adensity of — cellsml apoptotic cells were stainedwith propidiumiodide and annexinvfitc bd biosciences flow cytometry analysis was performed by facscalibur cytometer bd biosciences in which a minimum of events were recordedsphere formation assaycells were collected and rinsed to remove serum thendissociated to singlecellsuspension in serumfreedmemf12 medium supplemented with iumlpenicillin μgml streptomycin ngml human recombinant epidermal growth factor hregf ngmlhuman recombinant basic fibroblast growth factorbfgf and b27 supplement invitrogen cells weresubsequently cultured in ultralow attachment 24wellplates at a density of cells per well then treatedwith aspirin all the time until the spheres were formedanimal studyfemale balbc mice at “ weeks were separated randomly into several groups n ‰¥ — 4t1luci cellswere inoculated sc into each mouse at the right axillafor lung metastasis assay at days after injection micewere treated intraperitoneally with aspirin mgkg aspirin mgkg every days and dmso used as thecontrol for chemoresistance assay at days after injection mice were first treated intraperitoneally with cisplatin mgkg then treated with aspirin mgkgaspirin mgkg every days and dmso used as thecontrolnodscid mice at “ weeks were separated randomly into several groups n ‰¥ — a549luci cellswere inoculated sc into each mouse for inhibitor treatment assay days after tumor cells injection micewere first treated intraperitoneally with aspirin mgkgor kdm6ab inhibitor gsk j1 mgkg or src inhibitor a new specific highefficient src inhibitor [ ] mgkg or pi3k inhibitor ly294002 mgkg orlifitegrast mgkg dmso used as the controltumor volume mm3 was measured with calipers andcalculated by using the standard formulalength —width22 the individual measuring the mice was unaware of the identity of the group measured primarytumor tissues were harvested and separated into threeparts one was formalin fixed paraffin embedded andsectioned for ihc staining the other two were brokenby the tissue homogenizer then used for rna trizoland protein ripa lysis buffer extraction animal usecomplied with nankai university and jining medicaluniversity animal welfare guidelines western blottingthe western blot steps were described previously [ ]the special primary antibodies used in this assay arelisted in supplementary table s1 all western data wasthe representative image of three biologically independent repeats the results were quantified using image jsoftware national institutes of health baltimore mdand analyzed by graphpad prism5 software graphpadsoftware san diego ca usanuclear fractionation analysiscells were harvested and the cytoplasmic and nuclearfractions were separated and extracted with an nepernuclear and cytoplasmic extraction kit thermo fisherscientific inc ma usa h3k3me marker proteinswere detected by western blotimmunofluorescencecells grow on glass slides and tumor tissue slices werefixed in paraformaldehyde and labeled with primaryantibodies overnight at °c followed by incubation withspeciesappropriateroomtemperature for h nuclei were stained with dapi andimages were obtained using a leica dm4000 uprightmicroscope or confocal fluorescence microscopy nikontokyo japan [ ]antibodiessecondaryatchromatin immunoprecipitation assaythe assay was performed with an ezzyme chromatinprep kit millipore according to the manufacturer™sprotocol antihistone modification marker antibodieswere used to precipitate dna crosslinked with histone modification markers respectively and normal rabbitigg was used in parallel as a control enriched dna wasthen used as a template to assess the binding intensity of 0czhang stem cell research therapy page of histone modification markers to putative binding sitesin the icam3 promoter primers used in this assay arelisted in supplementary table s2immunohistochemistryimmunohistochemistry was performed on tumor tissuesections from the mice primary antibodies raise againstthe target proteins at a dilution overnight the expression levels of the proteins were evaluated accordingto the percentage of positive cells in each tumor tissuesections the images were recorded by olympus bx51epifluorescent microscopy under a — or — objective olympus co tokyo japan changes in apoptosis in the three cancer cell lines usingthe cell apoptosis assay after cisplatin ddp treatmentour results indicated that apoptosis increases in theaspirintreated groups versus controls and thereby reduces cisplatin chemo resistance fig 1e f in orderto determine the effects of aspirin on cancer cell stemness we next investigated the changes in cell sphere formation in the three cancer cell lines using the sphereformation assay our results showed that sphere formation decreases in the aspirintreated groups versus controls fig 1g h the abovementioned findings suggestthat aspirin diminishes cancer cell stemness propertiesin vitrostatistical analysisall data were analyzed using graphpad prism5 softwaregraphpad software san diego ca usa values wereexpressed as means ± sem p values were calculatedusing a twotailed student™s t test two groups or oneway anova more than groups unless otherwisenoted a value of p was used as the criterion forstatistical significance an asterisk indicates a significantdifference with p two asterisks indicate a significant difference with p and three asterisks indicatea significant difference with p [ ]resultsaspirin diminishes cancer cell stemness properties in vitroin order to establish the proper working concentrationsof aspirin in various cancer cells we determined theic50 of aspirin in a549 lung cancer cells mdamb231breast cancer cells and hepg2 liver cancer cells using acytotoxicity assay our results showed a 107mm ic50in a549 lung cancer cells a 43mm ic50 in mdamb breast cancer cells and a 97mm ic50 in hepg2liver cancer cells fig s1 based on the ic50 we choseworking concentrations of and mm aspirin fora549 lung cancer cells and mm aspirin formdamb231 breast cancer cells and and mmaspirin for hepg2 liver cancer cells in our studiesthe aldh subpopulation decreasesin order to determine the in vitro effects of aspirin oncancer cell stemness we investigated aldh subpopulation changes in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing the aldh staining assay our results indicatedthatin theaspirintreated groups versus controls fig 1a b inorder to determine the effects of aspirin on cancer cellstemness we next investigated the changes in the sidepopulation in the three cancer cell lines using the sidepopulation assay our results indicated that the sidepopulation decreases in the aspirintreated groups versuscontrols fig 1c d in order to determine the effects ofaspirin on cancer cell stemness we next investigated theaspirin diminishes cancer cell metastasis and stemnessproperties in vivoin order to determine the effects of aspirin on cancercell metastasis and stemness in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice seven days after implantation we ip injected themice with mgkg aspirin mgkg aspirin ordmso control group times per week fig 2a ourresults showed thattumor volume decreases in theaspirintreated groups versus the control fig 2b however we found that the body weight did not change inthe aspirintreated groups versus the control fig 2c inaddition we found that the survival time increases in theaspirintreated groups versus control fig 2d with respect to the effect of aspirin on cancer cell metastasiswe found thatlung metastasis decreases in aspirintreated groups versus the control fig 2e“g with respect to the effect of aspirin on cancer cell stemnessproperties we found thatthe immunocytochemicalstaining of sox2 and oct4 stemness markers decreasesin the aspirintreated groups versus dmso controlsfig 2h i the abovementioned findings suggest thataspirin diminishes cancer cell metastasis and stemnessproperties in vivoaspirin reduces cancer cell chemoresistance in vivoin order to determine the effects of aspirin on cancercell chemoresistance in vivo we implanted 4t1luciferase cells into the fourth fat pad of female balbcmice eight days after implantation we ip injected themice with mgkg cisplatin mgkg aspirin mgkgcisplatin mgkg aspirin or dmso control groupevery days fig 2j our results showed that tumor volume decreases in the cisplatinaspirintreated versus thedmso control fig 2k however we found that thebody weight did not change in the cisplatinaspirintreated versus the dmso control fig 2l in additionwe found that the survival time increases in the cisplatinaspirintreated groups versus the dmso controlfig 2m we also found that the rate of tumor growth 0czhang stem cell research therapy page of absllec evitisop hdladeabctrl2mm5mm 4mm10mm ctrleddp10ugml2mm5mm 4mm10mm agpehagpeha549ctrl 5mm 10mm ctrl 2mm 4mm sllec evitisop hdlasllec evitisop hdlahepg2fctrl 5mm 10mm slliec ssotpopaa549 slliec ssotpopactrl 5mm 10mm ctrl 2mm 4mm hepg2 slliec ssotpopactrl 5mm 10mm cblockctrl2mm5mm 4mm10mm agpeha549d noitlaupopeds ia549 noitlaupopeds ictrl 5mm 10mm ctrl 2mm 4mm fig see legend on next pagehepg2h noitlaupopeds ictrl 5mm 10mm rebmun erehpsgctrl2mm5mm 4mm10mm agpehhepg2ctrl 5mm 10mm ctrl 2mm 4mm rebmun erehpsrebmun erehpsctrl 5mm 10mm 0czhang stem cell research therapy page of see figure on previous pagefig aspirin restrains cancer cell stemness properties in vitro a aldh staining assay was performed to check aldh subpopulationpercentage in the three cancer cell lines with or without aspirin treatment b statistic results of aldh subpopulation percentage were shown cside population assay was performed to detect sp percentage in three cancer cell lines with or without aspirin treatment d statistic results of sppercentage were shown e facs was performed to detect cell resistance to cisplatin and the percentage of apoptotic cells was shown f statisticresults of apoptosis cells percentage were shown g sphere formation assay was performed to check the cell sphere formation ability in the threecancer cell lines with or without aspirin treatment scale bars μm h statistic results of sphere amounts were shownwas slower in the cisplatinaspirintreated versus thedmso control fig 2n with respect to the effect of aspirin on cancer cell stemness properties we found thatthe immunocytochemical staining of sox2 and oct4stemness markers decreases in the cisplatinaspirintreated groups versus dmso controls fig 2o p theabovementioned findings suggest that aspirin reducescancer cell chemoresistance in vivoaspirin inhibits the expression of inflammationrelatedstemness genes in vitro and in vivoour previously published report established a mediumthroughput sirna screening platform that identifies inflammation genes that regulate cancer cell stemness specifically we identified several novel candidate genesthat decrease oct4 expression and the aldh subpopulation both of which characterize stemness fig 3ain order to determine whether aspirin decreases theexpression of these novel candidate genes to further diminish cancer cell stemness we investigated the expression of novel candidate genes and stemness markerssox2 and oct4 in a549 lung cancer cells mdamb231 breast cancer cells and hepg2 liver cancer cellsusing western blot our results showed that icam3ccl16 pde3a prtn3 sox2 and oct4 protein expression decreases in the aspirintreated groups versuscontrols fig 3b fig s2a we also found that icam3ccl16 pde3a prtn3traf6 bcar1 il1a il1bnfkb1 ikbkb sox2 and oct4 mrna expression decreasesin the aspirintreated group versus controlfig 3c moreover in icam3 ccl16 pde3a prtn3traf6 bcar1 il1a il1b nfkb1 sox2 and oct4the protein expression decreasesindicated byimmunofluorescencestainingaspirintreatedmbamd231 fig 3d and a549 cells data not shownversus the controlasin thein order to confirm the above in vitro results we theninvestigated mrna and protein expression in tumorsfrom and 46day aspirintreated miceversus control using qpcr and western blot our resultsdemonstrated that icam3 pde3a prtn3 traf6bcar1 il1a il1b nfkb1 ikbkb sox2 and oct4mrna expression decreasesin the aspirintreatedgroups versus control fig 3e in addition we foundthat icam3 pde3a prtn3 traf6 bcar1 il1ail1b nfkb1 sox2 and oct4 protein expressionsimilarly decreases in the aspirintreated groups versuscontrol fig 3f fig s2b the abovementioned findingsexpression ofsuggesttheinflammationrelated stemness genesin vitro andin vivoaspirin decreasesthataspirin mediates histone methylation to regulate targetgenes expressionin order to determine the mechanism underlying the action of aspirin we explored the regulatory effect of aspirin on histone methylation markers in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using western blot our resultsindicated thatthe expression of h3 trimethylationmarkersie h3k43me h3k93me h3k273meh3k363me and h3k793me increases in the aspirintreated higher concentration groups versus controlfig 4a s3a we also found that the expression of histone demethylases ie kdm6a and kdm6b decreasesin the aspirintreated higher concentration groups versuscontrol fig 4a in addition we studied the protein expression of h3k43me h3k93me h3k273meh3k363me h3k793me and h3 in a549 lung cancercells mdamb231 breast cancer cells and hepg2 livercancer cells using immunofluorescence ourresultsshowed that the protein expression of the h3 methylation markers within the nucleus increases in the aspirintreated groups versus control fig 4b to further support this we extracted the nuclear proteins of eachgroup and detected these h33me markers the resultsalso showed that the expression of h33me markers wasincreased within the nucleusin the aspirintreatedgroups versus control fig 4c s3bin order to identify the role of h3 methylation in regulating selected inflammationrelated stemness genes wemeasured the amount of icam3 dna fragments in h3modification marker pulldowned dnas in a549 lungcancer cells mdamb231 breast cancer cells andhepg2 liver cancer cells using the chipqpcr assaywe selected icam3 since our previous studies demonstrated that icam mediates cancer cellinflammationand stemness ourtheamount of icam3 dna fragments in the various h3methylation marker pulldowned dnas decreases in alllines fig 4c the abovementionedthree cancer cellfindingssuggesthistoneresults demonstrated thatreducesthataspirin 0cbmcemuovl romutdliavvrus noitcarfctrlasp 50mgkgasp 100mgkgdayscg thgew ydobictrlasp 50mgkgasp 100mgkg days of treatmentectrlasp50 asp100ctrlasp 50mgkgasp100mgkggsedoni ssatsat emgnulfhctrlasp asp ctrlasp asp ——xostcoi sllec xos sllec tcoctrl asp50asp100ctrl asp50 asp100ctrl asp50 asp100zhang stem cell research therapy page of a4t1luci injectionaspirin injectionsurvivalday timej4t1luci injectionaspirin cisplatin 2mgkg injectionsurvivalday kmcemuovl romutctrlcisplatinasp 25cisplatinasp50cisplatin dayslg tihgew ydobctrlcisplatinasp 25cisplatinasp50cisplatin days of treatmentmliavvrus noitcarfncisplatin treatedctrlasp asp50yadyadctrlcisplatinasp 25cisplatinasp50cisplatintimefig see legend on next pageop sllec xoscisplatin treatedctrlasp asp50xostcoctrl asp25 asp50cisplatin treated sllec tcoctrl asp25 asp50cisplatin treated 0czhang stem cell research therapy page of see figure on previous pagefig aspirin suppresses cancer cell metastasis and stemness in vivo a schema of the metastasis model established by subcutaneousimplantation of 4t1luci cells into the 4th pair of mammary fat pad of balbc mice b tumor growth curve of 4t1luci with or without aspirintreatment c the body weight of balbc mice in the course of aspirin treatment d the survival curve of balbc mice inoculated with 4t1luciwith or without aspirin treatment e the representative luciferase images showing the 4t1luci tumors at the primary site and lung metastasissites with or without aspirin treatment f representative he staining images of 4t1luci tumors metastasis to the lung with or without aspirintreatment scale bars lower panel μm g statistic results of metastasis loci of 4t1luci tumors metastasis to the lung with or without aspirintreatment h immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment representativeimages with — magnification were shown scale bars μm i statistic results of sox2 or oct4 positive cells in 4t1luci primary tumors withor without aspirin treatment j schema of the chemoresistance model established by subcutaneous implantation of 4t1luci cells into the 4thpair of mammary fat pad of balbc mice k tumor growth curve of 4t1luci with or without aspirin treatment in the presence of cisplatin l thebody weight of balbc mice in the course of aspirin treatment in the presence of cisplatin m the survival curve of balbc mice inoculated with4t1luci with or without aspirin treatment in the presence of cisplatin n the representative luciferase images showing 4t1luci tumors at theprimary sites with or without aspirin treatment on day before cisplatin administration and day after cisplatin administrationo immunohistochemistry staining of sox2 and oct4 in 4t1luci primary tumors with or without aspirin treatment in the presence of cisplatinrepresentative images with — magnification were shown scale bars μm p statistic results of sox2 or oct4 positive cells in 4t1luciprimary tumors with or without aspirin treatment in the presence of cisplatindemethylase ie kdm6a and kdm6b expressionwhich mediates histone methylation and thereby inhibits gene expression in vitroaspirin mediates h3 methylation to regulate icam3expression in vivoin order to confirm the above in vitro results we nextexamined h3 methylation marker expression in tumorsfrom aspirintreated mice versus control using immunocytochemistry our results demonstrated that the h3methylation marker immunostaining within the nucleusincreases in the aspirintreated group versus controlfig 4d e we also found that the amount of icam3dna fragments in the various h3 methylation markerpulldowned dnas decreasesin the aspirintreatedgroup versus control indicating that icam3 expressionis blocked fig 4f these findings suggest that aspirinmediates h3 methylation and thereby regulates icam3expression in vivoside populationaspirin mediates h3 methylation to regulate icam3expression via a coxindependent mannerin order to determine the role of cox in aspirinmediated h3 methylation and targeted gene expressionwe knocked down cox1 and cox2 expression in a549cells respectively fig 5a s3c and examined thealdh populationand chemoresistance the results showed that the aldh population fig 5b e and side population fig 5c f were decreased in shcox1 or shcox2 cells treated with aspirincompared to shcox1 or shcox2 cells treated withdmso and also the aldh population and side population were decreased in shcox1 or shcox2 cellstreated with aspirin compared to shctrl treated with aspirin moreover the apoptosis was increased in shcox1or shcox2 cells treated with ddp and aspirin comparedto shcox1 or shcox2 cells treated with ddp anddmso ctrl and also the apoptosis was increased inshcox1 or shcox2 cells treated with ddp and aspirincompared to shctrltreated with ddp and aspirinfig 5d g in addition western blot results displayedthat the h3 trimethylation markers were increased andthe histone demethylases ie kdm6a and kdm6bwere decreased in shcox1 or shcox2 cells treated withaspirin compared to shctrl treated with aspirin fig 5hs3d accordingly as the new target genes icam3 expression was decreased in shcox1 or shcox2 cellstreated with aspirin versus shctrl treated with aspirinfig 5i s3e these findings suggest that aspirin mediates h3 methylation and thereby regulates icam3 expression via a coxindependent manneraspirin combined with hdm kdm6ab or icam3signaling inhibitors diminish cancer progression in vivoour previous work proved that icam3 could mediatesrcpi3k signaling to promote cancer cell stemness inorder to investigate the use of aspirin combined withhdm kdm6ab or icam3 signaling inhibitors as thetherapeutic strategies we implanted a549luciferasecells into the fourth fat pad of male nodscid micetwentythree days after implantation we injected ip themice with mgkg aspirin mgkg aspirin mgkg kdm6ab inhibitor gsk j1 mgkg aspirin mgkg src inhibitor mgkg aspirin mgkg pi3kinhibitor ly294002 mgkg aspirin mgkg lifitigrast icam3 inhibitor or dmso control group every days fig 6a our results showed that tumor size andtumor volume decreases in the aspirintreated groupand the aspirin inhibitortreated groups versus dmsocontrol fig 6b c however we found that the bodyweight did not change significantly in the aspirintreatedgroup and the aspirin inhibitortreated groups versusdmso control fig 6din addition we found that the survival time increasesin the aspirintreated group and the aspirin inhibitortreated groups versus dmso control fig 6e these 0czhang stem cell research therapy page of fig see legend on next page 0czhang stem cell research therapy page of see figure on previous pagefig aspirin inhibits the expression of inflammationrelated stemness genes in vitro and in vivo a schematic representation of the sirna screenleft summary of the results from the rnai screen right b western blot examining the expression of inflammatory candidates and stemnessproteins sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirin treatment c quantitative pcr examining the mrnaexpression of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cells with or without aspirintreatment d immunofluorescence staining of inflammatory candidates and stemness genes sox2 oct4 in a549 mdamb231 and hepg2 cellswith or without aspirin treatment scale bars μm e quantitative pcr examining the mrna expression of inflammatory candidates andstemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treated with aspirin for different survival days f western blotexamining the expression of inflammatory candidates and stemness genes sox2 oct4 in 4t1luci tumors separated from balbc mice treatedwith aspirin for different survival dayssuggestresultsthat aspirin combined with hdmkdm6ab or icam3 signaling inhibitors diminishcancer progression in vivo and may serve as the therapeutic strategiesproposed model of aspirin inhibits cancer cell stemnessand cancer progressionbased on our findings we propose the following modelfig aspirin inhibits histone demethylase hdm expression which then mediates histone methylationh3k43me h3k93me h3k273me h3k363meh3k793me respectively these h3 methylations theninhibit the expression of various inflammationrelatedstemness genes previously identified by highthroughputsirna screening il1a il1b icam3 ccl16 traf6pde3a prtn3 nfkb1 ikbkb bcar1 using theicam3 gene as a representative of the inflammationrelated stemness genes by the aspirinmediated h3modifications restrain icam3 promoter activity andcause icam3 expression is inhibited thus aspirin maydiminish cancer cell stemness properties and cancer progression in vitro and in vivo by inhibiting the expressionof various inflammationrelated stemness genes mostinterestingly the above process was not depending oncox expression as the therapeutic strategies aspirincombined various inhibitors suppressed tumor progression effectivelydiscussionthe widely recognized anticancer potential of aspirin aclassical nonsteroidal and antiinflammatory drug hascreated a broad interest to explore the clinical benefitsof aspirin in cancer therapy [“] previous findingsby many investigators have establishe

" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [“] an rln countof ‰¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ‰¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ‰¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ‰¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1“t3“tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ˆ’ ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes ˆ’ positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd student™s t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor ci““p“reference“““age ‰¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ‰¥ glasa ‰¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3“rlnslymph node metastasislnr lodds ˆ’ ptnm stage ‰¥ iiilauren subtypeintestinaldiffusemixedunknown ci““““““““““reference““““reference““““““““reference“““p 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range “ and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were × ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ‰¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ‰¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ‰¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ‰¥ characteristicsunivariateormultivariateor cipreference““ ““age ‰¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ‰¥ glasa ‰¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3“lymph node metastasisptnm stage ‰¥ iiilauren subtypeintestinaldiffusemixedunknown ci““““““““““reference““““reference“““““reference“““p bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ‰¥ deng proposed an optimal rln count of ‰¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[“] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [“] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors™ contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ™ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol “son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer “li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol “ aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol “koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet “zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer “chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol “deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncol“amin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more œpersonalized approach tocancer staging ca cancer j clin “okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol “ deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res “kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer “ wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg “ dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg “ hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol “tóth d bíró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg “ maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer “liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg “sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer “ zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol “ alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg “ 0c"

specialty sectionthis was submitted tomolecular and cellular oncologya section of the frontiers in cell and developmentalbiologyreceived april accepted july published august citationchen w yang j fang h li l andsun j relevance functionof lincror in the pathogenesisof cancerfront cell dev biol 103389fcell202000696cancer is a serious disease that aï¬ects human health being one of the main causes of death all overthe worldwide according to research in of new tumor cases and of the cancerassociated deaths occurred in lowincome and developing countries kumar and sharawat noorolyai owing to a shortage in eï¬ective screening methods and lack of identificationof early symptoms most patients were already in advanced stages when they were diagnosedwith cancer bray koo additionally some clinical studies have shownthat polarity and adhesion of cancer cells was decreased leading to heir increased mobility andinvasion which is a key step in the development of cancer yan therefore studies haveshown that the high mobility of cancer cells is the main factor leading to high mortality rates inpatients with cancer currently there are many ways employed in the treatment of cancer includingsurgery radiotherapy chemotherapy biotherapy and targeted therapy nie howeverin the past years the survival rate of patients with cancers remains dismal nakashima abbreviations bc breast cancer cenas competing endogenous rna emt epithelial“mesenchymal transition hcchepatocellular cancer ipscs induced pluripotent stem cells lincror long intergenic nonprotein coding rna regulatorof reprogramming lncrnas long noncoding rnas pc pancreatic cancerfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorin the process of developing human antitumorthereforestrategiesto find new earlyitbiomarkers and thus identify potential regulatory mechanisms toimprove the survival rate of patients with cancersis particularly importantover the past decades ncrnas constitute more than of the rnas made from the human genome but mostof the known noncoding rnas ncrnas havebeen discovered and remain largely unstudied bhan slack and chinnaiyan transfer rna trna and ribosomal rna rrna constitute themajority of ncrnasfollowed in abundance by messengerrnas mrnas thus the remaining ncrnas includingcircular rna circrna small nuclear rna snrna smallnucleolar rna snorna microrna mirna and long nonfor ˆ¼ ofcoding rna lncrna together accounttotalncrna despite their low abundancethese ncrnas havebeen reported to play critical roles in transcription posttranscriptional processing and translation such as epigeneticsposttranscriptional regulation chromatin modification andregulation of the cell cycle huarte kondo peng 2017b additionally because ncrnas can be packagedinto extracellular vesicles ev including exosomes meldolesi they have been shown to provide a mechanism forintercellular communication through the transfer of mirnaand lncrna to recipient cells both locally and systemicallysun it is important to note that the expressionof ncrnas their posttranscriptional modification particularlylncrnas and their subcellular distribution have been shownto be important to when assigning their potential functionpalazzo and lee recently nextgeneration sequencingand bioinformatics technology have revealed that circrnasplay crucial role in diagnosis and prognosis of various diseasespamudurti briefly circrnas are singlestrandedtranscripts generated by backsplicing jeck and sharpless with covalently linked headtotail closed loop structures withneither 5cid48“3cid48 polarity nor a polyadenylated tail memczak that range in length from a few hundred to thousandsof nucleotides and are widely expressed in mammals therebyshowing higher stability compared to that in linear rnas chenj and exhibiting a celltype or developmentalstagespecific expression pattern barrett and salzman wang j j many functions of circrnas have alsobeen identified including their role as mirna sponges bindingto rnabinding proteins and protein decoys and functioningas regulators of transcription hansen du yang y interestingly many circrnas havebeen shown to be dysregulated in pathophysiological processesand circrnas are known to regulate the expression of geneby acting as mirna sponges in a mechanism that is termedas competitive endogenous rna cerna mechanism zheng wang j j for example circmto1have been demonstrated to harbor conventional mirna bindingsites and has been identified as an inhibitor of mirna9 inhepatocellular carcinoma hcc han additionallyour previous study has demonstrated that mirna plays arole in limiting the development of liver fibrosis by markedlyblocking the activation and proliferation of hepatic stellatecells hscs suggesting that mirnas might be involved inthe development and progression of several forms of cancersyang j yang of notelncrnaswhich are mainly transcribed by rna polymerase ii are anew kind of ncrna that are longer than nucleotidesma owing to the lack of open reading frameslncrnas have extremely limited or no protein coding capacityruan li j these new regulatorswere initially regarded as transcriptional noise with no specificbiologicalfunctions kim and sung recently ourlaboratory found that epigenetic silencing of lncrna anrilpromoted the progression of liver fibrosis thereby indicating thatlncrnas were associated with the progression of cancers yang interestingly increasing evidence have shown thatcellular events including diï¬erentiation proliferation invasionapoptosis and migration have all been associated with lncrnasguttman additionally there has been new evidencesuggesting that lncrnas may regulate a variety of biologicaland disease processes from gene transcription and translationto posttranslational modifications davalos and esteller pang more importantlylncrnas have beenreported to be used as tumor suppressor genes or oncogenesthus aï¬ecting the proliferation and metastasis of various typesof tumors during tumorigenesis chen q n lu subsequent studies have demonstrated thatlncrnas may serve as cernas for mirnas and in chromatinremodeling during the development of cancers huang wang c j figure illustrates the functionsof lncrnas at the molecular level regarding certain cancerassociated human lncrnasit was demonstrated that lincror was demonstrated to be predominantly upregulated intumors peng 2017a the abnormal expression of lincror in tumors has been suggested to be one of the mainleading factors driving the development the main ways torevert this eï¬ect would be to aï¬ect cell growth migrationand invasionthus leading to the inhibition of epithelialmesenchymal transition emt enhancement of the sensitivityto chemotherapy etc chen 2016a zhao forexample the expression level of lincror in hcc tissues wasinhibited compared with the adjacent tissues at the same timethe downregulation of lincror was linked to the aggressiveprocess of the disease in patients with hcc furthermore theability of migration and invasion of hcc cells may be delayedby the low expression level of lincror in this review weattempted to introduce the latest research on the biologicaleï¬ects potential clinical applications and molecular mechanismsof lincror in human tumors and discuss its prognostic andtherapeutic valuesoverview of lincrorlincror isamong lncrnasand importantcarcinogenic kb lncrna located in chromosome which was initially identified as a highly expressed transcriptof pluripotent and embryonic stem cells chen 2016bstudies found that the octamerbinding transcription factor a novelfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure paradigms for the function of long ncrnas recent studies have identified a variety of regulatory paradigms for the mechanism by which long ncrnasfunction many of which are highlighted here transcription from an upstream noncoding promoter pink can negatively or positively affect the expression ofthe downstream gene purple by inhibiting the recruitment of rna polymerase ii or inducing chromatin remodeling respectively an antisense transcript orangeis able to hybridize to the overlapping sense transcript purple and block the recognition of the splice sites by the spliceosome thus resulting in an alternativelyspliced transcript alternatively hybridization of the sense and antisense transcripts can allow dicer to generate endogenous sirnas by binding to specificprotein partners a noncoding transcript blue can modulate the activity of the protein serve as a structural component that allows the formation of a largerrnaprotein complex or alter where the protein localizes in the cell long ncrnas green can be processed to yield small rnas such as mirnaspirnas and other less wellcharacterized classes of small transcriptsoct4 srybox transcription factor sox2 and nanoghomeobox nanog key pluripotency factors could regulatelincror wang howeverlincror was alsofound to be expressed in several ans including lungliver breast and colon since its discovery research in thisfield has been extensively expanded during the past yearsrevealing the important role of lincror in tumorigenesislincror has been suggestedadditionally upregulation ofto mediate the reexpression offetal and cardiomyocytehypertrophyrelated genes wang li inmany reportsrecent years have revealed thatlincror is positively correlated with the clinicopathologicalcharacteristics and poor prognosis of tumorsincluding thestages of advanced tumor node metastasis tnm positivelymph node metastasis lnm and lower survival rate buthigher recurrence rateregarding lincror and tumorigenesisthe upregulation ofcurrent evidence have strongly indicated thatlincrormay exert an impact on a variety of cancers pan furthermore both tumorigenesis and metastasis havebeen shown to be induced by lincror via activation of theemt in various cancers hou huang zhan for example lincror was demonstratedto be upregulated thereby promoting emt in hcc li j besides it was also reported that selfrenewal anddiï¬erentiation of glioma stem cells was significantly aï¬ectedby lincror zhang feng moreimportantly the chemoresistance of pancreatic cancer pcand breast cancer bc li as well as radioresistance of colorectal cancer crc cells were observed to beelevated by lincror yang p moreover lincror has also been shown to exert a significantly eï¬ect onthe stem celllike characteristics and tumorigenic potential ofpc recentlyit was also reported that lincror could beused as a biomarker in the field of diagnosis and prognosisof bc and oral cancer arunkumar zhao notably increasing studies showed that lincror couldbe used as a cerna thus exerting its impact in the posttranscriptional network of tumor pathogenesis for examplein triplenegative bc lincror has been shown to serve asa cerna therefore promoting the migration and invasion ofbc cells signal overall lincror is a typicallncrna that plays important regulatory roles in interactionwith mirnas and maintenance of stem cell pluripotencytriggering the emt as well furthermore lincror has alsobeen involved in various key roles under hypoxia and in thepromotion of tumorigenesis figure therefore lincrormay be considered as an oncogene aï¬ecting the progressionof tumor and a promising predictor for the poor prognosis inpatients with cancer the transition of lincror from basicresearch to clinical application requires further investigations asearly as possiblefrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure lincror is a typical lncrna that plays important regulatory roles in interacting with mirnas and maintaining stem cell pluripotency as well astriggering the emt as well lincror is also involved in various key roles under various stresses and in epigenetic regulationregulatory role of lincror invarious types of cancerincreasing evidence has shown that the lincror was abnormalexpression in many cancers and its dysregulation was associatedwith cellular functions fu spinelli additionally studies found that the expression level of lincror was substantially upregulated in samples of papillarythyroid carcinomas ptcs and ptcs cell lines as well as inmetastatic ptcs samples and ptcs cell lines zhang simultaneously cell migration and invasion could be regulatedby lincror via aï¬ecting emt pastushenko and blanpain more importantly studies demonstrated that lincrorwas abnormally expressed in several cancers and led to elevatedthe invasion and metastasis of cancer cells to promoting theprogression of tumors hashemian li 2020bcthis review summarizes the status of lincror research invarious human cancers and discusses its mechanism and clinicalsignificance in the development and progression of tumor theexpression pattern functional role and regulatory mechanism oflincror are summarized in table and depicted in figure breast cancerbc which accounts for a quarter of all female cancer casesis the most commonly diagnosed cancer and the leading causeof cancerassociated deaths among women worldwide li z in it was estimated that there would be million or so newly diagnosed cases of female bc bray hannafon the main risk factors forbc which is the difficult to change due to prolonged exposureto endogenous hormones is difficult to control rudel bray however comprehensive treatmentapproaches have resulted in relatively good clinical outcomesfor some patients with bc rudel goel kumler nevertheless it has been reported that aboutonethird of the patients with bc have the potential for cellmetastasis chemotherapy resistance and even recurrence goel kumler hence there is an urgent need todevelop new therapies targeting various molecular mechanismsof tumorigenesis for the treatment of bcthe level ofthe level ofhou the expression ofinvestigated the expression level of lincrorin patients hou their results revealedlincror was increased in bc tissuesthatmoreoverlincror in theperipheral blood ofthe patients with bc was shown tobe closely related to tnm phase and lnm in additionthe woundhealing assay showed that overexpression of lincror increased bc cells mcf10a mobility transwell assayrevealed that lincror overexpression remarkably increased themigration ability hou more importantly theyfound that ectopic expression of lincror induced an emtprogram in mcf10a cells fluorescence activated cell sorteranalysis demonstrated that the subpopulation of the stem cellphenotype cd44highcd24low was elevated in mcf10a cellstransfected with lincror plasmid mechanistically the resultsof bioinformatic analysis and rna immunoprecipitation analysisfrom hou demonstrated thatlincror functions asa cerna to regulate mir205 activity toward prevention ofthe degradation of transcripts of mir205 target genes suchas zeb1 and zeb2 from degradation additionallyit wasshown that the expression levels of mir205 members weredecreased upon lincror overexpression in mcf10a cellshou more importantly zhao recentlydemonstrated that crisprcas9generated brca1knockdownadiposederived stem cells stimulated a more aggressive behaviorfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlateillateoaglatenahzlatenehclatecillateihzlateilnusdnanaylatenapalategnahzlateuohsecnereferbezrmrorcniilmkppbtprmrorcniilgonanrmrorcniilnirehdacehzerorcnilsosfdmnlegatsmnttmeinosavnitmeinosavninoitargminoitarefilopicnegocnoldetaugerpuamoncraciraulllecotapehllseuceomyrotaugerllnoitaerroclacniilcleorlanoitcnufleorinosserpxesepytrecnacsrecnacnamuhnirorcnlielbatirmrorcnilevruccormnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnactsaerbtmeinosavnipbezrorcnilliavvrusroopmnlegatsmntnoitargminoitarefilorphtwicnegocnoldetaugerpurecnaccitaercnapmxofprmrorcniilncsfrmrorcniprmrorcniililirmrorcnilecnatsserigurdtmeinosavnitmelecycllecinosavniliavvrusroopmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnacdoryhtisosfdmnlegatsmntinoitargmecyclllecissotpopaicnegocnoldetaugerpurecnacgnulecnatsseriyparehtodaritmeinosavnisosfdmnlegatsmntnoitargmiissotpopanoitarefilorpicnegocnoldetaugerpurecnaclatcerooclin bc cells than wildtype adiposederived stem cells zhao therefore we believe that crisprcas9 may beused to in the treatment of bc by inhibiting the expressionof lincror during the progression of bc conclusively thelincrormirnas axis has been reported to closely aï¬ect theoccurrence and development of bcpancreatic cancerpc is the fourth most common cause of cancerrelated mortalityworldwide leading to approximately deaths annuallysiegel sabater the ˆ’year relativesurvival of patients with pc remained at approximately for“ siegel hence pc has been proposedto be one of the top two cancers in terms of fatalities in thenext decade rahib surgical resection remainsthe exclusive potential curative treatment xiong however approximately half ofthe patients present withmetastasis at the time of diagnosis missing the opportunityfor an eï¬ective treatment vincent xiong a growing body of literature has demonstrated that bothmetastasis and limited eï¬ective biomarker for the diagnosis andtreatment are the main obstacles for the efficient medical therapyof pc vincent boj basuroy thus it is an absolute necessity to identify potential biomarkersand therapeutic targets in pczhan have highlighted the oncogenic eï¬ectsof lincror in the initiation and progression of pc theirstudy demonstrated that the level of lincror was significantlyelevated in pc tissues zhan moreoverthewoundhealing assay and boyden™s chamber assay results showedthat lincror silencing reduced the migratory capability andmetastasis of pc cells zhan another study bychen showed that the proliferation rates of shrorcellsin which the level of lincror was suppressed were evidentlylower than those of shnccells this result was confirmed bycolony formation assay suggesting that lincror accelerated thegrowth of pc cells chen 2016a interestingly silencingof lincror was shown to result in increased levels of theepithelial markers ecadherin and αcatenin and decreased levelsof mesenchymal markers ncadherin and vimentin indicatingthat lincror plays an important role in the regulation ofemt in pc cells zhan chen moreimportantly microarray analysis identified zeb1 as potentialtarget gene of lincror further the expression of lincrorand zeb1 were observed to be negatively correlated with thatof p53 suggesting that lincror might mediate migration andmetastasis in pc cells may partly via activation of zeb1 throughthe inhibition of the expression of p53 zhan interestingly the fluorescence in situ hybridization and luciferasereporter assay results showed that the expression of lincrorwas demonstrated to be negatively correlated to that of mir145mir145 can induce posttranscriptional silencing of its targetedgenes by binding to the mrna ™utr or lincror specificsites indicating that lincror can act as a cerna to decreasemir145 in pc cells thereby activating expression of nanogthus leading to the proliferation of pancreatic cancer stem cellspcscs gao additionally li furtherfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorfigure underlying molecular mechanisms of lincror in multiple cancers a lincror binds to mir205 to upregulate zeb2 while it also regulated theexpression of emt markers b lincror could interact with mir145 to inhibit fscn1 and upregulated emtassociated proteins while it decreases g0g1 arrestand facilitated drug resistance c lincror facilitated emt through upregulate ezhz and regulated zeb2 by competitively binding to mir145 and zeb2overexpression leads to increased emt in addition lincror binds to mir8765p to upregulate foxm1 d lincror downregulated through emt production andrepress the expression of mir145 e lincror binds to mir68333p while also regulating the process of emt f lincror upregulated zeb1 to attenuate theexpression of p53 while also decreasing the expression of mir145 to increase the level of nanog and reduce that of mir124 to suppress pkm2 detailedmechanisms of lincror in other cancers are provided in the reviewproved that the impact of lincror can be partly reversed byoverexpression of mir124 consistently lincror was observedto exhibited a negative correlation with the expression of mir li hence a lincrormir124ptbp1pkm2axis was identified in pc shedding new light on the lncrnabased diagnosis and therapeutic approaches in pc li 2020b notably recent studies showed that pc cellderivedevs could be used as eï¬ective carriers of paclitaxel to theirparental cells thereby bringing the drug into cells through anendocytic pathway and increasing its cytotoxicity saari additionally it was demonstrated that vesiclecontainingncrnas could serve as evassociated pc detection markersworst thus the presence of lincrors in evs frompatients with pc could serve as a potential diagnostic biomarkerand a novel target for the therapy of patients with pc this isworthy of further and wider research attentionhepatocellular carcinomaas the sixth most international commonly occurring cancer in hcc has become the fourth cause of cancerassociateddeaths worldwide it has been estimated that new casesand deaths will occur each year bray brieflyhcc has been reported to account for “ of all the livercancer cases half of which have been detected in china omata bray as such hcc poses a huge threatto the worldwide health especially that of the chinese peopleomata about of the patients is expected torecrudescent within years after hepatectomy and of thepatients will die from this tumor vigano thereforeon the basis of studying the pathogenesis of hcc it is apparent tolook for more eï¬ective molecular markers and therapeutic targetsfor the management of hccli c and chen reported that theexpression level of lincror was obviously elevated in hcctissues and four cell lines compared to the corresponding nontumor tissues and normal liver cell lines respectively suggestingthat lincror might be critical regulator in the progression ofhcc furthermore biological function assay demonstrated thatlincror could play promoting role in regulating migration andinvasion of hcc chen moreover downregulationof lincror could result in a significant increase in g1g0phase and an obvious decrease in s phase li c more importantly silencing of lincror could lead to theincreased expression of eˆ’cadherin and decreased expressionlevel of nˆ’cadherin in hcc cell lines li c furtherconfirmed that lincror could bind to the zeste homolog ezh2 thereby aï¬ecting the expression of ecadherin furtherindicating that lincror could regulate the progression of emtmoreover lincror was further determined to be associatedwith dna repair currently mounting studies have identifiedreliable indicators of dna damage such as phosphorylatedhistone h2ax γh2ax chen uncover thatfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorlincror could obviously decrease theoverexpression ofexpression level of γh2ax illuminating the suppressive eï¬ectsof the overexpression of lincror on dna repair in hccfurther research demonstrated that lincror could interactwith mirˆ’ and dramatically downregulate the expression ofmirˆ’ in hcc cells li c the abovementionedresults revealed that lincror might play a promoting role inthe proliferation migration invasion and emt of the hcc cellwhich was contrary to the influence of mirˆ’ enrichmentli c it suggested that overexpressed mirˆ’could eï¬ectively reverse the promotion of hcc tumorigenesisinduced by the overexpression of lincror li c liand his colleagues proposed a mechanistic model that lincrorpromotes hcc tumorigenesis and autophagy partly throughnegatively regulating the expression of mirˆ’ aside fromthat it has been reported that mir145 represses emt tumormigration and invasion by directly targeting the ™utrs ofzeb2 in the tumor the decrease in mirˆ’ and increase inzeb2 can obviously reversed the inhibition of cell migrationand invasion mediated by the lincror knockdown thereforeit was suggested that targeting the lincrormirˆ’145zeb2axis might represent a novel therapeutic application in hccli c similarly zhi similarly showedthat the migration and invasion of cells was reduced by theknockdown of lincror moreover they further confirmedthat foxm1mediated activation of lincror contributes tothe poor sensitivity of hcc cells to sorafenib via partiallyregulating the mir8765pfoxm1 axis which forms a positivefeedback loop further evaluation of the regulatory mechanisminvolving this axis may provide new insights for exploringa potential therapeutic strategy for the management of hcczhi consequently these studies may oï¬er newinsights regarding the pathology of hcc and provide potentialstrategies for lncrnadirected treatment however both thein vivo influence and other underlying mechanisms of lincrorstill remain to be determined and clarified in the future researchthe prognosiscolorectal cancerthere are approximately million new crc cases and crcrelated deaths worldwide each year thus making crc thethird most common cancer in the world torre although the treatment of crc has significantly improvedin recent decadesremains unsatisfactoryespecially in case of advanced tumors with distant metastasesbogousslavsky torre current studiesresults showed that approximately of cases with crchave synchronous liver metastases during the time of diagnosiskawaguchi these patients have inherently lowsurvival rates of less than within years with an even worseprognosis hu kawaguchi thus there isan urgent need to better understand the progression of crc andto identify novel and sensitive biomarkers for the diagnosis andtreatment of patients with crcyang detected the expression of lincror in crctissues compared to normal tissues by using qrtpcr theyfound that the expression of lincror was remarkably increasedin crc tissues compared with normaltissues similarlylincror was shown to be overexpressed in five crc cell linesyan and sun li 2020a then they also performeda series of functional assays to clarify the biological eï¬ects ofthe aberrant expression of lincror on proliferation viabilityapoptosis migration and invasion of crc cells knockdownof lincror was shown to eï¬ectively inhibit the proliferationof crc cells whereas its overexpression obviously increasedthe proliferative capacity of crc cells accordingly silencing oflincror strongly inhibited the migratory and invasive abilitiesof crc cells compared with that in the control cells li 2020a in contrast the migratory and invasive ability of cells wasactivated following the overexpression of lincror the mts345dimethylthiazol2yl53carboxymethoxyphenyl24sulfophenyl2htetrazolium assay results showed thatthelincror could enhance the viability ofoverexpression ofcrc cells furthermore the flow cytometric analysis resultsrevealed that the percentage of apoptotic cells in lincroroverexpression group was reduced by ± indicatingthat the overexpression of lincror could inhibit apoptosisin the crc cell lines li 2020a more importantly arecent study revealed the role of lincror in the emt it wasrevealed that the upregulation of lincror could increase theexpression of ncadherin and vimentin as well as decrease thelevel of ecadherin leading to the promotion of the progressionof emt zhou yan and sun meanwhilethe high expression of lincror in crc was also confirmedby hu mechanistically li 2020a provedthat that lincror could bind to mir68333p which wasdetermined to be significantly downregulated in crc tissuesadditionally a negative correlation was exhibited between theexpression of lincror and mir68333p in bc tissues antiago2 rna immunoprecipitation assay further confirmed theseresults li 2020a besides rescue assays demonstratedthat downregulation of mir68333p could partly reversed theinhibition of tumorigenesis induced by lincror knockdown inbc cells li and his colleagues uncovered that lincror exertedits oncogenic role through negatively regulating the expressionof mir68333p during the progression of crc which mightgive new insights into molecular diagnosis and treatment li 2020a in addition li 2020a further uncoveredthat lincror could mediate the expression level of smc bysponging mir68333p in crc cells thus promoting crcprogression as for the eï¬ects of lincror on radiotherapyresistance yan and sun showed that overexpression oflincror increased the ability of crc cells for radiotherapyresistance collectively these findings indicated that lincrormight be engaged in the metastatic process of crc cells andcould promote the development of crc through a variety ofmolecular mechanismslung cancerlung cancer is the leading cause of cancerrelated deathsworldwide bray nonsmall celllung cancernsclc accounts for about of the lung cancer typesincluding squamous cell carcinomalung cancerand lung adenocarcinoma herbertz bray brainard and farver although there are variousapproaches for its diagnosis and treatments the 5year overallsurvival os rate for patients with advanced lung cancer is lesslarge cellfrontiers in cell and developmental biology wwwfrontiersinaugust volume 0cchen relevance function of lincrorthan zhou therefore in order to carry outan early diagnosis and treatment of lung cancer the search forvaluable and efficient tumor markers is very urgentin recent yearslincror has appeared as an importantregulator oflung cancer research from qu demonstrated that the expression of lincror was increasedin nsclc tissues compared to that in the normal tissuesthe overexpression of lincror was shown to be closely relatedto the poor prognosis of lnm histological grade and stageof tnm pan qu another study bypan demonstrated that the decreased expressionof lincror could obviously impair the proliferative capacityof lung adenocarcinoma lad cells cause a g0g1 phasearrest and increase the ratio of apoptotic lad cells moreoverdownregulation of lincror substantially inhibited the invasiveand metastatic ability of lad cells meanwhile forced expressionof lincror was observed to reduce the expression of ecadherinand βcatenin which are the characteristic biomarkers ofepithelial cells whereas it increased the expression of ncadherinand vimentinthus displaying a mesenchymal phenotypeconversely downregulation of lincror was demonstrated toresult in increased the expression of epithelial markers anddecreased the expression of mesenchymal markers this resultuncovered the fact that the prometastatic eï¬ects of lincrorwere induced by the regulation of the expression of a number ofgenes involved in cell metastasis and emt progress meanwhileoverexpression of lincror was shown to enhance the resistanceof lad to docetaxel dtx pan suggestingthat lincrorinduced resistance of lad cells to dtx andemt through regulation the expression of mir145 whichwas predicted to interact with lincror more importantlyfurther research confirmed that mir145 could bind to lincror and its downregulation could partly inhibit the resistanceof lad cells to dtx and emt aside from that pan andhis colleagues discovered that a decrease in the expression ofmir145 and increase in the expression fscn1 could obviouslyreverse the inhibition of cell proliferation chemoresistanceand emt mediated by lincror knockdown they identifiedthat dysregulation of the lincrormir145fscn1 axis wasassociated with the therapeutic resistance and emt transition inlad cells thereby providing potential therapeutic strategies formanaging drug resistance in patients with lad pan taken together these studies collectively suggested that lincrorcould activate the malignant phenotype of nsclc cells with theguidance of a mechanism involving mirnas hence more eï¬orts

Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedObjective To investigate whether preventive administration of a proton pump inhibitor PPI can reduce the occurrence anddevelopment of traumatic granuloma TG following type IVVI cordectomy Methods We retrospectively analyzed the statusof postoperative granulomas in patients who underwent type IVVI cordectomy due to glottic cancer and determinedwhether postoperative administration of a PPI had any impact on granuloma formation and development Results Thepercentage and number of patients with granuloma in the PPI treatment group experimental group at the 1st 2nd 3rd and6th month following surgery were and respectively The percentageand number of patients with granuloma in the noPPI group control group were and respectively The granuloma percentage of the PPI treatment group was lower than that of thecontrol group at all postoperative time points assessed The diï¬erences were not statistically significant at the 1st monthp but were statistically significant at the 2nd and 3rd months after surgery p p ConclusionPreventive use of a PPI in patients after type IVVI cordectomy can shorten the TG recovery duration and may reduce theseverity of TG but it cannot prevent TG from occurring Our results should be confirmed by prospective randomized controlledtrials with large sample sizes IntroductionLaryngeal squamous cell carcinoma LSCC is a commonhead and neck cancer It had an incidence of approximately in China between and [] and new cases were reported worldwide in [] Itis estimated that there will be new cases worldwidein [] Approximately twothirds of LSCCs originatein the glottic area The presence of hoarseness in patientswith earlystage glottic cancer GC prompts the patients toseek medical treatment Anatomically the larynx is surrounded by cartilage and has sparse lymphatic tissue As aresult patients with GC are mostly diagnosed at an earlystage which is clinically defined as T12N0M0 []In recent years surgery has been gradually abandoned in the treatment of earlystage GC and has beenreplaced with transoral microsurgery TM or radiotherapyTM has the advantages of being minimally invasive and having a high laryngeal preservation rate and high costeï¬ectiveness Transoral laser microsurgery TLM is the mostcommon surgical method used for GC although a few studies in the literature have adopted transoral coblation microsurgery TCM Although TM methods diï¬erthermalinstruments are often needed for the surgery [“]Traumatic granuloma TG is a common complication ofTM especially after type IVV cordectomy Mild TG mayonly manifest as hoarseness foreign body sensation and frequent throat clearing The severe granuloma may cause or becomplicated by perichondritis which leads to severe symptoms such as dyspnea Additionally this granulation isbelieved to be an important factor in the formation of glotticweb and larynx stenosis [“] Reflux is an important 0cBioMed Research Internationalfactor that aï¬ects granuloma formation Proton pump inhibitors PPI are often used empirically in the treatment ofpatients with postoperative granulomas [ ]We found in our previous clinical practice that patientswith a wide range of resections such as type IVVI cordectomies have a higher risk of postoperative granuloma Thiscan sometimes be very severe even requiring temporary tracheotomy to alleviate dyspnea which greatly and adverselyimpacts patients™ quality of life PPIs have been empiricallyused for the treatment of patients with postoperative granuloma and evidence of its eï¬ectiveness has been published[] However there has not been any research on the prevention of postoperative granuloma formation with PPIsTherefore we initiated PPI treatment for patients who hadundergone type IVVI cordectomies and compared theresults with those of patients who had also undergone thistype of surgery but were not treated with PPIs to evaluatethe eï¬ect of PPI treatment for preventing postoperative granuloma formation in this patient population Materials and Methods Patients This was a retrospective study Patient inclusioncriteria i patients with vocal cord cancer who agreed toundergo type IV V and VI cordectomies ii patients whodid not undergo preoperative and postoperative radiotherapy Exclusion criteria i patients who were complicatedwith diabetes and were not treated routinely ii patientswho used PPIs regularly iii patients who continued tosmoke and drink after surgeryA total of patients between January and December were recruited as the control group who did not use PPIsimmediately after surgery and did not take PPIs persistentlyA total of patients between January and June were recruited as the experimental group PPI treatment group who took PPIs immediately after the surgeryand routinely Since this was a retrospective study only thepatients in the experimental group underwent preoperativereflux symptom index RSI and reflux finding score RFSassessments Patients with an RSI score points andoran RFS ‰¥ points were considered to have laryngopharyngeal reflux disease LPRD [ ]All patients signed an informed consent form prior to thesurgery All clinical experiments conformed to the guidelinesissued by the committee on clinical research of Peking UnionMedical College Hospital PUMCH Ethics Committeeapproval was obtained at PUMCH and all patients providedspecific written informed consent Surgical Procedure According to the European Laryngological Society classification endoscopic cordectomies includethe following types type IV total cordectomy type Vaextended cordectomy encompassing the contralateral cordtype Vb extended cordectomy encompassing the arytenoidtype Vc extended cordectomy encompassing the ventricularband type Vd extended cordectomies encompassing thesubglottis and type VI extended cordectomies encompassing the anterior commissure [ ] During the surgerythe larynx was fully exposed with a selfretaining laryngo°scope Karl Storz Tuttlingen Germany and the tumor°was resected en bloc under direct visualization of a orlaryngoscope Karl Storz Tuttlingen Germany using amodel coblator ArthroCare Corp Sunnyvale CA witha coblation level of and a coagulation level of Postoperative Treatment Procedure and FollowUp Thepatients in the experimental group were treated with intravenous omeprazole mg per day before the recovery of oralfeeding cases recovered oral feeding on the first day aftersurgery and cases recovered oral feeding on the third dayThen they were given mg oral omeprazole twice daily minutes before breakfast and dinner for consecutiveweeks The patients in the control group were not treatedwith PPIs but if severe granulomas formed during followup and required intervention they were also given PPIs routinely patients in the control group developedgranulomas But only patients developed severe granulomas on the 2nd 3rd and 3rd month after surgery respectively and began to be given PPIs for weeks the same asthe experimental group while the other cases in the control group were not given PPIs throughoutthe wholefollowup period All patients were treated with cefuroximeat mg twice daily for week to prevent infection Afterthe surgery the patients were required to quit smoking anddrinking and engage in reasonable vocal useThe followup procedure included regular checkups at and months after surgery If a granuloma was detectedduring the 3month checkup the patients were followedmonthly until the granuloma disappeared Postoperativegranuloma was defined as relatively smooth tissue in the surgery region that protruded from the surrounding area thatmay be attached to a pseudomembrane The granulomaand the surrounding area did not have obvious vascularhyperplasia The proportion of patients with postoperativegranuloma was used as an observation indicator Additionally the number of unscheduled visits and the rate of reoperationincluding tracheotomy and granulectomy wererecorded as indicators of granuloma severityDuring followup if the granuloma was found to severelyimpact vocalization andor breathing or if patients were suspected of having a recurrent tumor the granuloma was surgically resected and the specimen was sent for examination Statistical Analysis Data were statistically analyzed withSPSS software SPSS Chicago IL Nonnormally distributed quantitative data are represented as median and interquartile range and were subjected to the Wilcoxon ranksumtest Normally distributed measurement data are representedas mean ± standard deviation and were subjected to theindependent sample t test Count data were subjected to thechisquared test Diï¬erences with p were consideredstatistically significant Results Baseline Characteristics The baseline data of the patientsin the PPI treatment group and the control group are shownin Table Among the patients in the PPI treatment 0cBioMed Research InternationalTable General information DiscussionVariableSex MFAge yearsTumor staging T1aT1bT2Surgery type IVVVIPPIn ± Controln ± p valuegroup patients were male and patient was female withan average age of ± years patients were at theT1 stage and patients were at the T2 stage patients underwent type IV surgery patients underwent type V surgeryand patients underwent type VI surgery Among the patients in the control group patients were male and patient was female with an average age of ± years patients were at the T1 stage and patients were at theT2 stage patients underwent type IV surgery patientsunderwent type V surgery and patients underwent typeVI surgery The two groups of patients did not diï¬er significantly in the baseline conditions Table Postoperative Granulation The numbers and percentageof patients with granuloma in the PPI treatment group atthe 1st 2nd 3rd and 6thmonth followup were and respectively The numbers and percentage of patients with granuloma in the control group at those time points were and respectively Although the percentage of granuloma in the PPItreatment group was lower than that of the control group ateach stage only the diï¬erences at the 2nd and 3rd monthsafter surgery were statistically significant p and p respectively Only one patient in the PPItreatment group required a second surgery due to persistentgranulation patients in the control group underwent a second surgery among whom patients had granuloma complicated with chondronecrosis and required totracheotomy due to dyspnea Figure However the diï¬erence between the two groups was not statistically significantp In the PPI treatment group only patients hadtwo unscheduled visits In the control group patients had unscheduled visits among them one patient had visitsdue to dyspnea The diï¬erence in the number of unscheduledvisits was not statistically significant p Table Eï¬ects of PPI Treatment in Patients with LPRD in theExperimental Group Among the patients in the experimental group were diagnosed with LPRD according to preoperative RSI and RFS scores and patients did not have LPRD The numbers of LPRDpatients with granuloma at the 1st 2nd 3rd and 6th monthsafter surgery were and respectively and the numbers of LPRD patients with granuloma at these time pointswere and While the data showed that the percentageof granuloma in the LPRD patients was higher than that inthe LPRD patients only the diï¬erence at the 3rd month aftersurgery was statistically significant p Table Transoral microsurgery TM for earlystage glottic cancerGC can achieve oncological therapeutic eï¬ects similar tothose of radiotherapy TM has the advantages of being minimally invasive and having a high laryngeal preservation rateand a low tracheotomy rate its disadvantage includes postoperative complications such as bleeding infection airwayburns and granuloma formation Therefore currently thetreatment selected for patients with earlystage GC is determined by the disease conditions as well as patient needs[ ] Postoperative traumatic granuloma TG is a common complication during the healing process after TLM surgery Severe TG may cause or be complicated by chondritis orchondronecrosis leading to severe complications includingdyspnea With the increase in the range of cordectomy theincidence of TG is also significantly increased Therefore itis necessary to investigate ways to reduce the incidence andseverity of postoperative TG [ ] Like TLMthecoblationassisted endoscopic cordectomy or TCM used inour study is also based on thermal damage whose working°C much lower than laser™s workingtemperature is °°temperature which is C1000C and as a result its healing process and the mechanism of TG formation are alsosimilar to those of TLM Current literature indicates thatreflux may be an important factor in the occurrence of postoperative TG [ “] Therefore we aimed to investigatewhether antiacid therapy could reduce TG through an analysis of the eï¬ects of PPI treatment in patients who underwenttype IVVI cordectomy which has rarely been reported°C70Our study showed that the incidences of TG after transoral surgery were as high as and in the twogroups which were much higher than the incidences of reported by Nerurkar and Shah and reported by Wang The reason for this discrepancy may be that the patients in our study all underwent typeIV or higher surgeries Enlarged wounds and damage to theperichondrium or cartilage may lead to increased TG andchondronecrosis Nerurkar and Shah reported that the incidence of TG in patients who have undergone type IV TLMwas and the study by Wang showed that the incidences of TG in patients who have undergone type IV andtype V TLM were and respectively [ ]Meanwhile the incidence of chondronecrosis was not rare especially on whom the surgeon had to expose thethyroid cartilage during tumor resection in TLM [] Thesestudies suggest that type IV or higher surgeries lead to a highlikelihood of TG occurrenceOur study showed that PPI treatment did not suppressthe formation of TG at the 1st month after surgery but thepercentage of TG in the PPI treatment group graduallybecame lower than that of the control group and the diï¬erences were statistically significant at the 2nd and 3rd monthsafter surgery At the 6th month granulomas disappeared inboth groups Our findings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a finding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure a“f Shows a typical case in the control group who was a male patient for years old with glottic cancer T2N0M0 at the left sidefollowed by type V cordectomy a One month after surgery a granuloma was found in the left vocal cord under a fibrolaryngoscope b months after surgery the granuloma enlarged and the right vocal cord become edema obviously c months after surgery d monthsafter surgery e Computerized tomography CT scan taken before surgery f months after surgery chondronecrosis was found in the CTscan where the white arrow points out g“i Shows a typical case in the experimental group who was a male patient for years old withglottic cancer T2N0M0 at the right side followed by type V cordectomy g h and i were taken under a fibrolaryngoscope in month months and months after surgery respectively A granuloma could be found in g but disappeared in h and idyspnea in the PPI treatment group however TG in patients in the control group caused or was complicated bychondritis or chondronecrosis which led to severe dyspneaThere were only unscheduled visits among the PPI treatment group compared to unscheduled visits in the controlgroup Unfortunately the diï¬erences in the two indicators ofTG severity between the two groups were not statistically significant although we believe PPI treatment did alleviate the 0cBioMed Research InternationalTable Percentage and severity of granuloma in the two groups1st month2nd month3rd month6th monthResurgeryNumber of unscheduled visits asmedian and interquartile rangePPI treatment groupControl groupp valueNote aindicates that the diï¬erence is statistically significant0005a0037a Table Percentage of granuloma in patients with or without LPRDin the experimental group1st month 2nd month 3rd month 6th month”LPRD n LPRD n p valueNote aindicates that the diï¬erence is statistically significant0029aseverity of TG In this study postoperative PPI was used for weeks referring to the antiacid duration weeks in amedical routine of vocal process granulation and laryngopharyngeal reflux disease J R Lechien™s report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reflux disease in [] The resultsshowed that PPI could shorten the recovery time and potentially prevent severe complicationsOur study showed that the percentage of TG did not differ significantly between the LPRD and LPRD patientsp However it took longer for granulomas to disappear in LPRD patients than in LPRD patients and the difference in the number of patients with granulomas wasstatistically significant between the two groups at monthsafter surgery p The reason for this phenomenonmay be that during the early stage of recovery after vocalcord injury changes in the extracellular matrix are the mainmanifestation and injury impacts a lot while acid reflux hasa little eï¬ect Acid reflux may impact the repair process in themiddle and late stages [ ]This was a retrospective nonrandomized controlledstudy with a small sample size We only analyzed granulomaformation in the patients and did not assess their oncologicaloutcome We based the diagnosis of LPRD on RSI and RFSscores and did not perform dualprobe 24hour pH monitoring Therefore we were unable to determine whethernonacid reflux had an impact on the formation and development of postoperative granuloma ConclusionThe preventive use of PPI in patients who have undergonetype IVVI cordectomy cannot reduce the incidence of TGwhile it can shorten the TG recovery duration and may alsoreduce the severity of TG Our findings should be confirmedby prospective randomized controlled studies with largersample sizesData AvailabilityAccess to these anonymized data will be made available bythe corresponding author Dr Jian Wang upon reasonablerequestConflicts of InterestThe authors declare that they have no conflicts of interestfinancial or nonfinancial to discloseAuthors™ ContributionsXiaofeng Jin and Yanyan Niu contributed equally to thisstudyAcknowledgmentsThis study was funded by the Nature Science Foundation ofBeijing China Grant No References[] L B Du W M Mao W Q Chen œIncidence and mortality of larynx cancer in China during  ZhonghuaLiu Xing Bing Xue Za Zhi vol no pp “ [] F Bray J Ferlay I Soerjomataram R L Siegel L A Torreand A Jemal œGlobal cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA A Cancer Journal for Clinicians vol no pp “ [] B Gupta N W Johnson and N Kumar œGlobal epidemiology of head and neck cancers a continuing challenge Oncology vol no pp “ [] M F Vaculik C A MacKay S M Taylor J R B Trites R DHart and M H Rigby œSystematic review and metaanalysisof T1 glottic cancer outcomes comparing CO2 transoral lasermicrosurgery and radiotherapy Journal of Otolaryngology Head and Neck Surgery vol no p [] W Steiner œResults of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp “ [] M S Strong œLaser excision of carcinoma of the larynxLaryngoscope vol no pp “ [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza œRadiofrequency coblation for the resection of head and neck malignancies Otolaryngology and Headand Neck Surgery vol no pp “ [] M Lee M A Buchanan F Riï¬at and C E Palme œComplications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987“E990 [] B Liu L Cheng H Ming and C Zhong œTreatment of theearlystage glottic cancer using lowtemperature radiofrequency coblation Journal of Cancer Research and Therapeutics vol no pp “ [] Y Zhang B Wang G Sun G Zhang L Lu and G LiangœCarbon dioxide laser microsurgery versus lowtemperatureplasma radiofrequency ablation for T1a glottic cancer asingleblind randomized clinical trial BioMed Research International vol Article ID pages [] M Remacle H E Eckel A Antonelli œEndoscopic cordectomy A proposal for a classification by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp “ [] L Wang S Sun S Wang D Liang and W Ji œClinical observation of traumatic granuloma after CO‚‚ laser cordectomy andlaryngopharyngeal reflux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp “ [] M Canis F Ihler A Martin C Matthias and W SteinerœTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp “ [] A Galli L Giordano D Sarandria D di Santo and M BussiœOncological and complication assessment of CO2 laserassisted endoscopic surgery for T1T2 glottic tumours clinicalexperience Acta Otorhinolaryngologica Italica vol no pp “ [] N K Nerurkar and R Shah œFactors responsible for the development of carbon granuloma post transoral laser cordectomy Lasers in Medical Science vol no pp “ [] P C Belafsky G N Postma and J A Koufman œThe validityand reliability of the reflux finding score RFS Laryngoscopevol no pp “ [] P C Belafsky G N Postma and J A Koufman œValidity andreliability of the reflux symptom index RSI Journal of Voicevol no pp “ [] M Remacle C van Haverbeke H Eckel œProposal forrevision of the European Laryngological Society classificationof endoscopic cordectomies European Archives of OtoRhinoLaryngology vol no pp “ [] J Yoo C Lacchetti J A Hammond R W Gilbert and Headand Neck Cancer Disease Site Group œRole of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp “ [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo œComplications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp “ [] J R Lechien F Mouawad M R Barillari œTreatment oflaryngopharyngeal reflux disease a systematic review WorldJournal of Clinical Cases vol no pp “ [] M K Wani and G E Woodson œLaryngeal contact granuloma Laryngoscope vol no pp “ [] C Ling M Yamashita J Zhang D M Bless and N V Welham œReactive response of fibrocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp “ 0c'

netosis is a type of regulated cell death dependent on the formation of neutrophil extracellular traps net where netlike structures of decondensed chromatin andproteases are produced by polymorphonuclear pmn granulocytes these structuresimmobilise pathogens and restrict them with antimicrobial molecules thus preventing theirspread whilst nets possess a fundamental antimicrobial function within the innate immunesystem under physiological circumstances increasing evidence also indicates that netosisoccurs in the pathogenic process of other disease type including but not limited to atherosclerosis airway inflammation alzheimer™s and stroke here we reviewed the role ofnetosis in the development of an injury including injury to the brain lung heart kidneymusculoskeletal system gut and reproductive system whilst therapeutic agents in blockinginjuries induced by netosis in its primitive stages were also discussed this review providesnovel insights into the involvement of netosis in different an injuries and whilstpotential therapeutic measures targeting netosis remain a largely unexplored area thesewarrant further investigationkey words netosis neutrophil an injury cell death inflammation cell death is commonly segregated into necrosisand apoptosis apoptosis being programmed cell death anaesthetics pain medicine and intensive care department of surgeryand cancer faculty of medicine imperial college london chelsea andwestminster hospital fulham road london sw10 9nh uk department of anesthesiology shanghai fengxian district central hospital shanghai jiao tong university affiliated sixth people™s hospitalsouth campus fengxian district shanghai china to whom correspondence should be addressed at anaesthetics painmedicine and intensive care department of surgery and cancer faculty of medicine imperial college london chelsea and westminsterhospital fulham road london sw10 9nh uk emaildmaimperialacukfor instance during development and physiological cellular turnover whilst necrosis predominantly takesplace in an unregulated manner netosis like necrosis is a mode of cell death that involves the loss ofmembrane integrity during netosis decondensationof chromatin is thought to be initiated by peptidyl arginine deiminase pad4 its subsequent releasetogether with granule contents is vital in the innateimmune response to infection and inflammation recentstudies suggest that net formation is of central topathogenesis of an injury this review will summarise the current understanding of the molecular mechanisms of netosis and the therapeutic approaches underdevelopment targeting netinduced an injury the authors this is an open access publication 0cmolecular mechanism of netformationalthough netosis is closely associated with netformation not all net formation requires the process ofcell death to take place beforehand according to nomenclature committee on cell death the term ˜netosis™should only be used in the context of cell death and notjust based on the presence of net formation two main pathways of net formation have beendescribed and categorised according to their dependenceon the activity of nicotinamide adenine dinucleotide phosphate nadph oxidase pathway fig nadph oxidasedependent net formationthe nadph oxidasedependent molecular pathwayof net formation begins with activation of neutrophilsurface receptors by stimuli derived from pathogenic ornonpathogenic sources such as cholesterol or urate andends with cellular lysis these stimuli trigger calciumrelease from the endoplasmic reticulum er resulting inthe activation of protein kinase c pkc and the assemblyof the nadph oxidase complex generating reactive oxygen species ros following this neutrophil elastasene a protease stored in the cytoplasmic granules migrates to the nucleus in a myeloperoxidase mpodependent manner and cleaves histones to initiate chromatindecondensation this is promoted by the citrullinationof histone arginine residues by peptidylarginine deiminaseiv pad4 finally mixing of the chromatin andgranule proteins takes place as cellular membranes arebroken down interestingly there have been reports ofmitochondrial dna mtdna instead of nuclear dnabeing the source of the dna fibres in nets with observations of mtdna being released from granulocytes inresponse to disease states such as trauma and systemiclupus erythematosus sle [ ] moreover it seemsthat histone citrullination is not always required for netformation as observed by kenny and colleagues in theirstudy of neutrophils activated by the pkc agonist phorbol12myristate 13acetate pma this highlights the diversity of pathways for net formation following their induction degradation of nets can take place through severalpathways for example by dnases or endocytosed bymacrophages factors that influence net formation include phco2 and hco3ˆ’ levels which modulate neutrophil activation this explains why nets form more readily in thecahilog zhao wu alam eguchi weng and maperiphery of an inflammatory site where the ph is morealkaline this may influence treatment efficacy asnets can seal off the affected area an acidic environmenthas been hypothesised to reduce nadph oxidasedependent net formation by reducing neutrophil glycolysis nadph oxidasedependent net formation also requires neutrophils to be in the cell cycle necessitating theactivation of cyclindependent kinases cdk phosphorylating the retinoblastoma proteinnadph oxidaseindependent net formationthis mechanism of net formation is more relevant inthe context of infection as inducers of netosis here arecalcium ionophore a23187 and the potassium ionophorenigericin which are products of streptomyceschartreusensis and streptomyces hygroscopicus respectively how this pathway leads to net release ispoorly understoodnetosis and inflammationnets under physiological conditions are central topathogen clearance when there is excessive formation orsuboptimal nets are able to initiate further destructivesignalling through interaction with other tissue constituentsand the immune system moreover the antimicrobial histones and peptides within the net structure impose adirect cytotoxic effect on tissues to date there havebeen numerous accounts of netosis being present indiseases of major ans understanding of netosis inpathophysiology may offer unique opportunities for clinical managementnetosis in an injurythere is an expanding body of research describingnetosis in infectious and noninfectious an injurysummarised in fig although it is valid that in thesescenarios nuclear dna released during necrotic cell deathcan contribute to tissue injury and exacerbate the extent ofan damage here we focus on the contribution by aberrant net formation and the implication of understandingits underlying pathogenesis for therapeutic interventions 0crole of neutrophil netosis in an injuryfig type of cell death for neutrophil in an injury during solid an injury neutrophils could be prompted to undergo caspasedependent apoptosiswhich results in controlled dissolution of cell into apoptotic bodies containing cellular debris to prevent immune and inflammatory responses neutrophilextracellular traps nets form via two pathways the first is through lytic netosis a cell death pathway characterised by nuclear delobulation anddisintegration of the nuclear envelope which precedes loss of cellular polarisation chromatin decondensation and plasma membrane rupture the secondmechanism involves the nonlytic form of netosis which is not associated with cellular death but prompts expulsion of nuclear chromatin together withrelease of granule proteins through degranulation these components can assemble in the extracellular space into nets leaving behind enucleated cytoplaststhat continue to ingest microanisms in addition neutrophils could undergo unregulated necrosis that does not involve specific molecular pathwayswith uncontrolled release of cellular debris acting as dangerassociated molecular patterns damps to trigger proinflammatory responsebrainalzheimer™s disease alzheimer™s disease ad is acommon disorder of neurodegeneration characterised bygradual loss of cognitive functions in ad patients neutrophils have been observed to invade the brain parenchyma and release nets causing destruction of neural cellsand the bloodbrain barrier stroke it is well known that the adaptive immunesystem is altered after a stroke predisposing patients toinfections [“] interestingly netosis has also beendescribed as significantly impaired up until on day inthose with an ischaemic stroke though netosis inhaemorrhagic stroke patients has yet to be documented ithas been noted that the generation of ros a keyrequirement for chromatin decondensation is suppressedin these patients for up to days lungcystic fibrosis it has been well established that chronic infections in cystic fibrosis cf patients are due to thehighly viscous mucus production harbouring microbialgrowth although impaired clearance of mucus has beenprincipally named responsible there is increasing evidencethat the high viscosity is also due vast amounts of freedna found in sputum samples which was in concordance with the high concentration of neutrophil and 0ccahilog zhao wu alam eguchi weng and mafig involvement of netosis in an injury accumulating evidences now point to an important role of netosis in infectious and noninfectious solidan injury neutrophil invasion into brain parenchyma and release of neutrophil extracellular traps nets have been established in the pathophysiology ofalzheimer™s disease to cause destruction to neural cells and bloodbrain barrier abnormal netosis activity and reactive oxygen species ros response akey element to netosis initiation were observed in stroke patients the degree of neutrophil infiltration net formationcomponent eg cellfreenucleosomes and netosis have been found to correlate with the severity of a range of lung diseases including cystic fibrosis acute lung injury aliacute respiratory distress syndrome ards and lung infection netosis was also shown to be involved in allergic asthma chronic obstructive pulmonarydisease and pulmonary hypertension wherein degree of net formation correlates with disease severity during liver ischaemiareperfusion tolllikereceptordependent net release has been suggested to mediate liver inflammation and injury conversely deficiency in net release was reported indecompensated cirrhotic liver disease and could explain susceptibility to bacterial peritonitis infection in those patients net formation and netosis havefurther been implicated in atherosclerosis and myocardial infarction wherein net was found in thrombi and infarct lesion and correlate with disease severityin rheumatoid arthritis enhanced net release and netosis are observed in synovial tissue rheumatoid nodules and skin whilst proinflammatory cytokinesand autoantibodies further aggravate neutrophil infiltration and netosis neutrophils could also be potently activated by monosodiumurate msu crystals ingout joints and point to a potential role of netnetosis in gout pathogenesis moreover neutrophil activation and net deposition were also observed incolon mucosa of ulcerative colitis excessive neutrophil activation net formation and netosis could also be responsible different pregnancyrelateddisorders including preeclampsia wherein net deposition and netosis in the intervillous space may damage maternal endothelium and impair foetaloxygen exchangenets found in cf lungs the source was believed to befrom necrotic neutrophils but is now considered to besecondary to netosis additionally net productionwas found to be promoted by bacterial infection in cfairways and was defective in clearance of the bacteriapseudomonas aeruginosa nets are also named as a facilitative factor for biofilm formation there is evidencethat surfactant protein d spd responsible foropsonising pathogens and dying cells for clearance byalveolar macrophages is essential for net clearancethrough binding directly to the chromatin within the netsspd levels are decreased in cf patients and the level ofdecrease is proportional to the degree of inflammationthrough accumulation of nets 0crole of neutrophil netosis in an injurylung infection neutrophils migrated into the affectedsite and initiate the cascade of antimicrobial mechanismsincluding net generation to combat microanismsthis happens more readily in the lungs compared with inother tissues with neutrophils found to exist in higherconcentrations in pulmonary vasculature compared withsystemic blood vessels a prominent pathway leadingto net formation in infection is through the interaction oflipopolysaccharide lps with tolllike receptor tlr4found on neutrophils in patients with communityacquired pneumoniacap increased levels of cellfree nucleosomes in serumsamples used as surrogate markers of netosis werefound this was associated with prolonged hospitalisationand a greater 30day allcause mortality this findingsuggests nets could function as a novel marker of prognosis in capacute lung injury and acute respiratory distresssyndrome the degree of neutrophil influx into the lungsand net release during ali and ards positively correlates with disease progression and severity with neutrophil depletion conferring protection in a transfusionrelated ali animal model netosis seems to be akey component of ventilatorinduced lung injury vili as evidenced by detection of citrullinated histone3suggesting that this was a mode of cell death independentfrom apoptosis and necrosis the authors suggestedthat this may be due to increased levels of il1β andhmgb1 which have been both shown to be able to inducenetosisallergic asthma patients with neutrophilic asthmahave a greater severity of disease and reduced response tocorticosteroid treatment compared with the eosinophilictype the increased expression of neutrophilchemoattractant il8 in airway smooth muscle cells couldbe contributing to disease severity through inducingnetosischronic obstructive pulmonary disease netosis hasbeen documented as an integral part of chronic obstructivepulmonary disease copd pathophysiology unlike asthma where neutrophils are important in certain subtypesnetosis has been directly linked to disease severity incopd [ ] tlr4 expression one of the main potentiators for net formation is increased during copd exacerbations pulmonary hypertension nets are also able to potentiate dysregulated angiogenesis as seen in patients withchronic thromboembolic pulmonary hypertension and idiopathic pulmonary hypertension as plasma levels of dnane and mpo levels are significantly elevated moreovernets also seem to destabilise intercellular junctions andincrease endothelial cell motility through direct contactwith endothelial cells nets were found to induce theactivity of the proinflammatory transcription factor nfκbby approximately 3fold moreover nets increase thesurface expression of von willebrand factor and plateletadhesion thereby producing a prothrombotic state kidneyglomerulonephritides nets have been visualised upon immunostaining renal biopsies from patients with sleand antineutrophil cytoplasmic antibodiesassociated vasculitis aav and may be at least partially responsiblefor activating complement pathways resulting in diseaseexacerbations these autoimmune conditions alsoseem to affect the patient™s ability to degrade nets amplifying their deleterious inflammatory effects increscentic glomerulonephritis neutrophilmediated glomerular damage is worsened by addition of extracellularmpo which could have been released during netosis netosis could also contribute to the loss of immunetolerance through further externalisation of crucialautoantigens during cell death haemolytic uraemic syndrome hus plasma from affected patients exhibited a greater capacity to undergonetosis compared with healthy patients the ensuingdamage has been linked to the proinflammatory cytokinesil6 and il8 released from glomerular epithelial cellsupon stimulation by nets this potentiates microvasculature inflammation and thrombosis precipitating renal failure liverdecompensated cirrhotic liver disease a deficiency innet release has been demonstrated to play a role in theonset of endstage liver disease as neutrophils incirrhotic patients are found to have defective ros production which commonly triggers net release thismay also partially explain why these have a predispositionto recurrent bacterial infections and increased rates ofdecompensatory complications such as spontaneous bacterial peritonitis sbp this is corroborated by defectivenet release from ascitic fluid neutrophils in cirrhoticpatients compared with controls in vitro cirrhoticpatients with sbp were also found to have an increase in 0cpro and antiinflammatory cytokines in peripheral bloodand ascitic fluid ischaemiareperfusion injury ischaemiareperfusioninjury iri is an inherent consequence of liver transplantation hypovolaemia or trauma and results in the release ofdamageassociated molecular patterns damps which inturn cause net formation in a tlrdependent mannerexacerbating inflammation treatment with apeptidylargininedeiminase pad4 inhibitor ordnase has been shown to be significantly hepatoprotectivefollowing liver iri cardiovascular systematherosclerosis nets are a wellknown constituent ofatherosclerotic lesions mpo has been strongly associated with diminishing the cardioprotective effects ofhighdensity lipoprotein cholesterol hdlc through oxidation reactions and an increased enzymatic activity islinked to increased plaque rupture other proteinsfound in nets such as cathelinrelated antimicrobial peptide cramp have also been shown to contribute todisease progression moreover in vitro studies showthat hypercholesterolemia triggers net formation alargescale study in patients with suspected coronary arterydisease revealed that the markers of netosis such asextracellular dna are independently associated with disease severity coronary specimens from patients afteran acute myocardial infarction mi showed the presenceof nets in both fresh and lytic thrombi therefore suggesting netosis happens in the early stages of thrombusevolution furthermore net burden was shown tobe positively correlated with the infarct size in patientsundergoing primary percutaneous coronary interventionspostmi this is supported by increased levels of mpodna and ne in the lesion site therefore nets couldpotentially be considered as a novel biomarker in atherosclerosis diabetes mellitusinduced vasculopathy it has beenshown that neutrophils form peripheral blood of diabetesmellitus dm patients showed increased spontaneousnetosis interestingly metformin reduces the deleterious effects of netosis in a mechanism independentlyfrom glucose control one recent study showed that month treatment with metformin in predm patients reduced levels of components of nets whereas glycaemiccontrol with other medication such as insulin saw nodifference when compared with placebo controls thiscahilog zhao wu alam eguchi weng and mahas been attributed to a direct effect of metformin oninhibiting the activation of nadph oxidase musculoskeletal systemrheumatoid arthritis neutrophils from the peripheralblood and synovial fluid of patients with rheumatoid arthritisra exhibit increased netosis compared with healthy controls and patients with osteoarthritis the externalisation ofcitrullinated proteins during the process of netosis wasfound to initiate and perpetuate the aberrant immune responsein ra moreover the autoantibodies and inflammatory cytokines commonly seen in ra promote netosis resulting in avicious cycle of tissue destruction gout gout is an inflammatory disease that involvesthe deposition of monosodiumurate msu crystals injoints during acute gout there is increased movement ofneutrophils into the synovial fluid msu is a known neutrophil stimulus and it has been shown that acute gout isassociated with an increase in il1β levels a keyplayer in net formationgutulcerative colitis there is prominent neutrophil infiltration in the colon mucosa in ulcerative colitis uc and this correlates with disease severity in uc the inflammatory environment promotes neutrophil activation andil1β expression in contrast nets do not seem toplay a key role in crohn™s disease this may explain whymesalazine a known inhibitor of il1β production and thefirstline treatment for uc flareups is not therapeutic incrohn™s patients per se reproductive systempreeclampsia placentas from women diagnosed withpreeclampsia showed increased neutrophil infiltration andnetosis when compared with nonhypertensive pregnantcontrols [ ] and are probably involved in causingwidespread damage to the maternal endothelium placental and endothelial injury during pregnancy aberrantneutrophil activity during pregnancy is also associated withother severe complications including recurrent foetal loss one recent study indicated neutrophils in pregnant womenseem to have an increased propensity to undergo netosissecondary to an increase in granulocyte colonystimulating 0crole of neutrophil netosis in an injuryfactor during pregnancy progesterone has been shown toattenuate neutrophilmediated ros production whereas 17βestradiol induces intracellular ros generation in a dosedependent manner associated an injury associated with netosis fig examples of recent publications on potential therapeutic compounds targeting netosis are summarised in table netosis as a therapeutic targettargeting critical steps in net formation and degradation is critical for developing treatment strategies for netosistlr inhibitorsdexamethasone dex has been shown in vitro toreduce netosis in neutrophils that are stimulated withstaphylococcus aureus but not in those stimulated withpma the tlrs involved in s aureusinduced net formation seem to be tlr2 and tlr4 as agonists of thesereceptors rescued dex inhibition interestingly althoughfig therapeutic strategies targeting net formation stimulation of neutrophil receptors eg fc γ receptor tolllike receptor by microanisms orsterile signals leads to release of calcium ca2 from the endoplasmic reticulum er cellular ca2 overload results in activation of protein kinase c pkcassembly of the nicotinamide adenine dinucleotide phosphate nadph oxidase complex andor mitochondrial activation thus stimulating reactive oxygenspecies ros production oxidative stress promotes myeloperoxidase mpodependent migration of granular neutrophil elastase ne into the nucleus tocleave histones subsequent activation of peptidylarginine deiminase pad induces histone citrullination to cause chromatin decondensation the last stepinvolves nuclear membrane degradation and extrusion of a mixture of chromatin and granular proteins into extracellular space whereby extracellular dnaseeventually digests and removes neutrophil extracellular traps nets in this regard modulation of critical steps in net formation and degradation shown byblocking arrows might be beneficial for the treatment of inflammatory disorders figure modified and reproduced with permission fcγr fc γ receptortlr tolllike receptor 0cdex reduced net formation it did not significantly affectros production calcineurin inhibitorscalcineurin is a calciumdependent serinethreonineprotein phosphatase that is important for neutrophil activity many stages of netosis induction depend upon calcium mobilisation hence modulators of the calcineurinpathway are potential pharmacological inhibitors of netformation cyclosporine a csa an antagonist of thecalcineurin pathway has been shown to reduce the effectof key physiological activators of neutrophils this effecton netosis may in part explain csa™s efficacy in ra and steroidresistant uc patients pmainducednetosis seems to be calciumindependent as this wasnot inhibited by csa cahilog zhao wu alam eguchi weng and mapad inhibitorsusing a murine model of atherosclerosis knight andcolleagues have shown that pharmacological inhibition ofpad4 using weeks of daily clamidine injections reduced netinduced vascular damage with delayed plaqueprogression in the carotid arteries the same groupalso showed that pad inhibitors reduce disease activity inmurine models of sle by reducing endothelial dysfunction it is worth mentioning that the possibility of padinhibition as a therapeutic avenue to be pursued in netinduced an damage in glomerulonephritis has beenrecently challenged by the work of gordon and colleagueson murine models on sle with pad4 deletion theyshowed that this did not reduce endan damage asmeasured by proteinuria suggesting that mechanismsother than net formation are implicated in this complexautoimmune conditionros scavengersdnase therapythe mitochondria are a powerful source of ros ros scavengers such as nacetyl cysteine nac reducenet formation and severity of sle in patients troloxand tempol are two antioxidants which have also beenshown to prevent netosis of pmastimulated humanneutrophils in a dosedependent manner and have beenrecommended for treatment of autoimmune and inflammatory pathologies dnase therapy has been proposed to improve outcomes in cf patients through reducing mucous viscosityhowever it appears that recombinant dnase does notreduce the load of dnaprotein complexes seen innetosis one solution to this is to combine elastase withdnase in order to degrade histones and provide dnaseaccess to chromatin this combination has been shown toenhance solubilisation of sputum drug classstudymain findingstable potential therapeutic approaches targeting netosistlr inhibitorswan t et al calcineurin inhibitorsgupta ak et al dexamethasone reduced netosis in neutrophils stimulated with s aureusagonists of tlr2 and tlr4 rescued dexamethasone inhibitionros production was unaffected by dexamethasonecyclosporine a reduced the effect of key physiological stimuli that activate neutrophilssuch as il8 and suppressed netosisros scavengerspatel et al vorobjeva nv and pinegin bvnacetyl cysteine reduced net formation and severity of sle in patientsantioxidants trolox and tempol prevent netosis of in stimulated human neutrophils in apad inhibitorsknight js et al weeks of daily clamidine injections reduced netinduced vascular damage and area ofdosedependent mannerlesions in a murine model of atherosclerosispad inhibition dampens disease activity by reducing endothelial dysfunction in a murinemodel of slednase therapypapayannopoulos v staab delastase combined with dnase therapy enhances solubilisation of sputum in cystic fibrosistetrahydroisoquinolines martinez ne et al tetrahydroisoquinolines selectively target net overproduction at micromolarzychlinsky a patientsconcentrations possibly at multiple stages of net formation without compromisingnormal neutrophil function 0crole of neutrophil netosis in an injurytetrahydroisoquinolinesin contrary to the aforementioned mechanisms ofnetosis inhibitors tetrahydroisoquinolines thiqs area new class of net formation inhibitors that do not targetros formation or granular protein activity as functionalneutrophils are paramount to maintaining immune reactivity this difference offers an advantage to selectively targetnet overproduction without impairing normal functionthe exact molecular mechanisms of thiqs are yet to bedetermined however it is known that thiq inhibition ofnetosis take place at micromolar concentrations and possibly at different stages of net formation conclusionwhen regulated as part of normal physiology netsare antimicrobial and fundamental to the innate immunesystem dysregulated net formation contributes to thepathogenesis of a plethora of diseases this review hassummarised the role of netosis in pathologies of multiplebody systems as well as highlighted the stages of netosisthat has so far been investigated as emerging pharmacological targets these putative strategies seem to hold therapeutic potential and warrant further investigationauthors™ contributionsdm designed and reviewed the manuscript zc andhz wrote the first draft of the paper all authors readrevised and approved the final manuscriptcompliance with ethical standardscompeting interests the authors declare that they haveno competing interestsethics approval and consent to participate notapplicableconsent for publication not applicableopen access this is licensed under a creativecommons attribution international license whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicateif changes were made the images or other third partymaterial in this are included in the 's creativecommons licence unless indicated otherwise in a creditline to the material if material is not included in the's creative commons licence and your intended useis not permitted by statutory regulation or exceeds thepermitted use you will need to obtain permission directlyfrom the copyright holder to view a copy of this licencevisit httpcreativecommonslicensesby40references vandenabeele p l galluzzi t vanden berghe and g kroemer molecular mechanisms of necroptosis an ordered cellularexplosion nature reviews molecular cell biology “ httpsdoi101038nrm2970 lewis hd j liddle je coote sj atkinson md barker bdbax kl bicker rp bingham m campbell yh chen cwchung pd craggs rp davis d eberhard g joberty kelind k locke c maller k martinod c patten o polyakovace rise m rüdiger rj sheppard dj slade p thomas jthorpe g yao g drewes dd wagner pr thompson rkprinjha and dm wilson inhibition of pad4 activity issufficient to disrupt mouse and human net formation naturechemical biology “ httpsdoi101038nchembio1735 galluzzi lorenzo ilio vitale stuart a aaronson john m abramsdieter adam patrizia agostinis emad s alnemri et al molecular mechanisms of cell death recommendations of the nomenclature committee on cell death cell death and differentiation “ 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"We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation MET amplification and KRAS/PIK3CA mutation. These findings represent a promising strategy for the treatment of gefitinib-resistant NSCLC and provide a strong basis for the design of clinical trials for this purpose. Competing interests The authors declare that they have no competing interests. Authors™ contributions YQQ conceived and designed the experiments. XXW YHY YY and HL performed the experiments. XXW YHY and DDM analyzed the data. XXW YHY and HL wrote the paper. YQQ supervised the whole experimental work and revised the manuscript. All authors read and approved the manuscript. She J Yang P Hong Q Bai C Lung cancer in China: challenges and interventions Chest 2013 143 1117 1126 23546484 Weir HK Thun MJ Hankey BF Ries LA Howe HL Wingo PA Jemal A Ward E Anderson RN Edwards BK Annual report to the nation on the status of cancer "

"Laryngology COVID19 Coronavirus Head and Neck Cancer Otolaryngology Key Points Journal Preproof 0c The landscape of ever evolving information about COVID19 during the pandemic has hindered the transition to normal clinical volume and efficiency COVID19 should not be a reason for delay in diagnosis or treatment with patients who have upper aerodigestive tract pathology or malignancy The approach to resection reconstruction and surveillance for patients with head and neck cancer may need to be altered to consider severity of disease patient comorbidity and prevalence of regional COVID19 infections amongst other factors In light of the significant number of prolonged intubations there may be an increase in the number of patients who develop early and late sequelae of treatment for COVID19 Tracheostomy should be performed in a safe and efficient manner when specific indications are met Synopsis This review explores the changes to practice associated with COVID19 for providers treating patients with head and neck cancer and laryngeal pathology The aim of the review is to highlight some of the challenges and considerations associated with treating this patient population during the pandemic Additionally it seeks to discuss some of the areas of concern related to ramping up clinical volume IntroductionHistoryDefinitionsBackground The downstream effects of COVID19 caused by severe acute respiratory syndrome coronavirus SARSCoV2 have now pervaded most aspects of society and have made an indelible mark on the way that medicine and specifically otolaryngology is being practiced Of note the Journal Preproof 0cdisease represents a threat to an aging population throughout the world but also has dangerous implications for providers Among the most œatrisk group of medical providers may be those within the fields of otolaryngology and ophthalmology An otolaryngologist was among one of the first providers to succumb to the illness in its early days as it spread through Wuhan China In light of the risk to both patients healthcare workers and society atlarge a push has been made to mitigate the risk of transmission within the field of Otolaryngology Head and Neck Surgery As of June 22nd there are a total of COVID19 cases reported worldwide with a total of deaths The United States has the highest number of cases at with the total number dead at Given the high mortality associated with the novel virus much of the world has enacted significant social distancing restrictions and facial covering mandates to curb the spread of the disease The origin of the virus is not well understood but it is thought that a bat or pangolin vector might have served as the primary reservoir The disease tends to be marked by fever of patients and cough of patients however a litany of other symptoms have also been described including gastrointestinal upset diarrhea shortness of breath headache loss of smelltaste among others Severe disease is characterized by an acute respiratory distress syndrome ARDS with a mortality for patients that require mechanical ventilation The disease has a slight male predominance at Severity of disease seems to correlate to age as patients who are aged have a mortality while those over present with a mortality approaching in early studies Journal Preproof 0cThe nasal cavity and nasopharynx seem to harbor the highest viral load concentration and thus the nasopharynx is the preferred location for acquisition of samples for diagnostic testing Nasal swabs oropharyngeal swabs bronchial alveolar lavage saliva and tracheal aspirates have also been suggested as possible testing sites The current preferred diagnostic assay is RTPCR which has a variable sensitivity of depending on the institution and type of test During the months of May and June many cities states and countries have focused on a return to normal activity and a ramp up of commercial activities During this time many otolaryngology practices have aimed to ramp up activity as well while employing telehealth social distancing and utilization of personal protective equipment PPE The American Academy of Otolaryngology recently published return to practice guidelines which are detailed below As the world continues to move forward during the COVID19 era considerations such as testing including preoperativepreprocedure COVID testing surgical triage clinic workflow and practice management continue to evolve as more information becomes available This review is intended to highlight some of the current recommendations for patient care within the Laryngology and Head and Neck Surgical Oncology scope of practice Discussion Laryngology Journal Preproof 0cAs cases continue to rise increased emphasis has been placed on protection for the provider in the clinical setting Over the last decade officebased management of many common laryngeal disorders has significantly expanded This includes but is not limited to officebased laser ablation of papilloma or dysplasia transoral or transcervical injection laryngoplasty for vocal fold paralysis and EMGguided injection of Botox for spasmodic dysphonia Given the high number of clinicbased aerosolgenerating procedures practiced by today™s laryngologists many providers have seen a marked reduction in their ability to treat patients and their clinical productivity Within the category of aerosolgenerating procedures is flexible fiberoptic laryngoscopy one of the most widely used diagnostic tools for all otolaryngologists and speech pathologists A consensus statement reported by Rameau et al from a virtual webinar attended by approximately participants in the American laryngology community recommended flexible laryngoscopy should be reserved for critical cases in which the findings may have an immediate impact on diagnosis or treatment œIndications include hemoptysis odynophagia limiting hydration and nutrition or airway compromise”notably secondary to infectious and malignant conditions Some have advocated for preclinic COVID testing prior to any aerosol generating procedure however given the high false negative rate of many available tests the use of universal personal protective precautions is recommended According to Givi and colleagues examinations should take place in negative pressure rooms if possible with avoidance of topical lidocaine spray although other groups recommend administration of a topical anesthetic to theoretically decrease the coughsneeze reflex unpublished online chats A substitute to standard aerosolized anesthesia may be pledgets soaked in lidocaine and Journal Preproof 0c oxymetazoline The group also suggests using videolaryngoscopy whenever possible to keep the practitioner and the patient farther apart Disposable laryngoscopes should be used whenever possible Most studies universally recommend the following personal protective equipment PPE N95 mask or powered airpurifying respirators PAPRs gloves gown eye shield or goggles and cap It has also been suggested that the patients wear a mask covering the mouth during flexible laryngoscopy to reduce aerosolization from phonatory maneuvers and in case of coughing or sneezing At this time transoral rigid and mirror laryngoscopy are discouraged unless flexible laryngoscopy cannot be performed due to the increased risk of gagging and coughing as well as the need for the patient to phonate with the mouth uncovered to allow visualization of the larynx Additionally universal masking is encouraged in all clinical spaces in accordance with many state policies Patients in the waiting rooms are encouraged to physically distance or wait in their car for a phone call prior to presenting for their appointment Crosby also suggests offering personal protective equipment for the friends and family accompanying the patient during laryngoscopy and other sites prevent friends and family from accompanying patients inside for the visit Some alternatives to flexible laryngoscopy have been raised including transcervical laryngeal ultrasound which has a reported concordance of in identifying vocal fold motion abnormalities Another key consideration for the laryngologist in the COVID19 era is the approach to sanitization and room turnover after aerosol generating procedures AGPs Laryngoscope turnover should include highlevel disinfection including the use of such chemical disinfectants as glutaraldehyde chlorine dioxide or orthophthaladehyde OPA Some authors recommend immediate placement of the scope after use into a covered receptacle for transport from the Journal Preproof 0cexamination room to the sterile processing areas After completion of laryngoscopy room sanitization with an EPAregistered hospitalgrade disinfectant is recommended with a hydrogen peroxide solution gL chlorine disinfectant or alcohol According to the CDC website it is unknown how long the air inside a particular examination room remains infectious and likely relates to the room size rapidity of air exchange patient factors like viral shedding amount of coughingsneezing and length of time patient was in the room The CDC suggests that rooms with air changeshour ACH take about and minutes to purify the air with and efficiency respectively As the number of air changeshour decreases the time between patients should be increased to allow for appropriate dissipation of theoretical infectious agents As such many hospitals have recommended a turnover time of 4x the time it takes to purify the air with efficiency which may be either minutes or minutes depending on the level of air turnover or could be no additional time if any additional HEPA filtration system and negative pressure has been added Limited data exists to support this approach for SARSCoV2 Laryngology patients are quite diverse with respect to their level of acuity Some patients require more urgent intervention while others may have their care deferred1221 Most guidelines advocate for a tiered approach to ramping up both clinicbased and surgical activity The American Academy of Otolaryngology published guidelines for ramping up clinical activity on May The AAO recommends limiting patient care to individuals with œtimesensitiveurgent and emergent medical conditions This approach is also echoed in the care of head and neck cancer patients see below According to the guidelines emergent conditions include œimpending airway obstruction due to infection neoplasm stenosis foreign body which may Journal Preproof 0cwarrant the following intervention œflexible and rigid laryngoscopy with intervention direct laryngoscopysuspension laryngoscopy bronchoscopy and tracheostomy Urgent conditions include œmoderate or impending airway obstruction progressive dysphonia progressive dysphagia glottic incompetence causing aspiration or impaired pulmonary toilet which warrant the previously described procedures in addition to œstroboscopy functional endoscopic evaluation of swallow esophagoscopy with or without intervention open airway procedures for cancer Time sensitive conditions include œT1 glottic carcinoma or carcinoma in situ stablemild dysphonia stable dysphagia which adds œtranscervical Botox injection to the above list of procedures Routine conditions which may be deferred for days or more include œmildmoderate dysplasia nonobstructive benignphonotraumatic lesions of the vocal folds glottic incompetence glottic incompetence with mild to moderate dysphonia gender affirmation globuscough without alarm signs Comparing acuity of patients also raises an important point about the subset of patients who are typically seen for benign phonotraumatic voice disorders Many live vocal performance venues have shut down concerts have been cancelled or postponed and some studies point to live singing as being a potential source of massive spread For this reason one might assume that the percentage of patients being seen for acute phonotraumatic voice disorders diminishes somewhat Conversely as patients continue to recover from hospitalizations related to COVID19 it is anticipated that there may be a number of patients with sequelae of prolonged intubation including posterior glottic stenosis vocal fold granulomas and trachealsubglottic stenosis Laryngeal surgery in the era of COVID has had to undergo some significant changes in the approach to patient triage surgical technique and management of the airway Preoperative Journal Preproof 0cevaluation of patients must weigh the risk of delaying surgery with the risk of complications related to COVID19 infection Lei et al studied a group of operative patients in whom all were COVID19 positive within the incubation period Mortality was for this group and required ICU admission25 Of note all patients in this study underwent surgery about days prior to demonstrating signs or symptoms of COVID19 pneumonia This suggests there is significant risk associated with elective surgery in seemingly asymptomatic patients who are infected with COVID19 For this reason many authors have suggested preoperative COVID testing although it is a subject of some debate Some advocate for a negative test within hours followed by selfquarantine until the time of surgery while others favor a negative test 48hrs from the time of surgery and a point of care negative test on the day of surgery17 This is not always possible given the limitations of the institution where the patient is undergoing surgery Just as discussed earlier with regard to personal protective equipment in clinic universal precautions should be taken including full PPE and all patients should be presumed positive Airway management in the COVID19 era has become a point of focus for quality improvement and safety groups Endotracheal intubation is cited as one of the procedures which seems to have the highest aerosol generating burden It is recommended that intubation be performed by the most experienced practitioner Additionally some recommend early intubation for patients that are high risk for decompensation while others have advocated delaying intubation in favor of noninvasive means of ventilation This may include high flow nasal cannula which actually has minimal dispersion of exhaled air if appropriately fitted according to Cheung It is recommended that flexible fiberoptic intubation be avoided whenever possible Journal Preproof 0cAdditionally excessive bagmask ventilation should be avoided due to the risk of dispersion of exhaled air Furthermore jet ventilation is considered particularly high risk and should be avoided if possible Management of the surgical airway and the topic of tracheostomy has been well represented in the recent literature During the SARS outbreak in open tracheostomy was the most common surgical procedure performed on infected patients Most studies seem to favor open tracheostomy over percutaneous tracheostomy however consideration may be given for percutaneous dilatation tracheostomy in some patients if the anatomy is favorable and the practitioner has sufficient expertise with the procedure Tay and colleagues advocate for use of PAPR during tracheostomy based on the experience of countries during the SARS crisis Other authors have suggested the use of an N95 mask appropriate eye protection gown double gloves and cap To decrease the risk of autocontamination some have recommended an infection control nurse be available to monitor donning and doffing procedures during tracheostomy Additional proposals include trach teams which may consist of a surgeon anesthetist and scrub nurse to increase efficiency and create an environment of consistent verbal and nonverbal communication especially important given the burdens of communicating through a mask or PAPR Portugal et al discuss a surgical safety checklist for performing tracheostomy in patients with COVID1932 This includes performing tracheostomy in the ICU whenever possible decreasing the number of personnel in the room and having a specific tracheostomy bundle in the ICU room to decrease the number of times providers and nurses need to break scrub to leave the room They also recommend donning inner gloves prior to gown and outer gloves after donning gown to maintain clean inner gloves for the removal and Journal Preproof 0cdisposal of the rest of the PPE Two universally agreed upon maneuvers include stopping ventilation prior to entrance into the airway and holding ventilation until after the tracheostomy tube cuff has been inflated Givi and colleagues suggest that a smaller tracheotomy cuffed may be preferred to decrease the spread of aerosolized particles Miles discusses the New York experience suggesting that for intubated patients the cuff pressure should be checked every hours with a goal of cm H2O greater pressure predisposes tracheal pressure necrosis33 The group also suggests delaying the timing of tracheostomy until days after onset of symptoms when feasible Finally some have advocated for the use of specific air containment setups including plastic draping smoke evacuator tubing or specifically designed negative pressure box The field of laryngology has had to undergo significant change in the setting of the COVID pandemic As the numbers of COVID19 patients have continued to increase during the month of June it is clear that practice of laryngology in the postCOVID era will need to be carefully ramped up to protect patients and providers alike Additionally one would expect a continued increase in the number of recovered patients being seen for sequelae of prolonged intubation Decisions to relax restrictions on flexible laryngoscopy and other AGPs will depend on the local incidence of COVID19 infection availability and accuracy of preprocedure testing sustainable supply of PPE the ability to properly sanitize rooms and ultimately development of an effective vaccine Head and Neck Surgical Oncology Journal Preproof 0cSimilar to laryngology the approach to head and neck surgical oncology continues to evolve as more information becomes available during the COVID era During the early weeks of the pandemic the aspect of cancer care most concerning to patients and providers involved potential delays in therapy Finley suggests that delaying cancer surgery should be done with extreme caution despite COVID1937 Additionally delays beyond weeks could significantly affect longterm outcomes and morbidity of treatment Among patients diagnosed with severe COVID19 requiring ICU admission patients with cancer deteriorated faster than noncancer patients Desai and colleagues discovered a higher risk of severe events in patients recently treated with chemotherapy or surgery in the past days compared to noncancer COVID16 patients38 Additionally patients with advanced stage cancer tended to have a higher rate of severe events when compared to early stage cancer Cancer patients undergoing active treatment are predisposed to COVID19 related complications and critically ill patients with cancer have a higher predisposition to death Head and neck cancer patients especially are considered a highrisk population for complications associated with COVID19 infection8 making safe coordination of care difficult but imperative Head and neck cancer patients are an atrisk group for a number of reasons Silverman points out that head and neck cancer patients tend to present with advanced age history of tobacco and alcohol abuse and cardiac and pulmonary comorbidities which are similarly found in COVID19 Risk of respiratory sequelae in patients who have received chemotherapy andor radiation therapy are high with increased rates of dysphagia aspiration and pneumonia Additionally head and neck cancer patients have an increased risk of respiratory infections and aspiration pneumonitis These factors may expedite deterioration to Journal Preproof 0csevere adverse events in patients with COVID19 Additionally head and neck patients who are actively receiving chemotherapy or immunotherapy may have depressed immune function malnutrition and older age For this reason the patients need to be carefully selected and comorbidities strongly considered when constructing a treatment plan for patients with head and neck cancer Within the United States mortality for patients of color African American and Latinx with COVID19 is significantly higher than for Caucasian patients40 Unfortunately this is a consequence of inequality within society and the healthcare system rather than a biological or pathological difference41 Correspondingly these communities also tend to present with more advanced disease and have significantly worse mortality compared to their Caucasian fellow citizens This pandemic has laid to bear some of the gross inequities within the American health care system and highlighted the need for equitable decision making for all patients with a diagnosis of head and neck cancer during the COVID19 era Another consideration for the head and neck cancer patient during the COVID19 era may include the financial burden and cost of survivorship associated with undergoing cancer treatment and financial hardship related to COVID19™s effect on the world economy and increasing levels of unemployment Given the significant job losses across the United States there is preliminary data to suggest that there will be at least million newly unemployed people who will also lose their insurance coverage in the wake of the pandemic Increased financial strain has been associated with decreased quality of life scores and subsequently mortality in head and neck cancer patients Journal Preproof 0c Recommendations for head and neck clinic are similar to what was previously discussed for laryngology Providers are expected to take universal precautions regardless of the patient™s COVID status Flexible fiberoptic laryngoscopy is considered a highrisk aerosol generating procedure Due to this laryngoscopy should be reserved for instances in which it is likely to change management One of the beneficial consequences of the COVID19 era is the increased access of care through the widespread adoption of telehealth clinics among most hospitals49 Providers may use telemedicine as an initial preoperative assessment or prescreen for patients that will later be seen in clinic or prior to surgery While telehealth is wonderful for obtaining a detailed history reviewing dataimaginglabs and discussing surgical optionsrisksbenefits a big drawback is the inability to perform a comprehensive head and neck physical exam Physical examination with or without fiberoptic laryngoscopy is important to define the extent of tumor and formulating an ablative and reconstructive plan Fortunately some workarounds include anatomic and physiologic imaging for ablative planning and CT angiography and virtual planning sessions for microvascular reconstruction Telemedicine also serves a vital role in triage of posttreatment head and neck cancer patients who may not be able to be seen as frequently due to the pandemic Telemedicine also serves a vital role in the coordination of care between multiple oncologic disciplines Dharmarajan and colleagues highlighted the University of Pittsburgh approach to a virtual multidisciplinary tumor board clinic MDC This strategy has been adopted by multiple institutions and works quite well to coordinate care between specialties In their study they found that of virtual tumor board participants preferred virtual multidisciplinary clinic to Journal Preproof 0cthe inperson format Additionally about of participants indicated that they would prefer to continue the virtual multidisciplinary format once in person meeting restrictions had been lifted Through multiple virtual meeting applications practitioners can share imaging and laryngoscopy which may assist with decision making for patients Similar to the guidelines published for laryngeal surgery the American Academy of Otolaryngology has published recommendations for ramping up clinical volume as it relates to triage for head and neck surgical oncology Setzen et al note that most head and neck cases fall within the urgent category The guidelines list emergent procedures as being tumor obstructing airway significant bleeding acute or impending neurological change and salivary gland or deep neck abscesses Urgent procedures within days include all head and neck squamous cell carcinomas of the upper aerodigestive tract benign tumors with impending complications or morbidity anaplastic thyroid cancer medullary thyroid cancer bulky differentiated thyroid cancer with regionaldistant metastasis locally aggressive or large nodules 4cm Bethesda high grade salivary malignancies skin cancers and parathyroid carcinomas with significant systemic effects Time sensitive procedures include lowrisk differentiated thyroid cancer lowgrade salivary neoplasms and slower growing basal cell carcinomas in favorable locations Routine procedures include benign thyroid pathology parathyroid disease without significant systemic effects benign salivary lesions and low risk skin cancers and posttreatment disorders Ranasinghe and colleagues recommend a tiered approach to surgical triage with more aggressive pathology being prioritized in a similar fashion to the AAO guidelines Similarly the review recommends considering alternatives to longduration microvascular reconstructive cases It is recommended that the focus shift to simplifying reconstruction and Journal Preproof 0creducing surgical duration when it™s feasible and appropriate However it is also acknowledged that this approach may lead to an increase in downstream complications Endocrine surgery is similarly tiered in a memo by Ashok Shaha which outlines a strategic approach to thyroid surgery during the pandemic Similar to other strategies anaplastic thyroid cancer medullary thyroid cancer and locally aggressive differentiated thyroid cancer specifically with impending concern for airway obstruction take precedent However some alternatives are also discussed for instance in patients with BRAF V600E mutations who may have surgery delayed while being treated with appropriate targeted therapies Additionally deescalation of surgical care is advocated for benign conditions like thyroid goiters that are nonobstructive and even papillary thyroid microcarcinoma which may be followed with serial ultrasonography until resource allocation has returned to preCOVID levels As institutions attempt to weigh the pros and cons of elective and essential surgery in the midst of the pandemic some authors have advocated for creating rating systems to allow for appropriate surgical triage during periods of limited clinical output and resource reallocation The medically necessary timesensitive MeNTS procedures scoring system aims to œethically and efficiently manage resource reallocation and risk to healthcare providers during the COVID19 pandemic51 The scoring system which uses procedural disease and patient factors within a 5point Likert scale to determine the potential risk of proceeding with surgery The cumulative MeNTS score may range between and with score above being considered within the high risk or resource heavy procedures either due to patient factors AgeComorbidities or procedure factors head and neck surgical site high anticipated blood loss ICU admission requirement Using scoring systems like MeNTS should help hospitals more Journal Preproof 0cappropriately and objectively triage elective and essential surgeries in the setting of a resurgence of caseslimiting of resources Given the significant lack of available knowledge regarding SARSCoV2 and its associated complications it is difficult to characterize risk for patients undergoing ablative and reconstructive head and neck surgery As mentioned earlier in asymptomatic COVID19 positive patients undergoing elective surgery mortality approached COVID19 has demonstrated myriad manifestations which might interfere with the success and management of patients undergoing head and neck surgery Tang demonstrated that coagulopathy was more common in patients with severe disease and nonsurviving COVID patients52 In this study Ddimer fibrin

neutrophils account for “ of circulating leukocytes and are the first immune cells recruitedto an ‚ammatory site they play an important role in the innate immune response topathogens as patients with neutropenia are highly susceptible to bacterial and fungal infections neutrophils perform numerous functions that target microbes including phagocytosis the releaseof antimicrobial peptidesproteases and netosis interestingly neutrophils have garneredconsiderable interest for their emerging and prominent roles in modulating cancer growth andmetastatic progression the roles played by neutrophils in the cancer setting are diverse andcomplex leading to the concept of neutrophil heterogeneityplasticity and the notion that distinctneutrophil subsets might existgranulopoiesisdiï¬erentiationand mobilization of maturefrom the bone marrow intocirculation this process begins with the commitment of granulocytemonocyte myeloidsegmented neutrophilsregulatedprocessthatinvolvestheisatightlyedited bybrahm segaluniversity at buffalo united statesreviewed byye liuniversity of texas md andersoncancer center united statesconnie jackamancurtin university australiacorrespondencepeter m siegelpetersiegelmcgillcaspecialty sectionthis was submitted tocancer immunity and immunotherapya section of the frontiers in immunologyreceived may accepted july published august citationhsu be shen y and siegel pm neutrophils orchestrators of themalignant phenotypefront immunol 103389fimmu202001778frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypegmps which progressthrough a series ofprogenitorsneutrophil progenitors myeloblast promyelocyte myelocytemetamyelocyte band cell untilthey become a matureneutrophil in cancer dysregulated granulopoiesis hasled to theidentification of diï¬erent neutrophil subsets that play a role intumor progression preneus comprise a neutrophil precursorpopulation that retain their proliferative capacity and expandin the bone marrow and spleen of tumor bearing mice preneus diï¬erentiate into immature and mature neutrophilswith the former found to accumulate in growing tumors anearly stage committed unipotent neutrophil precursor nep hasalso been identified and their adoptive transfer into humanizedmice promoted solid tumor growth by inhibiting t cellactivation two neutrophil subsets highdensity neutrophilshdns and lowdensity neutrophils ldns were identified invarious tumor models by diï¬erential density centrifugation hdns represent mature segmented neutrophils whereas ldnscomprise a heterogeneous mixture of mature and immatureneutrophils increasing mobilization of ldns into theperipheral blood was associated with enhanced tumor growthand metastasis “functionsand antitumorigenicin addition to the identification of distinct neutrophil subsetsneutrophils exhibit plasticity in response to tumorderivedfactors in a manner similar to macrophages neutrophils havebeen classified into two categories n1 and n2 to describetheir prorespectively in vivo evidence has shown that tumorassociatedneutrophils tans can change their function from a protumor phenotype n2 to an antitumor n1 phenotypewith the addition of a tgf inhibitor arguing that tgfis an important factor driving the n2 phenotype incontrast signals associated with an antitumor n1 phenotypeinclude type iinterferons and those propagated by themet receptor however this categorization is likelyto represent an oversimplification of neutrophil diversityneutrophil polarization similar to macrophages could alsorepresent a continuum of diï¬erent neutrophil phenotypespresent in the tumor microenvironment these advancesregarding the degree of neutrophil heterogeneityplasticityobserved in the cancer setting have sparked an intense andrenewed interestin this cell population while there areongoing discussions in the field regarding the relationshipsubsets we directbetween pmnmdscs and neutrophilto excellentthe readerthatfully discussthese we will briefly discuss antitumorrelationshipsneutrophilreview will primarilyroles of neutrophils and neutrophildiscussassociated functionsgrowth andmetastatic progressionfunctions howeverin promotingthe recentreviewstumorthisantitumor neutrophil functionscan participateantitumorneutrophilsmechanisms thattumor growth or eliminate cancercells a wellstudied neutrophilassociated function isin a variety oflimittheir ability to generate reactive oxygen species ros tolimit tumor progression upon tumor cell contact mousederived neutrophils can release hydrogen peroxide to eliminatemetastatic cancer cells in vitro subsequentlyit wasdemonstrated that expression of trpm2 transient receptorpotential cation channel subfamily m2 on tumor cells increasedtheirsensitivity to neutrophilmediated h2o2dependentcytotoxicity this occurred through a mechanism that involveda transient increase in ca2 mobilization within cancer cells trpm2 upregulation in tumor cells occurred followingan epithelialtomesenchymaltransition emt and cancercells that have undergone an emt were more susceptible toneutrophilmediated killing more recently an interactionbetween the receptor for advanced glycation end productsrage which is expressed on tumor cells and cathepsin gpresent on murine neutrophils was shown to mediate in vitrotumor cell cytotoxicity in a h2o2dependent manner the release of neutrophil ros is also dependent on the tumormicroenvironment in hypoxic tumor microenvironments theability of murine neutrophils to kill tumor cells in vivo throughthe release of ros is greatly diminished thus neutrophilshave the capacity to mediate rosdependent direct tumorcell killingcausingthe interplay of neutrophils with otherimmune celltypes can also indirectly limittumor progression tumorassociated neutrophils suppress the protumorigenic role ofil17 secreting Îδ t cells by inhibiting their proliferationlow glutathione levels in Îδ17 t cells rendered them sensitiveto neutrophilderived rosenhanced oxidativestress and reduced proliferation in earlystage humanlung cancer a subset ofimmature neutrophils have beenidentified as having antigenpresenting functions and act topromote antitumor immunity by stimulating the secretionofinaddition to neutrophilt cellinteractions communicationbetween neutrophils and monocytes can also elicit antitumoreï¬ects nonmetastaticifnÎproducing monocytes to the lungs ifnÎ release activatestmem173sting within neutrophils whichstimulatesneutrophilmediated killing of disseminated cancer cells in thelungs ‚ammatory cytokinesfrom t lymphocytescan mobilizecancercellsneutrophils have been shown to ltrate deposits of prostatecancer cells within bone metastases importantly neutrophilsimpaired bone metastasis progression by inhibiting stat5signal transducer and activator of transcription functionwithin prostate cancer cells resulting in their apoptotic cell death recently neutrophils have been reported to be involvedin antibodymediated trogocytosis a process that mechanicallydisrupts the plasma membrane of antibodyopsinized cancercells leading to a lyticnecrotictype cell death iga antibodiesagainst receptors expressed by cancer cells her2 egfr couldenhance neutrophilmediated trogocytosis of cancer cells if thecd47sirpα innate immune cell checkpoint was simultaneouslyblocked taken together these results demonstrate thatneutrophils can impair tumor growth and metastasis using acombination of direct and indirect cancer cell killing mechanismssupplementary table frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure neutrophil functions that promote tumor growth and metastasis to support primary tumor growth neutrophils can mediate t cell suppression and altermacrophage differentiation neutrophil release of timp1 enhances tumor cell invasion by inducing epithelialtomesenchymal transition once in circulation circulatingtumor cells interact with neutrophils which enables tumor cell proliferation secretion of various pro‚ammatory markers such as il8 il1 or mmps can mediateincreased tumor cell extravasation in addition neutrophils can inhibit intraluminal nkmediated killing of circulating cancer cells leading to increased extravasation atthe metastatic site various systemic and microenvironmental factors can promote neutrophil ltration neutrophils can awaken dormant cancer cells by promotingecm remodeling and angiogenesis lastly continued growth of the metastatic lesion is facilitated by key neutrophildependent mechanisms which includeangiogenesis proliferation immune suppression and immune exclusion csf1 colony stimulating factor timp1 tissue inhibitor of matrix metalloprotease pdl1programmed death ligand tgf transforming growth factor ros reactive oxygen species mmp matrix metalloproteinases gmcsf granulocyte macrophagecolony stimulating factor angptl2 angiopoetin like2 fgf2 fibroblast growth factor ltb4 leukotriene b4 inos inducible nitric oxide synthase net neutrophilextracellular trap caf cancerassociated fibroblastneutrophil functions thatpromote primary tumor growthneutrophils promote primary tumor growth by variousmechanisms figure netosis is a process that involvesthe extrusion of neutrophilderived chromatin structures thatare decorated with neutrophil granule constituents whichform extracellular structures called neutrophil extracellulartraps nets normally netosis and net productionhave been described in the context of a neutrophil™s ability tocapture and kill bacteria extracellularly however netshave been shown to play an important role in the growth of aprimary tumor tumor microenvironmental changes includingtumorassociated coagulation and enhanced thrombosishave been linked to enhanced tumor growth several recentstudies suggest that netosis may play an important role inthese processes lps stimulation was shown to increase c3arexpression within neutrophils enhance netosis and increasecoagulation these events were correlated with n2 neutrophilpolarization and increased tumor growth interestingly ithas recently been shown that immature neutrophils preferentiallyrespond to cancer cell derived c3a to promote their migration subsequently it was shown that breast cancer cells thatexpressed high levels of gcsf and il1 exhibited highneutrophil counts and tumorassociated thrombosis which wasdependent on net formation pharmacological blockadefrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeof il1 receptor signaling reduced net formation attenuatedtumorassociated thrombosis and impaired tumor growth nets can also directly ‚uence cancer cell proliferationneutrophil elastase ne present within nets activates tumorcells to increase mitochondria biogenesis and atp productionthereby further enhancing the growth of cancer cells in addition to the impact of nets neutrophils canalso interact with other immune cells through additionalmechanisms to promote tumor growth neutrophilderived roscan inhibit t cell proliferation creating an immunosuppressiveenvironment that is supportive of tumor growth phenotypiccharacterization and singlecell rna sequencing identified aneutrophil subset that is cd84hi which exhibited potent t cellsuppressive activity and increased ros production in amodel of gastric cancer neutrophils were activated by tumorderived gmcsf that resulted in elevated programmed deathligand pdl1 expression these pdl1 neutrophils wereable to suppress t cell function and promote tumor growth secretion of mmp9 matrix metalloproteinase fromltrating neutrophils activates latent tgf and induces tcell suppression and tumor growth in a colorectal cancer model siglecfhigh neutrophils in lung adenocarcinoma createdan immunosuppressive environment by promoting macrophagediï¬erentiation causing the release of high levels of rosand enabling tumor progression together these findingsindicate that neutrophils that ltrate diverse primary tumorscan modify the local environment in diï¬erent ways to favortumor growthneutrophil functions thatpromote metastasisthe ability of cancer cells to leave the primary tumor anddisseminate to distant ans represents the deadliest aspectof cancer progression indeed the emergence of metastaticcancer accounts for ˆ¼ of cancer related deaths themetastatic cascade represents a series of barriers to cancercells and neutrophils have been found to assist cancer cells insuccessfully navigating several of these distinct steps figure supplementary table local invasionintravasationltrating neutrophils within primary tumors are associatedwith an increase in emt enhanced metastasis and pooroutcomes mechanisticallyof matrixmetalloprotease timp1 secreted by neutrophils induced anemt and consequently increased the migration and invasion oftumor cells cancer cells that had undergone an emt expressedcd90 which enhanced timp1 secretion by neutrophils in acontactdependent manner inhibitortissuesurvival in circulationextravasationthe ability of circulating tumor cells ctcsto surviveis criticalfor metastasis formation the formation ofheterotypic cancer cell”neutrophil clusters was found to greatlyincrease metastatic fitness using a 4t1 breast cancer modelit was demonstrated that ctcneutrophil interactions reliedon vcam1 dependent adhesion which enhanced cancercell proliferation and increased metastasis indirectlyneutrophils can also inhibit nk cellmediated tumor clearancein circulation thereby increasing the intraluminal survival ofdisseminated tumor cells in this study 4t1 breast cancer cellswere injected subcutaneously to mobilize murine neutrophilsly6gfollowing which d2a1 breast cancer cells wereinjected intravenously mice bearing 4t1 cells exhibited reducedclearance of d2a1 cells from the lungs when compared to micethat were not injected with 4t1 cells depletion of nk cellsresulted in enhanced d2a1 cancer cell accumulation in the lungswhile neutrophil depletion had the opposite eï¬ect cancer cells that have survived in circulation must exitthe bloodstream and extravasate into tissue parenchyma neutrophils have been shown to regulate the extravasationprocessthrough several mechanisms neutrophilderivedfactors can diminish the integrity of the endothelial barrierpermitting cancer cellsil8il1 and matrix metalloproteasesmmp8 and mmp9released from neutrophils activated endothelial cells reducedendothelialtransendothelialmigration and accelerated the rate of cancer cell extravasation to extravasate more easilyincreasedfunctionbarriersites“netosis and net constituents can support cancer cellextravasation through enhanced trapping of ctcs withinmetastaticimportantly blocking netosisdecreases cancer cell adhesion and inhibits metastatic spread tothe lung and liver furthermore changes within specificmetastatic microenvironments such as exposure to ozoneor redox imbalance triggered netosis and led to increasedentrapment of cancer cells in the lung and enhanced metastasis collectively these studies show that neutrophils play animportant role in enhancing tumor cell survival and increasedextravasation which promote cancer metastasisrecruitmentearly seedingsurvivalsystemic and tumorderived factors have been implicatedin neutrophilin the premetastatic nichetumorderived il1 induces Îδ t cells to produce il17aand granulocytecolony stimulating factor gcsf whichresults in the recruitment of immunosuppressive neutrophilsto the lung gmcsf and il5 have been shown topromote the expansion and recruitment of prometastaticneutrophils in the lungs of obese mice which promotes lungmetastasis angiopoetinlike2 angptl2 secreted byosteosarcoma cells implanted in the tibia stimulates lungepithelial cells which led to the accumulation of neutrophilsin the lung and enhanced lung metastatic burden in the lung neutrophils secrete ltb4increases theinitiating cellsproliferation of ltb4rpositive metastasis activation of notch1 in colorectalcellsdrives tgf2dependent recruitment of immunosuppressiveneutrophils within the liver which enabled the formation of livermetastases cancerthatnets also support early cancer cell seeding and colonizationof metastases induction of nets by ovarian tumorderivedfactors has been shown to be important in promoting metastasisfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeto the omentum in the liver nets have also beenshown to promote metastasis by activating cancerassociatedfibroblasts growth in the metastatic siteneutrophils have been shown to promote the growth ofmetastases after seeding minor subclones of breast cancer cellsthat secrete il11 and figf cfosinduced growth factor cansupport the formation of polyclonal metastases composed ofdriver and passenger subpopulations these il11 producingsubclones activated il11 responsive mesenchymal stromal cellswhich induced chemokine secretion and subsequent recruitmentof prometastatic neutrophils tumor cellderived gmcsfwas shown to stimulate neutrophils to synthesize and secretetransferrin an iron transport protein which has mitogenicactivity that promotes lung metastatic growth when taken up bycancer cells a recurring function of prometastatic neutrophils is theirability to create an immunosuppressive microenvironmentthat support metastasis within lung metastasesinduciblenitric oxide synthase inos producing neutrophils havebeen shown to limit cd8 t cell dependent antitumorresponses by promoting immune suppression recently p53deficient cancer cells were found to increase the expressionof wnt ligands which in turn upregulated il1 productionfrom tumorassociated macrophages high il1 levelsengaged Îδ17 t cells which subsequently enhanced neutrophilrecruitment that promoted the formation of lung metastases furthermore loss of elf5 e74like transcription factorexpression in triplenegative breast cancer led to increased ifnÎ signaling resulting in the expansion of immunosuppressiveneutrophils in addition to tumorderived factors a lackof systemic testosterone levels can lead to an impairmentof antitumor neutrophil functions a shift toward immatureneutrophils was observed in castrated male mice leading toincreased neutrophilderived ros and suppression of nk cellactivation that promoted increased lung metastatic burden intwo melanoma models recently a role for net formationhas been described for the continued growth of establishedmetastases nets released during cancer progressionwas shown to limitthe ability of nk and cytotoxic tcells to eliminate cancer cells specifically net formationimpaired direct contact between the cancer cells and cytotoxicimmune cells nk and t cells inhibition of netosis with aprotein arginine deiminase pad4 inhibitor synergized withimmune checkpoint inhibitors to control tumor growth andmetastasis proangiogenic functions have long been ascribed forneutrophils which revealed that neutrophilderived proteasessuch as mmp9 could release stored angiogenic factors vegffgfs that were stored in the local environment to enable bloodvessel formation recently a diï¬erent mechanism bywhich neutrophils enhance angiogenesis has been describedthe synthesis and secretion of fibroblast growth factor fgf2 by neutrophils in the liver microenvironment drivesangiogenesis and growth of nascent colorectal cancerderivedhepatic metastases dormantresidual disease andtherapy resistanceneutrophils have also been implicated in awakening dormantcancer cells lpsinduced tissue ‚ammation led to metastaticoutgrowth of dormant tumor cells in a neutrophildependentmanner mmp9 produced by neutrophils can trigger thegrowth of dormant cancer cells by remodeling extracellularmatrix and releasing potent angiogenic factors ne andmmp9 which are enzymes associated with nets can cleavethe extracellular matrix ecm leading to integrinmediatedsignaling which awakens dormant cancer cells and promotescancer cell growth severalstudies have shown that neutrophils promoteresistance to therapy doxorubicin and paclitaxel resistant breastcancer cells express more il17 and cxcr2 ligands whichincreases neutrophil recruitment a neutrophilenrichedsubtype characterized in triple negative breast cancer tnbcdetermined that neutrophils were largely immunosuppressiverendering these tumors resistant to immune checkpoint blockadetherapy in a genetically engineered mouse model ofsarcoma neutrophils promote resistance to radiation therapyby activating mitogenactivated protein kinase mapk pathway in addition cd177 neutrophil ltrates in colorectalcancer patients are associated with adverse outcome in patientsreceiving bevacizumab [antivascular endothelial growth factora vegf a] furthermore lysyl oxidaselike loxl4expressing neutrophils that ltrated colorectal cancer livermetastases were found to identify patients that were resistant toantiangiogenic therapy metabolic programming inneutrophilsrecentinteresthasbeenconceptin thethereofimmunometabolism and the realization that altered cellularmetabolism in ltrating immune cells can have a significantimpact on tumor growth and metastasis neutrophilsare typically viewed as a cell type that is heavily reliant onglycolysis to perform their eï¬ector functions consistentwith this notion neutrophils have very few mitochondria andinhibitors of oxidative phosphorylation oxphos do notalter their rates of oxygen consumption howeverduring tumor progression neutrophils have been shown toundergo a metabolic switch which involves the upregulationof genes associated with oxphos fatty acid metabolism andglycolysis figure neutrophils isolated from lewis lungcarcinoma exhibit increased flux through oxphos glycolysisand increased atp production compared to naïve neutrophilssuggesting that multiple metabolic strategies are engaged intumor ltrating neutrophils recently upregulation offatp2 fatty acid transport protein in neutrophils was shownto increase lipid accumulation in these cells fatp2 regulated theuptake of arachidonic acid which was subsequently convertedto prostaglandin e2 neutrophilderived prostaglandin e2 wasfrontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypefigure metabolic changes in cancerassociated neutrophils neutrophils which possess few mitochondria are reliant on glycolysis to generate atp to fueleffector functions such as phagocytosis generation of reactive oxygen species and netosis in cancer neutrophils upregulate oxidative phosphorylation oxphosand fatty acid transporters to mediate many neutrophil functions including migration and t cell suppression under nutrient limiting conditions such as low glucoseneutrophils can reprogram their metabolism to break down fatty acids or utilize certain amino acids glutamate proline to fuel protumorigenicprometastaticfunctions ppp pentose phosphate pathway glut glucose transporter mct monocarboxylate transporter tca tricarboxylic acid cycle fatp2 fatty acidtransport protein aa arachidonic acid pge2 prostaglandin e2found to be important or neutrophilmediated cd8 t cellsuppression and tumor growth metabolic flexibility refers to the ability of a cell to shiftbetween one metabolic program to another in response tochanging metabolic demands or nutrient supply high metabolicflexibility increases the cell™s ability to survive various andeverchanging metabolic microenvironments neutrophilsubpopulations can also exhibit metabolic flexibility figure in breast cancer splenic neutrophils can engage mitochondrialdependent fatty acid oxidation as a predominate fuel sourceto support ros production and maintain t cell suppression under glucoselimiting conditions similar to certain tumormicroenvironments immature ldns have been shown to utilizeoxphos to generate atp that is required to support theirprotumorigenic functions indeed immature ldns can supportnetosis under nutrient limiting conditions via mitochondrialdependent amino acid catabolism which is importantforefficient breast cancer liver metastasis in addition thelongevity of neutrophils could also be altered due to the enhancedmetabolic flexibility the ex vivo halflife of mouse circulatinghdns and ldns was and h respectively suchobservations raise the intriguing possibility that under certainconditions distinct neutrophil subsets may not be as shortlived as previously thought these studies argue that increasedmetabolic flexibility in distinct neutrophil populations may beimportant for cellular functions that can ‚uence tumor growthand metastatic progressionclinical importance futureperspectives on treatmentin keeping with their protumorigenicmetastatic functions thepresence of neutrophils across diï¬erent cancers was shownto be strongly associated with adverse patient outcomes frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotypeamong certain subtypes of breast cancer er the presence ofa neutrophil ltrate in the primary tumor is also indicativeof worse patient outcomes furthermore in patients withadvanced cancers serum il8 levels and neutrophil ltrationare associated with worse overall survival and diminishedresponse to immune checkpoint inhibitors the mobilization of neutrophils into circulation also hasprognostic significance the neutrophiltolymphocyte rationlr is an important risk stratification and treatment selectiondiagnostic tool for cancer patients a high nlr is associated withpoor prognosis in many solid human cancers “ a highnlr is also associated with decreased overall survival in patientswith tnbc or metastatic breast cancer an important and unanswered question with respect to thenlr is the type of neutrophil that is being detected in thesepatients are they high or lowdensity neutrophils interestinglyldns have been identified in patients with breast cancer lungcancer head and neck cancers urologic cancers and lymphoma “ in patients with advanced lung cancerit wasreported that higher proportion of ldns predictedpoorer survival these observations are in keeping withthe protumorigenic and prometastatic functions associatedwith ldnn2 neutrophils while most studies reveal a negativeprognostic impact of neutrophils in cancer there was one studythat associated the presence of a cd16high cd62dim neutrophilsubset with increased survival of head and neck squamous cellcarcinoma patients these observations highlight the needfor better markers that are capable of discriminating betweenneutrophils that exert antitumor vs those that mediate protumormetastatic eï¬ectsmechanistic insights have greatly advanced our knowledgeoftumorderived factorstumor growth andmetastasis in a neutrophildependent manner additional studiesimpactthatreferences sipsas nv bodey gp kontoyiannis dp perspectives for the management offebrile neutropenic patients with cancer in the 21st century cancer “ 101002cncr20890 kolaczkowska e kubes p neutrophil recruitment and function in healthand ‚ammation nat rev immunol “ 101038nri3399inde visser ke neutrophilssb wellenstein md coï¬eltcancer neutral no more nat rev cancer 101038nrc201652 “ cowland jb borregaard n granulopoiesis and granules of humanneutrophils immunol rev “ 101111imr12440 evrard m kwok iwh chong sz teng kww becht e chenbone marrow neutrophilstraffickinganalysisspecializedpopulationsexpansioninofal developmentaljetrevealsand 101016jimmuni201802002functionseï¬ectorimmunity“79e8 zhu yp padgett l dinh hq marcovecchio p blatchley a wu r identification of an early unipotent neutrophil progenitor with pro tumoralactivity in mouse and human bone marrow cell rep “41e8 101016jcelrep201807097 sagiv jy michaeli j assi s mishalian i kisos h levy l phenotypicdiversity and plasticity in circulating neutrophil subpopulations in cancercell rep “ 101016jcelrep201412039focused on characterizing the phenotypic and functional role ofneutrophils in cancer it may be possible to develop strategies thatspecifically target those neutrophil subsets that actively promotetumor growth and metastasis while sparing those neutrophilsthat possess antitumor and antimicrobial functions finallythe emerging concept of metabolic flexibility that is exhibited bycertain neutrophil subsets may aï¬ord new ways of targeting theseprotumorigenicmetastatic neutrophilsauthor contributionsbh ys and ps wrote the review and prepared the figuresall authors contributed to the and approved thesubmitted versionfundingwork from the authors laboratory cited in this review wassupported by an operating grant to ps from the cancer researchsociety and the terry fox research institute and québecbreast cancer foundation grant bh acknowledgessupport from the charlotte and leo karassik foundation phdfellowship and the rolande and marcel gosselin graduatestudentship ys holds an entrance studentship from thegoodman cancer research centre ps is a mcgill universitywilliam dawson scholarsupplementary materialthe supplementary materialfor this can be foundonline at httpswwwfrontiersins103389fimmu202001778fullsupplementarymaterial coï¬elt sb kersten k doornebal cw weiden j vrijland k hau cs il17producing gammadelta t cells and neutrophils conspireto promote breast“ 101038nature14282cancer metastasis nature hsu be tabaries s johnson rm andrzejewski s senecal j lehuede c immature lowdensity neutrophils exhibit metabolic flexibility thatfacilitates breast cancer liver metastasis cell rep “15e6 101016jcelrep201905091 fridlender zg sun j kim s kapoor v cheng g ling l polarizationof tumorassociated neutrophil phenotype by tgfbeta œn1 versus œn2tan cancer cell “ 101016jccr200906017 ohms m möller s laskay t an attempt to polarize human neutrophilstoward n1 and n2 phenotypes in vitro front immunol 103389fimmu202000532jablonska j leschner s westphal k lienenklaus s weiss s neutrophilsresponsive to endogenous ifnbeta regulate tumor angiogenesis andgrowth in a mouse tumor model j clin invest “ 101172jci37223 finisguerra v di conza g di matteo m serneels j costa s thompsonaa met is required for the recruitment of antitumoural neutrophilsnature “ 101038nature14407 ostuni r kratochvill f murray pj natoli g macrophages and cancer frommechanisms to therapeutic implications trends immunol “ 101016jit201502004frontiers in immunology wwwfrontiersinaugust volume 0chsu neutrophils orchestrating the malignant phenotype brandau s moses k lang s the kinship of neutrophils and granulocyticmyeloidderived suppressor cells in cancer cousins siblings or twins semincancer biol “ 101016jsemcancer201302007 vols s sionov rv granot z always look on the bright side antitumor functions of neutrophils curr pharmac design “ granot z henke e comen ea king ta norton l benezra r tumorentrained neutrophils inhibit seeding in the premetastatic lung cancer cell “ 101016jccr201108012 gershkovitz m caspi y fainsodlevi t katz b michaeli j khawaled s trpm2 mediates neutrophil killing of disseminated tumor cells cancer res “ 10115800085472can173614 gershkovitz m fainsodlevi t khawaled s shaul me sionov rvcohendaniel l microenvironmental cues determine tumor cellsusceptibility to neutrophil cytotoxicity cancer res “ 10115800085472can180540 sionov rv fainsodlevi t zelter t polyansky l pham ct granotz neutrophil cathepsin g and tumor cell rage facilitate neutrophilanti8e1624129 1010802162402x20191624129cytotoxicity oncoimmunologytumor mahiddine k blaisdell a ma s crequergrandhomme a lowell caerlebacher a relief of tumor hypoxia unleashes the tumoricidal potentialof neutrophils j clin invest “ 101172jci130952 mensurado s rei m lanca t ioannou m goncalvessousa n kubo h etal tumorassociated neutrophils suppress protumoral il17 gammadeltat cells through induction of oxidative stress plos biol 16e2004990 101371 pbio2004990 singhal s bhojnagarwala ps o™brien s moon ek garfall al rao as etal origin and role of a subset of tumorassociated neutrophils with antigenpresenting cell features in earlystage human lung cancer cancer cell “ 101016jccell201606001et hagerling c gonzalez h salari k wang cy lin c roblescooperationtumor prevents metastatic progressionaliinduced byof breast cancer proc natl acad sci usa 101073pnas1907660116eï¬ector monocyteneutrophilimmunethe primary “ costanzogarvey dl keeley t case aj watson gf alsamraae myu y neutrophils are mediators of metastatic p

Millions of people are suffering from cancers but accurate early diagnosis and effectivetreatment are stilllong noncoding RNAslncRNAs have been proven to play an important role in diseases especially cancersThese lncRNAs execute their functions by regulating gene expression Thereforeidentifying lncRNAs which are related to cancers could help researchers gain a deeperunderstanding of cancer mechanisms and help them find treatment options A largenumber of relationships between lncRNAs and cancers have been verified by biologicalexperiments which give us a chance to use computational methods to identifycancerrelated lncRNAs In this paper we applied the convolutional neural network CNNto identify cancerrelated lncRNAs by lncRNA™s target genes and their tissue expressionspecificity Since lncRNA regulates target gene expression and it has been reportedto have tissue expression specificity their target genes and expression in differenttissues were used as features of lncRNAs Then the deep belief network DBN wasused to unsupervised encode features of lncRNAs Finally CNN was used to predictcancerrelated lncRNAs based on known relationships between lncRNAs and cancersFor each type of cancer we built a CNN model to predict its related lncRNAs Weidentified more related lncRNAs for kinds of cancers Tencross validation has beenused to prove the performance of our method The results showed that our method isbetter than several previous methods with area under the curve AUC and areaunder the precision“recall curve AUPR To verify the accuracy of our results casestudies have been doneKeywords long noncoding RNA lncRNA cancer convolutional neural network CNN deep belief network DBNmachine learningINTRODUCTIONFour to nine percent of the sequences™ transcription are long noncoding RNAs lncRNAs inmammalian genomes Canzio Ji lncRNA was regarded as the noise ofgenome transcription and did not have biological functions at first However an increasing numberof studies have reported that lncRNA is widely Robinson involved in chromosomeEdited byLei DengCentral South University ChinaReviewed byHao LinUniversity of Electronic Science andTechnology of China ChinaInner Mongolia University ChinaJuan WangCorrespondenceNan Dudunan05aliyuncomGanfeng Xiexiegfaliyuncom These authors share first authorshipSpecialty sectionThis was submitted toMolecular Medicinea section of the journalFrontiers in Cell and DevelopmentalBiologyReceived June Accepted June Published August CitationLiu Z Zhang Y Han X Li C Yang XGao J Xie G and Du N Identifying CancerRelated lncRNAsBased on a Convolutional NeuralNetwork Front Cell Dev Biol 103389fcell202000637Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsgenomicimprintingchromatin modificationsilencingtranscriptional activationinterference andnuclear transport Cheng 2018a Recently it has beenproven to be associated with many kinds of cancerstranscriptionalThe secondary structure spliced form and subcellularlocalization of most lncRNAs are conserved Karner which is very important for lncRNA to execute functionsHowever compared to the functions of microRNAs miRNAsand proteins the function oflncRNA is more difficult todetermine According to the position of lncRNA in the genomerelative to proteincoding genes it can be divided into five typessense antisense bidirectional intronic and intergenicMany researchers have found lncRNAs play an important rolein cancers Avgeris Cheng 2018b Zhao and neurodegenerative diseases Peng and Zhao as other biological molecules Zhang T Bai Cheng 2019a Liang Although manyresearchers have verified many associations between lncRNAsand cancers by biological experiments compared with ourknowledge about diseaserelated genes we still do not knowenough about diseaserelated lncRNAs Considering the timeand money cost of finding diseaserelated lncRNAs more andmore researchers tend to use computational methods to identifydiseaserelated lncRNAs These methods could be divided intothree categories machine learning methods network methodsand other methodsMachine learning methods build models based on thesimilarities of diseases orlncRNAs and their biologicalcharacteristics Cheng Cheng 2019b Zeng Zou Lan developed thelncRNA“disease association prediction LDAP which is amethod based on bagging support vector machine SVM toidentify lncRNA“disease associations They used similarities oflncRNAs and diseases as the features Yu developedcollaborative filtering naive Bayesian classifier CFNBC based onnaive Bayesian They integrated miRNA“lncRNA associationsmiRNA“disease associations and lncRNA“disease associationsto infer more lncRNA“disease associations Considering thediscriminative contributions of the similarity association andinteraction relationships among lncRNAs disease and miRNAsXuan 2019a developed a dual convolutional neuralnetwork CNN with attention mechanisms to predict diseaserelated lncRNAsNetwork methods are the most common way to identifyassociations between diseases and lncRNAs nowadays Gu Yu Zhang J Kuang Wang L Liu Thiskind of method would build one or multiple networks toinfer new information Wang L built a lncRNA“miRNA“disease interactive network and used their novel methodœLDLMD to predict associations between lncRNAs and diseasesSumathipala used a multilevel network topologywhich includes lncRNA“protein protein“protein interactionprotein“disease relationship to use network diï¬usion algorithmto predict diseaserelated lncRNAs The graph convolutionalnetwork GCN and CNN were used on a lncRNA“miRNA“disease network by Xuan 2019b Deng builtlncRNA similarity network disease similarity network miRNAsimilarity network and their associations Then they calculatedthe metapath and feature vector for each lncRNA“disease pair inthe heterogeneous information networkOther methods may borrow the feature extraction methodor similarity conjecture of network methods but the core ofthis method is matrix decomposition or matrix completionLu developed the geometric matrix completionlncRNA“disease association GMCLDA which is a methodbased on geometric matrix completion They calculated diseasesimilarity based on Disease Ontology DO and calculatedthe Gaussian interaction profile kernel similarity for lncRNAsThen they inferred diseaserelated lncRNAs based on theassociation patterns among functionally similar lncRNAs andsimilar diseases Wang Y proposed a weightedmatrix factorization to capture the interintraassociationsbetween diï¬erent types of nodes Then they approximated thelncRNA“disease association matrix using the optimized matricesand weights to predict diseaserelated lncRNAs Localityconstrained linear coding label propagation Latent DirichletAllocation LLCLPLDA was developed by Xie Firstly localconstraint features of lncRNAs and diseases wereextracted by localityconstrained linear coding LLC Thenthey predicted diseaserelated lncRNAs by label propagationLP strategyHowever previous methods did not consider the regulatingtarget gene expression of lncRNA which is an important functionof lncRNA and plays an important role in associations betweenlncRNAs and diseases In addition deep learning methods arean important tool and have shown their power in bioinformaticsChen Lv Wei Wu Zhao 2019abc Therefore in this paper we used thisinformation as features of lncRNA In addition the expressionof lncRNA in diï¬erent tissues were also used as the featuresof lncRNA Then the deep belief network DBN was used toencode and the CNN was used to classifyMETHODSFeature ExtractionTissue Expression Specificity of Long NoncodingRNACompared with proteincoding geneslncRNA shows strongtissue specificity The specificity of lncRNAs in diï¬erent kindsof tissues and cell types has been proven by many biologicalexperiments The diï¬erent expression also plays an importantrole in essential cellular processes Sasaki testedthe expression of lncRNAs in diï¬erent tissues and found lncRNAs exhibited tissuespecific expression and oflncRNAs were only expressed in one discrete tissue Thereforethe expression of lncRNAs in diï¬erent tissues were used asthe featuresWe obtained the expression of lncRNAs in diï¬erenttissues which included adipose adrenal breast colon heartkidney liver lung lymph node ovary placenta prostate testisand thyroidTherefore the dimension of each lncRNA™s expression featureis ˆ— Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsTherefore the dimension of each lncRNA™s target gene featureis ˆ— Deep Belief NetworkThe DBN can eï¬ectively learn complex dependencies betweenvariables Zhao 2019d The DBN contains many layers ofhidden variables which can eï¬ectively learn the internal featurerepresentation of the data and can also be used as an eï¬ectivenonlinear dimensionality reduction methodWhen the observable variables are known the joint posteriorprobabilities of the hidden variables are no longer independentof each other so it is difficult to accurately estimate the posteriorprobabilities of all hidden variables The posterior probability ofearly DBN is generally approximated by Monte Carlo methodbut its efficiency is relatively low which makes its parameterlearning difficult In order to eï¬ectively train the DBN weconvert the sigmoid belief network of each layer to a restrictedBoltzmann machine RBM The advantage of this is that theposterior probabilities of the hidden variables are independentof each other which makes it easy to sample In this way theDBN can be regarded as being stacked from top to bottom bymultiple RBMs and the hidden layer of the Lth RBM is used asthe observable layer of the L 1th RBM Further the DBN canbe trained quickly by layerbylayer training that is starting fromthe bottom layer and training only one layer at a time until thelast layer The specific layerbylayer training process is to trainthe RBM of each layer in turn from bottom to top Assuming wehave trained the RBM in the first L1 layer we can calculate theconditional probability of the bottomup hidden variablesphihiˆ’ σ bi Wihiˆ’where bi is the bias of ith layer of RBM Wi is the connectionweight hi is the ith layer of RBMThe process of training DBN is as followsFIGURE The number of target genes for each long noncoding RNAlncRNAFIGURE The distribution of the number of target genes lncRNA longnoncoding RNAreverseTarget Gene of Long Noncoding RNAQuantitativechainreaction qRTPCR and Western blot were used to testthe diï¬erentexpression genes after knocking down oroverexpressing lncRNAstranscriptasepolymeraseWe obtained target genes of lncRNA from LncRNA2TargetInput train dataset ˆvn learning rate λJiang As we can see in Figure there are kinds of lncRNAsOne lncRNA has more than target genes Then we drawthe distribution of the number of target genes correspondingto lncRNAAsshown in Figure most ofthe target genes arecorresponding to less than five lncRNAs Therefore if we usedthem to be the features of lncRNAs the features would be sparseTherefore we only select the most common target genes to bethe features The genes which are corresponding to more thanfive lncRNAs were selected as the features of lncRNAs There are kinds of genes Then we need to encode these genesF [G1 G2 · · · G45]where G1 denotes the first gene of these genes and F denotesthe feature of lncRNA For each lncRNA if G1 is the target geneof it then G1 otherwise G1 Output weight matrix Wl bias al and blFor l 1LInitialization Wi al bi Sample from train dataset ˆh0For i lˆ’Sample hi based on phi ˆhiˆ’EndSet hi1as the train sample to train lth layer ofRBMEndSince the dimension of expression feature and target genefeature are diï¬erent we should reduce the dimension of targetgene feature and make it the same as the expression feature™sTherefore in this paper two layers of RBM were used to builda DBN modelThe number of nodes oftheand respectively Sigmoid function was used astwo layers was theFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alactivation functionσ x eˆ’xTherefore the dimension of final features is ˆ— F cid20 G1 G2 · · · G13E1 E2 · · · E13 cid21A Method to Identify CancerRelated lncRNAsConvolutional Neural NetworkThe power of CNN in dealing with bioinformatic problems hasbeen proven by many researchers We selected CNN as theclassifier based on two reasons The dimension of features is ˆ— which can be regarded as an image The outstandingperformance of CNN in image classificationThere are five layers in our CNN model The structure of CNNis shown as Table where G1 G2 · · · G13 denotes target gene feature after DBNand E1 E2 · · · E13 denotes the expression of lncRNAs in diï¬erent tissuesTABLE The structure of convolutional neural network CNNLayersParameterConvolutional layerPooling layerConvolutional layerPooling layerFully connected layerOutputFilter kernel size Activation function tanhpool size Activation function tanhFilter kernel size Activation function tanhpool size Activation function tanhUnits Activation function tanhUnits Activation function sigmoidWork FrameFigure shows the work frame of our method œDBN“CNNThere are three steps of our methods Firstly we should extractfeatures of lncRNAs There are two parts of features expressionfeature and target gene feature Then DBN was used to encodethe target gene feature After encoding the two kinds of featureswere combined together Finally CNN was used to classifyRESULTSData DescriptionThe known associations between lncRNA and diseases wereobtained from LncRNADisease database Bao Wetotally obtained kinds of cancerrelated lncRNAs The numberof their corresponding lncRNAs is shown as Figure As shown in Figure People™s understanding of cancerrelated lncRNAs varies widely We have known more than lncRNAs for some cancers but few lncRNAs are known for somecancers To better build our model we only selected cancerswhich have more than related lncRNAs Therefore kindsof cancers were selectedFIGURE Work frame of deep belief network DBN“convolutional neural network CNN lncRNA long noncoding RNAFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsFIGURE The number of long noncoding RNAs lncRNAs for each cancerTABLE The performance of deep belief network DBN“convolutional neuralnetwork CNN in cancersCancerArea undercurve AUCArea under precisioncurve AUPRCervical cancerBreast cancerColorectal cancerStomach cancerUrinary bladder cancerLung cancerOvarian cancerThyroid cancerProstate cancerLiver cancerPancreatic cancerOvarian epithelial cancerGallbladder cancerEndometrial cancerColon cancerEsophageal cancerThetargetgenes oflncRNAs were obtained fromLncRNA2Target database We have discussed about this insection Target Gene of Long Noncoding RNAFIGURE The receiver operating characteristic ROC curves of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisFIGURE The area under the precision“recall curve AUPR of the threemethods DBN deep belief network CNN convolutional neural network PCAprincipal component analysisThe expression oftissues wasobtained from NONCODEV5 Zhao We only usedhuman datalncRNAs in diï¬erentThe Performance of Deep BeliefNetwork“Convolutional Neural NetworkWe did 10cross validation on each cancer Area under the curveAUC Cheng Dao Zhang and areaunder the precision“recall curve AUPR were used to evaluatethe performance of DBN“CNN The results are shown in Table As we can see in Table the performance of DBN“CNN isquite diï¬erent in diï¬erent cancers This may be caused by thediï¬erent sample sizes The average AUC is and AUPR is Comparison ExperimentsTo verify the superior of DBN“CNN we compared it with similarmethods Since the main function of DBN is to reduce dimensionprincipal component analysis PCA has the same functionTherefore instead of using DBN to encode we used PCA thistime and CNN was used to classify the features after PCA We callthis method PCA“CNN In addition we also used the deep neuralnetwork DNN to replace CNN so this comparison method wascalled DBN“DNNWe used these three methods to test on cancers andsummarized the results to get a final AUC and AUPR for eachmethod The receiver operating characteristic ROC curves areshown in Figure As shown in Figure the blue curve denotes the results ofDBN“CNN The red and black curves denote PCA“CNN andDBN“DNN respectively As we can see DBN“CNN performedbest among these three methods The AUC of DBN“CNN is which is better than and for PCA“CNN andDBN“DNN respectivelyFrontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsAs shown in Figure the AUPR of DBN“CNN is the highestwith the least standard errorCase StudyLiu found down syndrome cell adhesion molecule antisense RNA DSCAMAS1 is associated with breast cancerby constructing two suppression subtracted cDNA librariesMartensUzunova reported the associationbetween H19 and bladder cancer They also pointed out that H19could be the biomarker of bladder cancerShi measured the expression level of lncRNAsLoc554202 in breast cancer tissues and found that Loc554202was significantly increased compared with normal control andassociated with advanced pathologic stage and tumor sizeCONCLUSIONSIncreasing evidence has shown the relationship between lncRNAsand cancers lncRNAs could be the biomarkers to help diagnosecancer and also help researchers understand the mechanismof cancers Compared with people™s knowledge of diseaserelated protein coding genes we knew few about diseaserelated lncRNAs However the biological experiments for findingdiseaserelated lncRNAs are timeconsuming and expensiveTherefore in this paper we proposed a novel method foridentifying cancerrelated lncRNAs We called this methodœDBN“CNN which is a fusion of DBN and CNN Two kindsof features were used based on the biological background SincelncRNAs have tissuespecific expression and the expression ofcancer tissues is diï¬erent from normal tissues the expressionoftissues could provide importantin diï¬erentlncRNAsREFERENCESAvgeris M Tsilimantou A Levis P K Tokas T Sideris D C StravodimosK Loss of GAS5 tumour suppressor lncRNA an independentmolecular cancer biomarker for shortterm relapse and progression in bladdercancer patients Br J Cancer “ 101038s4141601803206Bai Y Dai X Ye T Zhang P Yan X Gong X PlncRNADBa repository of plant lncRNAs and lncRNARBP protein interactions CurrBioinform “ Bao Z Yang Z Huang Z Zhou Y Cui Q and Dong D LncRNADisease an updated database of long noncoding RNAassociateddiseases Nucleic Acids Res D1034“D1037 101093nargky905Canzio D Nwakeze C L Horta A Rajkumar S M Coï¬ey E L Duï¬y EE Antisense lncRNA transcription mediates DNA demethylationto drive stochastic protocadherin α promoter choice Cell “653e15 101016jcell201903008Chen X Shi W and Deng L Prediction of disease comorbidity usinghetesim scores based on multiple heterogeneous networks Curr Gene Ther “ Cheng L Computational and biological methods for gene therapy CurrGene Ther Cheng L Hu Y Sun J Zhou M and Jiang Q 2018a DincRNA afor exploring disease“comprehensive webbased bioinformaticsassociations 101093bioinformaticsbty002ncRNA functionBioinformaticstoolkitandCheng L Jiang Y Ju H Sun J Peng J Zhou M 2018busingcrossontologyInfAcrOntsimilaritiescalculatingtermtheirexecutelncRNAsinformation for us to identify cancerrelated lncRNAs Inadditionregulation function byinteracting with their target genes Therefore the target genesof lncRNAs can also be the features of lncRNAs To encode thefeatures DBN was used to reduce the dimension Finally CNNwas used to identify real cancerrelated lncRNAs based on thefinal featureTo verify the eï¬ectiveness of our method we comparedDBN“CNN with PCA“CNN and DBN“DNN since PCA canalso reduce the dimension of features and DNN can also doclassification The results showed that DBN“CNN performedbest Finally case studies have been done to verify the accuracy ofour results We found potential lncRNAs for kinds of cancerswhich can be a kind of guidance for researchers finding novelcancerrelated lncRNAsDATA AVAILABILITY STATEMENTThe datasets presented in this study can be found in onlinerepositoryrepositoriesrepositories Theandnumbersbethesupplementary materialaccessionnamesfoundcantheofinAUTHOR CONTRIBUTIONSND and GX designed the research ZL performed the researchand wrote the manuscript YZ and XH acquired the dataand reviewed and edited the manuscript CL XY and JGanalyzed the data All authors reviewed the manuscript andprovided commentsinformation flow by a random walk BMC Genomics 19Suppl 101186s1286401743386Cheng L Yang H Zhao H Pei X Shi H Sun J 2019a MetSigDisa manually curated resource for the metabolic signatures of diseases BriefBioinform “ 101093bibbbx103Cheng L Zhao H Wang P Zhou W Luo M Li T 2019bComputational Methods for identifying similar diseases molecular therapyNucleic Acids “ 101016jomtn201909019Dao F Y Lv H Zulfiqar H Yang H Su W Gao H Acomputational platform to identify origins of replication sites in eukaryotesBrief Bioinform 101093bibbbaa017 [Epub ahead of print]Deng L Li W and Zhang J LDAH2V Exploring metapaths acrossmultiple networks for lncRNAdisease association prediction IEEEACMTransac Comput Biol Bioinform 101109TCBB20192946257 [Epubahead of print]Gu C Liao B Li X Cai L Li Z Li K Global network randomwalk for predicting potential human lncRNAdisease associations Sci Rep 101038s4159801712763zJiJ TangJ Xia KJandJiang Rtumorigenesis microenvironment CurrBioinformLncRNA in“Jiang Q Wang J Wu X Ma R Zhang T Jin S LncRNA2Targeta database for diï¬erentially expressed genes after lncRNA knockdown oroverexpression Nucleic Acids Res D193“D196 101093nargku1173Karner H Webb CH Carmona S Liu Y Lin B Erhard M Functional conservation of lncRNA JPX despite sequence and structuraldivergence J Mol Biol “ 101016jjmb201909002Frontiers in Cell and Developmental Biology wwwfrontiersinAugust Volume 0cLiu et alA Method to Identify CancerRelated lncRNAsKuang L Zhao H Wang L Xuan Z and Pei T A novel approachbased on point cut set to predict associations of diseases and LncRNAs CurrBioinform “ Lan W Li M Zhao K Liu J Wu FX Pan Y LDAP a webserver for lncRNAdisease association prediction Bioinformatics “ 101093bioinformaticsbtw639Liang C Changlu Q He Z Tongze F and Xue Z gutMDisorder acomprehensive database for dysbiosis of the gut microbiota in disorders andinterventions Nucleic Acids Res Liu D Rudland P Sibson D and Barraclough R Identification ofmRNAs diï¬erentiallyexpressed between benign and malignant breast tumourcells Br J Cancer “ 101038sjbjc6600456Liu X Hong Z Liu J Lin Y Alfonso RP Zou Q Computational methods for identifying the critical nodes in biologicalnetworks Brief Bioinform “ 101093bibbbz011Lu C Yang M Li M Li Y Wu F and Wang J Predicting humanlncRNAdisease associations based on geometric matrix completion IEEE JBiomed Health Inform 101109JBHI20192958389 [Epub ahead of print] Protein function predictionto deep learning Proteomics 19e1900119Lv Z B Ao C Y and Zou Qfrom traditionalclassifier 101002pmic201900119MartensUzunova E S Böttcher R Croce C M Jenster G Visakorpi T andCalin G A Long noncoding RNA in prostate bladder and kidneycancer Eur Urol “ 101016jeururo201312003Peng J and Zhao T Reduction in TOM1 expression exacerbatesAlzheimer™s disease Proc Natl Acad Sci USA “ 101073pnas1917589117Robinson E K Covarrubias S and Carpenter S The how and why oflncRNA function an innate immune perspective Biochim Biophys Acta GeneRegul Mech 101016jbbagrm2019194419Sasaki Y T Sano MIdeue T Kin T Asai K and Hirose T Identification and characterization of human noncoding RNAs withtissuespecific expression Biochem Biophys Res Commun “ 101016jbbrc200704034Sumathipala M Maiorino E Weiss S T and Sharma AShi Y Lu J Zhou J Tan X He Y Ding J Long noncodingRNA Loc554202 regulates proliferation and migration in breast cancer cellsBiochem Biophys Res Commun “ 101016jbbrc201402144Network diï¬usion approach to predictlncRNA disease associationsusing multitype biological networks LION Front Physiol 103389fphys201900888Wang L Xuan Z Zhou S Kuang L and Pei T A novel modelassociations based on the LncRNA“for predicting LncRNAdiseaseMiRNAdisease interactive network Curr BioinformWang Y Yu G Wang J Fu G Guo M and Domeniconi C Weightedmatrix factorization on multirelational data for LncRNAdisease associationprediction Methods “ 101016jymeth201906015Wei L Su R Wang B Li X Zou Q and Gao X Integrationof deep feature representations and handcrafted featuresto improvethe prediction of N 6methyladenosine sites Neurocomputing “ 101016jneucom201804082Wu B Zhang H Lin L Wang H Gao Y Zhao L A similarity searching system for biological phenotype images using deepconvolutional encoderdecoder architecture Curr Bioinform “ Xie G Huang S Luo Y Ma L Lin Z and Sun Y LLCLPLDA a novelmodel for predicting lncRNA“disease associations Mol Genet Genomics “ 101007s00438019015908Xuan P Cao Y Zhang T Kong R and Zhang Z2019a Dualconvolutional neural networks with attention mechanisms based methodfor predicting diseaserelated lncRNA genes Front Genet 103389fgene201900416Xuan P Pan S Zhang T Liu Y and Sun H 2019b Graph convolutionalnetwork and convolutional neural network based method for predictinglncRNAdisease associations Cells 103390cells8091012Yu G Fu G Lu C Ren Y and Wang J BRWLDA birandomwalks for predicting lncRNAdisease associations Oncotarget “ 1018632oncotarget19588Yu J Xuan Z Feng X Zou Q and Wang L A novel collaborativefiltering model for LncRNAdisease association prediction based on the NaïveBayesian classifier BMC Bioinform 101186s1285901929850Zeng X X Wang W Deng G S Bing J X and Zou Q Prediction ofpotential diseaseassociated microRNAs by using neural networks Mol TherNucleic Acids “ 101016jomtn201904010Zhangand Deng LJ Zhang Z Chen ZIntegratinglncRNAdisease associationIEEEACM Transac Comput Biol Bioinform “multiple heterogeneous networks for novelinference 101109TCBB20172701379Zhang T Tan P Wang L Jin N Li Y Zhang L RNALocate aresource for RNA subcellular localizations Nucleic Acids Res D135“D138 101093nargkw728Zhang Z M Tan J X Wang F Dao F Y Zhang Z Y and LinH Early diagnosis of hepatocellular carcinoma using machinelearning method Front Bioeng Biotechnol 103389fbioe2020Zhao T Cheng L Zang T and Hu Y 2019a Peptidemajor histocompatibilitycomplex class I binding prediction based on deep learning with novel featureFront Genet 103389fgene201901191and Cheng LIdentifyingAlzheimer™s diseaserelated proteins by LRRGD BMC Bioinform 101186s1285901931247Zhao T Hu Y Zang T2019bZhao T Hu Y Zang T and Cheng L MRTFB regulates the expressionof NOMO1 in colon Proc Natl Acad Sci USA 101073pnas2000499117Zhao T Hu Y Zang T and Wang Y 2019c Integrate GWAS eQTLand mQTL Data to Identify Alzheimer™s diseaserelated genes Front Genet 103389fgene201901021Zhao T Wang D Hu Y Zhang N Zang T and Wang Y 2019d IdentifyingAlzheimer™s diseaserelated miRNA based on semiclustering Curr Gene Ther “ Zhao Y Li H Fang S Kang Y Wu W Hao Y NONCODE an informative and valuable data source of long noncoding RNAs NucleicAcids Res D203“D208 101093nargkv1252Zou Q Xing P Wei L and Liu B Gene2vec gene subsequenceembedding for prediction of mammalian N6methyladenosine sites frommRNA RNA “ 101261rna069112118Conflict of Interest The authors declare that the research was conducted in theabsence of any commercial or financial relationships that could be construed as apotential conflict of interestCopyright Liu Zhang Han Li Yang Gao Xie and Du This is an openaccess distributed under the terms of the Creative Commons Attribution License CCBY The use distribution or reproduction in other forums is permitted providedthe original authors and the copyright owners are credited and that the originalpublication in this journal is cited in accordance with accepted academic practiceNo use distribution or reproduction 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" apolipoprotein l1 apol1 is a trypanosome lytic factor present in human and certain other primatesapol1 gene variants present in individuals of recent subsaharan african descent increase risk for glomerulardisease and associate with the disease progression but the molecular mechanisms have not been definedobjectives we focus on the mechanism how apol1 variant proteins enhance podocyte injury in the stressedkidneymethods first we investigated the expression of apol1 protein isoform and the localization of apol1 protein inthe kidney next we examined the role of apol1 in the podocyte stress and the inflammatory signaling in thekidney after heminephrectomyresults we identified a novel rna variant that lacks a secretory pathway signal sequence and we found that thepredicted apol1b3 protein isoform was expressed in human podocytes in vivo and by bacapol1 transgenicmice apol1b3g2 transgenic mice carrying a renal risk variant manifested podocyte injury and increased proil1 mrna in isolated glomeruli and increased il1 production in the remnant kidney after uninephrectomyapol1b3 interacted with nlrp12 a key regulator of tolllike receptor signalings these results suggest a possible mechanism for podocyte injury by which one of the apol1 proteinisoforms apol1b3 and its renal risk variants enhances inflammatory signalingkeywords apol1 protein isoform interleukin1 beta nlrp12 podocytes kidney chronic kidney diseaseinflammatory stresstermed g1 and g2resistance antigen sra which binds apol1 and prevents pore formation the human apol1 coding variantsto the morewidespread g0 isoform elude binding by sra and killt b rhodesiense the g1 variant has two nonsynonymous coding variants s342g and i384m and in theg2 variant n388 and y389 are deletedin contrastintroductionafrican americans have increased rates of chronic kidney disease and a major cause of this health disparityare apol1 genetic variants present exclusively in individuals of african descent apol1 kill trypanosomabrucei likely via formation of pores that serve as ionchannels t b rhodesiense possesses a protein serum correspondence jbkoppnihgov1kidney disease section niddk nih kdb center dr 3n116 bethesdamd usafull list of author information is available at the end of the apol1 gene variants are strongly associated with riskfor focal segmental glomerulosclerosis fsgs hivassociated nephropathy hivan and hypertension attributed kidney disease arterionephrosclerosis the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwakashin bmc nephrology page of apol1 risk allele status in deceased donor kidneys affects transplant kidney survival while the apol1 riskallele status of the recipient does not affect transplantkidney survival these data suggest that intrarenalapol1 expression rather than circulating apol1 is associated with kidney disease as most apol1 nephropathies are either podocyte diseasessegmentalglomerulosclerosis hivassociated nephropathy lomerular and vascular diseases arterionephrosclerosisit appears that apol1 risk variant expression inpodocytes and possibly renal vascular cells contributesto kidney diseasefocalchronic inflammation contributes to progression ofglomerular diseases in an experimental model datafrom the neptune study of glomerular disease patientsindicated a role for apol1 variants in enhancing glomerular expression of cxcl9 and cxcl11 chemokinesinduced by proinflammatory cytokines such as tnf andil1 nodlike receptors nlr are pattern recognition receptors that sense intracellular pathogens and hostderived danger signals particular nlrs may amplify orsuppress the inflammatory response nlrp3 is a component of the inflammasome which activates caspase1and in turn produces mature il1 proinflammatorysignaling via both nlrp3 inflammasome and nfκbpathways is involved in the pathogenesis of experimentalglomerular disease [ ] nlrp12 is a negative regulator of the proinflammatory signaling pathway inducedby tolllike receptors tlr and the tumor necrosis factor tnf receptor nlrp12 which contains pyrinnacht and leucinerich repeat domains associates withinterleukin1 receptor associated protein kinase irak1in canonical nfκb signaling and associates withtnf receptorassociated factor traf3 and nfκb inducing kinase in noncanonical nfκb signaling [ ]nlrp12 negatively regulates the nfκb and mapk pathways as shown in an experimental colitis model nlrp12 is also a negative regulator of nfκb signaling intcells and absence of nlrp12 enhances severity of experimental autoimmune encephalomyelitis seven exonsand isexpressed in most ans including the kidney [“]after cleavage of the secretory signal sequence locatedin exon mature apol1 is secreted and binds to hdlps isoform c lacking a signal sequence ispredicted to be retained within the cell the expressionpattern of specific apol1 splice variants in ans andcells is unknownapol1 mrna compriseswe have examined whether apol1 regulates proinflammatory signaling in glomerular cells we identified anovelintracellular protein isoform apol1b3 andfound that this isoform associated with nlrp12 further apol1b3g2 enhanced podocyte damage inmurine nephropathy this proinflammatory pathwayrepresents a possible therapeutic targetfor apol1nephropathymethodsethics approvalsparaffinembedded autopsy kidney sections were usedfor immunostaining human subjects gave written consent to participate under research protocols approved inadvance by the niddk institutional review boardcell cultureconditionally immortalized human podocytes a generous gift from moin saleem bristol uk were cultured at °c and differentiated at °c with rpmi completemedium with fetal bovine serum insulin transferrin selenium penicillin and streptomycin helacells were cultured at °c with dmem completemedium with fetal bovine serum penicillin andstreptomycinantibodiesapol1 was detected using the mouse monoclonal antibody clone cl0171 rabbit polyclonal antibody fromsigma aldrich hpa st louis mo and rabbitmonoclonal antibody clone rpr2907 from abcam allof which are predicted to recognize isoforms a b and cand by rabbit polyclonal antibody produced at niddkthat is specific for isoform b3 affinity purified with immunizing peptides using sulfolink immobilization kitfor peptides thermofisher carlsbad ca antibody details are provided in the supplementary tablequantitative analysis of p57 staining of mouse kidneycortexformalinfixed paraffinembedded tissue sections werestained with recombinant antip57 kip2 rabbit monoclonal antibody sigma sab5500158 which highlightspodocyte nuclei with dab detection on an automatedstainer lab vision autostainer grand island nysections were counterstained with periodic acid schiffpas podocyte numbers were assessed as follows usingwhole slide scans nanozoomer hamamatsu bridgewater nj a glomeruli were randomly selected in eachcortical profile and p57 positive podocytes were enumerated b using the pas stain as the delimiter of theglomerular area glomerular area was measured usingndpiview2 hammamatsu and c the podocyte number was indexed to the glomerular area μm2 the intensity of podocyte staining was evaluated using thequpath qupathgithubio digitalimage analysis platform which permits quantification of the stained tissueas follows a at least glomeruli were selected in eachcortical region b the software identified the podocytes 0cwakashin bmc nephrology page of and graded the diaminobenzidine dab chromogen intensity for each stained pixel on a continuous scale alonga predefined spectrum considered as œpositive stain bythe user c multiple thresholds were tested to explore arange of sensitivities and specificities and the finalthresholds were defined based on the highest accuracyto find the region of interest across all the images whichpermits the comparison of the relative intensities ofstains between the images d the average dab intensityfor all pixels inside a podocyte was used to compare thestaining intensity of the p57positive podocytes in theglomerulus across different imagesthe locustransgenic micehuman apol1 gene locustransgenic mice bacapol1 mice were generated using a bacterial artificialchromosome bac which containsforapol1 resulting in apol1g0 ˆ’g1 and “g2 mice a kb human dna encompassing only the apol1gene with ² and ² flanking regions including exons and of apol2 and ² region including exons “ ofpart of myh9 gene was isolated and subcloned fromhuman bac clone enst00000397278 which corresponds to nm_003661 bac subclone was injected into129svjb6n f1 embryos and the founders were subsequently backcrossed into 129svj the subclone was sequenced and found to be identical to the referencegenome sequences in the ncbi and ensembl databasesexcept for the g1 and g2 polymorphisms for cagapol1b3 transgenic mice linear dna encoding cagpromotorapol1b3 g0 or g2 flagsv40 polya signal was introduced into fvbn mouse embryos by pronuclear injectionmouse nephropathy model experimental procedure“ weeks old and independent lines of apol1b3g0transgenic mice and lines of apol1b3g2 transgenicmice were studied details in supplementary tables following unilateral nephrectomy random urine was harvested before and at days after the surgery glomeruliwere isolated from the remnant kidney at days after thesurgery and from intact kidney by using dynabeads m tosylactivated invitrogen animals wereeuthanized by exposure to compressed carbon dioxide peran nihapproved animal care and use protocol and peranimal facility standard operating proceduresresultskidney cells expressed apol1 splice variants in vitro andin vivoapol1 has several mrna splice variants predicted toencode diverse protein isoforms it has not been fullyclarified which apol1 splice variants are expressed inthe kidney and which of those contribute to kidney disease first we examined the expression of apol1mrna splice variants in immortalized human podocytesby tacloning we identified six mrna variants fourof them have been described as v1 a v3 a v2“b1 and v4 c with the protein isoform precursor inparentheses fig 1a we identified two novel mrnavariants containing exon which we have named v2“ b2 and v2“ b3 with regard to v2“ b2 we isolated an mrna encoding exon to exon and partiallysharing exon to exon with a variant that has beendescribed in ncbi aceview database ncbi aceviewapol1 variant l aug10 further the protein isoformshave predicted variants as shown by the first numberafter the letter œv exon is unique to the v2 variantsthe apol1b3 and apol1c protein isoforms are derived from isoform precursors of b3 and c respectivelyand the localization of these proteins are different fromisoform a as they lack a cleavable signal sequencethus apol1 mrna splice variants are predicted toproduce three different proteins apol1a apol1b3and apol1c fig 1bnext we examined the expression of apol1 splice variants in human tissues by rtpcr using caucasian cdnasamples with no disease in kidney we observed foursplice variants v1 v4 fig 1c v2“ and v2“ whilelung also expressed v2“ fig 1d we confirmed thepresence of apol1 v2“ in human kidney by pcr usingthe v2“ specific primer pair supplementary figure 1aand confirmed the dna sequence of v2“ pcr productsupplementary figure 1c we then asked whetherapol1 mrna splicing could also be observed in themouse kidney we generated bacapol1 transgenicmice that carry the human apol1 gene and upstreamnoncoding regions and we examined the expression ofapol1 splicing variants in the kidneys of the mouse asshown in supplementary figure 1b apol1 v1 v2“ andv2“ mrna splice variants were expressed in the kidneyand by using pcr with the v2“ and v2“ specificprimers we confirmed the presence of respective dna sequences supplementary figure 1cstatisticsin vivo data were analyzed by the kruskalwallis testwith posttesting of selected pairs of data sets by dunnett multiple comparison test prism graphpad sandiego ca in the figures group medians are shown ap was accepted as significantapol1b3 was expressed in kidney cells but was absentin human serumhuman kidney expressed apol1 v1 v2“ and v2“the circulating form which ismrnas apol1amainly expressed in and secreted from liver tissueispresent in human serum and is part of the trypanosome 0cwakashin bmc nephrology page of fig kidney tissue expressed apol1 splice variants a b apol1 splice variants were cloned from differentiated immortalized human podocytesby tacloning the exons of each of the six mrna splice variants are shown a as are the three predicted protein isoforms following signalsequence cleavage b exon lengths are not drawn to actual size c d apol1 splice variant expression in human tissues was analyzed by using ahuman cdna library rtpcr was performed with two sets of primers as shown by arrows in the above exon schema apol1 splice variant tavectors were used for pcr positive controls the major splice variant v1 encoding isoform a is seen in all examined tissues c human lung andkidney expressed the apol1 splice variant v2“ d lower band encoding protein isoform b3lytic factor howeverlocalization and function ofapol1b3 were unknown to investigatecellularlocalization of apol1b isoforms in kidney tissue wetherefore produced a polyclonal rabbit antibody againsta amino acid sequence mrfkshtvelrrpcsd thatis encoded by exon and exon is specific to apolbisoforms and is absent from isoforms apol1a andapol1“c apol1b antibodies were affinity purifiedfrom the serum of immunized rabbits we then characterized the specificity of apol1b antibody by probingit against the immunizing peptides and apol1b protein isoforms for that we transfected human podocyteswith 3x flagtagged flagtagged expression constructs encoding b1 b2 and b3 isoforms apol1protein isoforms were then immunoprecipitated withantiflag antibodies and analyzed by immunoblottingusing apol1b and mouse antiapol1 cterminalantibodies clone cl0171 a signal for apol1b wasnot observed when the apol1b antibody was first preincubated with immunizing peptides this demonstratedthat the apol1b antibody was specific for the immunizing peptides supplementary figure 2a top all threeproteins were recognized by the antiapol1 cterminalantibody but only apol1b3 was detected by bothapol1b and apol1c antibodiessupplementaryfigure 2a second from top this suggests that apol1b1 and apol1b2 were cleaved probably in the signalsequence encoded by exon and consequently lost exon 0cwakashin bmc nephrology page of apol1b3 lacked exon and therefore retained exon thus we concluded that the apol1b antibody specifically recognized apol1b3we examined the cellular localization of apol1b3in vivo using apol1b and mouse antiapol1 cterminal antibodies cl0171 in human serum apol1was not detected by the apol1b antibody supplementary figure 2b but was detected with an apol1 antibody thatis predicted to recognize the cterminalregion of apol1 a b and c protein isoforms supplementary figure 2c apol1 was originally identified inhuman serum as two species polypeptides with apparentmolecular weights of kda and kda aminoacid sequence analysis had shown thatthe kdaapol1 was the result of cleavage at exon which encodes for the apol1 signal peptide the kda apol1species was amino acids shorter at the n terminusthan the kda form and glycosylated the lowerapol1b3 protein band was not recognized by apol1b antibody supplementary figure 2a suggesting it wascleaved similar to isoform a the biological significanceof this species is undeterminednext we examined the localization of apol1b3 inkidney tissue we first selected wildtype mouse andbacapol1 transgenic mouse kidneys for these studiessince mice do not express apol1 and thus wildtypemice can be used as a negative control bacapol1g0mouse kidneys which expressed v2“ mrna weresubjected to immunohistochemical analysis using therabbit polyclonal apol1b antibody and the rabbit antiapol1 monoclonal antibody clone epr2907 in bacapol1 mouse kidney we found apol1 immunoreactive signalin podocytes and tubular cells using themonoclonal apol1 antibody epr2907 supplementaryfigure 2d and also with the polyclonal apol1b antibody supplementary figure 2e suggesting that bothpodocytes and tubular cells expressed apol1b3 theseantibodies did not stain glomeruli in wildtype mousekidney fig 2d e the polyclonal apol1b antibodyproduced staining of tubular cells in bothwild type and transgenic mice next we examined theexpression of apol1 in autopsy samples from humankidneys similar to the pattern seen in the bacapol1mouse kidney in human kidney both the monoclonalapol1 antibody epr2907 fig 2a and the polyclonalapol1b antibody fig 2b stained the cytoplasm ofpodocyte and tubular cells while the antibody againstthe podocyte marker wt1 stained podocyte nucleirabbit igg was used as negative control fig 2c themonoclonal apol1 antibody epr2907 stained vascularwallin the autopsy kidney in summary our resultsgathered from kidneys of transgenic mice and humansindicated that podocyte and tubular cells expressedapol1b3 in vivoapol1 mrna expression was enhanced by il1it has been shown that apol1 mrna expression increases upon proinflammatory stimulation [ ]we examined the expression of apol1 mrna variantsin human podocytes that were differentiated for threedays quantitatively using v2“ and v1 specific primerswe observed that expression of both v2“ and v1 variants was increased following il1 stimulation fig this was consistent with a previous report [ ]however the role of apol1 in the regulation of inflammatory signaling is unknownexpressedflagtagged apol1b3g0cagapol1b3 transgenic mice expressed apol1b3 inpodocytes and tubular cellsfollowing the results shown in fig we asked whetherthe apol1b3 renal risk variant induces podocyte damage by promoting inflammatory stress in vivo for thispurpose we generated cagapol1b3 transgenic micewhichorapol1b3g2 expression of apol1b3 protein in themouse kidney was confirmed by western analysisapol1b3 protein from whole kidney was immunoprecipitated with antiflag antibody and characterizedusing apol1b and antiflag antibodies showing thatapol1b3 was expressed in the transgenic mouse kidney fig 4a in the immunoblot the lower flag signalband corresponded to truncated apol1b3 as discussedaboveantiapol1 monoclonalwe examined the cellular localization of apol1b3 inmouse kidney sections by immunohistochemistry usingrabbitcloneepr2907 immunoreactive signal was present in thepodocytes and tubular cells of apol1b3g0 and g2expressing mice fig 4b there were no remarkablepathologicalfindings in the kidneys from 8weekoldapol1b3 transgenic mice as judged by light microscopy supplementary figure antibodyapol1b3g2 transgenic mice manifested enhancedalbuminuria and il1 production followinguninephrectomywe hypothesized that apol1b3g2 might be involvedin podocyte damage we evaluated podocyte damageand glomerular proil1 mrna levelin apol1b3mice after uninephrectomy three days following uninephrectomy acr was significantly higher in apol1b3g2 mice compared to wildtype and apol1b3g0 micefig 5a to elucidate mechanisms of podocyte injurywe isolated glomeruli from the remnant kidney and bonemarrow cells as a positive control glomerular apol1mrna levels did not differ among mouse lines supplementary figure 4aexpressednephrin that was as expected absent in isolated bonemarrow cells and both glomerular[add] cells and boneisolated glomeruli 0cwakashin bmc nephrology page of fig human kidney cells expressed apol1b3 in vivo ac to examine the presence of apol1 isoforms in human kidney human autopsy kidneyspecimens were used for immunohistochemical analysis using apol1b antibody and rabbit monoclonal antibody rpr2907 podocytesexpressed apol1 as shown by staining with rpr2907 antibody a and with apol1b antibody recognizing apol1b3 b control rabbit igg didnot stain podocytes c wilms tumor1 wt1 was used as a podocyte marker all glomeruli were derived from identical kidney scale bar indicates μm in regular and μm in high magnification images arrow and arrowhead indicate podocytes and tubular cell respectively 0cwakashin bmc nephrology page of fig apol1b3 regulated proinflammatory signaling in vitro immortalized human podocytes were stimulated by il1 ng ml h theexpression of the apol1 splice variant was examined by qpcr using v2“ primers and v1 primers as indicated by arrows in the above exonschema the graph shows the relative expression of il1 to gapdh student™s t test p fig cagapol1b3 transgenic mice expressed apol1b3 in podocytes and tubular cells a cag chicken ßactin promoter apol1b3flagtransgenic mice were generated in a fvbn mouse to confirm the expression of apol1b3 in the kidney extracts from wholekidneys were subjected to western analysis using indicated antibodies showing that apol1b3 was expressed b localization of apol1b3 in thekidney was determined by immunohistochemistry using rabbit monoclonal antibody epr2907 podocalyxin was used as a podocyte markerscale bar shows μm 0cwakashin bmc nephrology page of fig apol1b3g2 enhanced podocyte damage and il1 production in vivo a three days after uninephrectomy the urine acr was increasedin apol1b3g2 mice compared to wildtype mice and apol1b3g0 mice b following uninephrectomy proil1 mrna from isolatedglomeruli was upregulated in apol1b3g2 but not in apol1b3g0 mice data shown as expression of proil1 relative to actin c il1 levelsin the urine standardized by urine creatinine cr concentration were found to be significantly higher in apol1b3g2 transgenic mice comparedto baseline data shown as il1 pgmlcr mgdl bars are shown median asterisk shows p marrow cells expressed tlr4 mrna supplementaryfigure 4b tlr4 signaling has been associated with proteinuria and podocyte tlr4 signaling contributesto podocyte injury in experimental animal models further podocyte nfκb pathway activation has beenassociated with proteinuria in a glomerulonephritismodel [ ] glomerular expression of tlr4 mrnawas significantly upregulated in apol1b3g2 mice following uninephrectomy supplementary figure 4c andproil1 mrna levels were higher at day in apol1b3g2 mice than in the other groups fig 5b moreover il1 in the urine was increased following uninephrectomy and the change reached significance inapol1b3g2 mice suggesting enhancement of il1processing in the kidney fig 5c these data indicatedthat the uninephrectomy was associated with inductionof tlr4 signaling proil1 mrna expression and il1 production in the remnant kidney possibly involvingan activation of the inflammasomewe further investigated the effects of apol1b3g0and apol1b3g2 expression on podocytes in kidneysections of mice prior to and following uninephrectomywe quantitated podocyte numbers with p57 stainingand determined p57 staining intensity as a surrogate forpodocyte injury supplementary figure 4d i we observed that the number of podocytes differed little whencomparing pre and postuninephrectomy kidney samples for wildtype fvb mice and apolb3g0 micesupplementary figure 4e in contrast for the risk variant apol1b3g2 mice podocyte number decreased by following nephrectomya difference whichreached statistical significance p we next measured glomerular areas supplementary figure 4f inapol1b3 variant samples glomerular areas changedlittle and without reaching statistical significance incontrast in wildtype samples there was a increasein glomerular area p however the ratio of thepodocyte number per glomerular area did not changesignificantly for any group supplementary figure 4gfinally we investigated p57 staining intensities as a surrogate for podocytespecific changes in cellular statussuch as observed during podocyte injury 0cwakashin bmc nephrology page of supplementary figure 4h i when comparing preand postuninephrectomy samples mean p57 stainingintensities declined both per glomerular area and per podocyte only for the apol1b3g2risk variant p differences were not significantfor p57 staining intensities when calculated ˜per glomerulus™ for wildtype and apol1b3g0 mice howeverp57 intensity values increased for both wildtype andapol1b3g0 mice and respectively significantly when evaluated ˜per podocyte™ p andp respectively this observation that podocytenumbers and podocyte p57 expression were lower inapol1b3g2 mice following uninephrectomy is consistent with the presence of greater microalbuminuriasuggesting increased podocyte injuryin summaryapol1b3g2 expression aggravated podocyte damageand enhanced the proinflammatory response in glomerular cells after uninephrectomyapol1b3 associated with nlrp12we wished to determine whether apol1b3 modulatesproinflammatory signaling via either the il1 receptor ortlr4 we investigated apol1b3 binding partners byusing mass spectrometry analysis of proteins that coprecipitated with apol1b3flag from apol1b3flagstably transfected hela cells we identified a 12aminoacid sequence tvvmqgaagigk specific to nlrp12suggesting an interaction between apol1b3 andnlrp12 to identify apol1 isoformsthat bindnlrp12 we cotransfected nlrp12 and flagtaggedapol1 isoforms into hela cells and immunoprecipitated apol1 with antiflag antibodies transfectednlrp12 was coprecipitated with apol1b3flag andapol1cflag but not with apol1b1flag fig or apol1aflag supplementary figure 5a thesedata indicated that nlrp12 specifically associated withthe predicted intracellular forms of apol1 namely b3and c but not the secreted forms of apol1 namely b1or a in concordance with the notion that presence orabsence of a signal peptide determines the localizationof apol1 isoforms and hence their cellular interactionsnext we examined the interaction of apol1b3 renalrisk variants with nlrp12 all wildtype and renal riskvariants of apol1b3 associated with nlrp12 as shownin pulldown experiments supplementary figure 5bhowever the reason for the differences in the observeddegree of coprecipitation of nlrp12 by the apol1variants remains to be determinedfinally we examined which domains of apol1b3were responsible for the interaction with nlrp12 wefig apol1b3 and c isoforms uniquely interacted with nlrp12 shown are the structure of flagtagged apol1 isoforms expression vectorsalso shown is the design of the nlrp12 expression vector highlighting the three major domains pyrin domain pyd nacht domain and leucinerich repeat lrr of this protein the constructs were transiently transfected into hela cells apol1b3 and apol1c isoforms coimmunoprecipitated with nlrp12 while the secreted apol1b1 isoform did not apol1b3 interacted with nlrp12 but not apol1b1 which isidentical to apol1a in its amino acid sequence 0cwakashin bmc nephrology page of made apol1b3g0 constructs in which the cterminalsra binding domain cdel orthe membraneaddressing domain mdel was deleted nlrp12 wascoprecipitated with fulllength apol1 and both domain deleted apol1b3 polypeptides the cterminaldomain seemed to be dispensable for association withnlrp12 supplementary figure 5cis not clearwhether apol1b3 and nlrp12 interacted directly itremains to be shown how renal risk variants located inthe cterminal affect binding to and to inhibition ofnlrp12ittoemptyapol1b3 regulated nlrp12 functionwe addressed the consequences of apol1b3 association with nlrp12 in hela cells stably transfected withempty vector apol1b3g0 or apol1b3g2 constructs we examined the effect of apol1b3enhancedil1 receptor signaling in the setting of nlrp12 overexpression achieved by transient transfection of hela cellscompared to control hela cells apol1b3 transfectedcells g0 and g2 both showed enhanced erk phosphorylation at min after stimulation with il1 comparedoverexpressiondownregulated phosphorylation of erk in each groupempty vector apol1b3g0 and apol1b3g2 stablytransfectedhela cells as expected for a negative regulator of the immune process supplementary figure these datatransfectednlrp12 attenuated il1 receptor signaling and thatapol1b3 g0 and g2 did not overcome this inhibitory function at supraphysiologic levels of nlrp12 inthese data the effect of overexpressed apol1b3 onsignaling was similar for the g0 and g2 variants in helacellsvector nlrp12indicated thatexogenousour data suggest that overexpressed apol1b3 g0and g2 functionally antagonized endogenous nlrp12in the early phase of il1 receptor activation in vitrohow apol1b3g2 and nlrp12 are functionally linkedin the kidney remains unclear as we did not investigatethe consequences of overexpression of nlrp12 in thekidney of apol1b3 transgenic micediscussionwe report an apol1 isoform apol1b3 that regulatesproinflammatory signaling and associates with nlrp12a tolllike receptor signaling regulator as previously reported apol1 has multiple mrna splice variants and we confirmed the expression of these variants in immortalized human podocytes and in human and bacapol1 mouse kidney we identified a novel mrnavariant apol1v2“ that lacks the signal sequencewhich suggests that apol1b3 is a cytoplasmic proteinand not secreted as is the case for the apol1a isoform khatua reported that immortalized humanpodocytes express apol1 mrna which include splicevariants thatlack exon and which correspond toapol1b3 and apol1c kidney transplant studies suggest that kidney injury isthe result of kidneyexpressed apol1 and not that ofcirculating apol1 [ ] kidneyexpressed apol1mrna v1 a v2“ b1 v2“ b3 and v4 c arepredicted to produce apol1 isoform a b3 and c priorreports [ ] on podocyte apol1 expression haveused antibodies that were not specific for individualapol1 splice isoforms a b3 and c using anapol1b3 specific antibody we showed that apol1b3is present in human podocytes human and bacapol1mouse kidneywe demonstrated that transgenic apol1b3g2 expression enhanced proinflammatory response in theglomeruli and albuminuria following uninephrectomynot only in apol1b3g2 glomeruli but also in wildtype and apol1b3g0 mice glomeruli tlr4 mrnawas numerically upregulated after uninephrectomy butproil1 mrna was enhanced only in apol1b3g2this suggest that apol1b3g2 might interfere withnegative regulation of tlr4 signaling the resulting prolonged tlr4 signaling and cellular inflammation wouldpromote progressive podocyte dysfunction and fibrosisthis agrees with the observation that apol1 gene variants are associated with glomerular disease which tendsto manifest in a more rapid progression to endstagekidney disease tlr4 signaling has been implicatedin the pathogenesis of glomerular diseaseincludingcryoglobulinemia in mice and diabetic nephropathyin humans data from the neptune study ofglomerular disease supports a role for apol1 variantsin enhancing inflammatory signaling thus two of thethree glomerular transcripts that are upregulated inapol1 highrisk genotype subjects compared to lowrisk subjectschemokinescxcl9 and cxcl11 which are enhanced by tnfil1 and tlr signaling [ ]the proinflammatoryareit is wellestablished that nephrectomy activates thereninangiotensin system in addition studies have shownthat angiotensin ii induces tlr4 expression [“]here we showed that glomerular proil1 mrna andurine il1 levels were increased in apol1b3g2 miceapol1b3g2 promoted podocyte damage followinguninephrectomy possibly by increasing glomerular synthesis and processing of proil1 however we have notevaluated the contribution of podocytederived il1 inpodocyte damage in this settingstudyin thisconstitutivelyexpressedtransgenicapol1b3 did not induce glomerulosclerosis at steadystate however doxycyclineinduced renal riskapol1overexpression in podocytes caused severe podocytedamage glomerulosclerosis and resulted in enhanced 0cwaka

natural killer nk cells are innate lymphocytes that play an important role in immune surveillanceagainst tumors and virusinfected cells and are an emerging target for tumor immunotherapynk cell immune surveillance is mediated by direct eï¬ector functions such as ifnÎ productionand cytotoxicity as well as immuneregulatory functions [such as interactions with dendriticcells “] of nk cells nk cells develop from precursors generated from hematopoietic stemcells hscs in the bone marrow and then begin the process of maturation before they egressto the periphery for immune surveillance since the initial discovery of nk cells in enormous progress has been made in our understanding of nk cell development and maturationas well as in identifying the cytokines and transcription factors that are important for theseprocesses nk cells that reach maturation represent the optimal functional status of the nk cellpopulation at steadystate and also represent the optimal functional status at the singlecell levelas evidenced by the upregulation of genes encoding cytotoxicityrelated eï¬ector molecules duringnk cell maturation compromised nkdependent immune surveillance usually accompaniesimpaired nk cell maturation therefore it is important to understand the molecularregulatory mechanisms underlying nk cell maturation which could potentially be exploited forthe development of novel therapeutic strategies in this review we aim to provide a framework ofthe current knowledge of nk cell maturation and the factors that regulate this process includingtranscription factors and cytokines and to discuss the importance of nk cell maturation inhealth and diseaseedited byzhigang tianuniversity of science and technologyof china chinareviewed byxi yanguniversity of manitoba canadajian zhangshandong university chinacorrespondencejiacheng bijcbisiataccnxuefu wangwangxuefuahmueducnspecialty sectionthis was submitted tonk and innate lymphoid cell biologya section of the frontiers in immunologyreceived june accepted july published august citationbi j and wang x molecularregulation of nk cell maturationfront immunol 103389fimmu202001945frontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationan overview of nk cellmaturationilc precursornk cell maturation in micein thein adult mice nk cells begin their developmentbone marrow and go through a stepwise cell diï¬erentiationprocess including hscs common lymphoid progenitors clpsprenk cell progenitors prenkps nk cell precursors nkpsimmature nk cells inks and mature nk cells mnksfigure clps are linˆ’il7rαckitsca1flt3 andcan generate prob and pret cells as well as the earliestcommon innate lymphoid cellcilcpprenkps representing an intermediate stage between clps andnkps are linˆ’cd244ckitlowil7rαflt3ˆ’cd122ˆ’ andcomprise both early nkcommitted precursors and il nkpsalso called refined nkps rnkps are linˆ’nk11ˆ’dx5ˆ’il7rαcd122nkg2d and diï¬erentiate into nk cells before the final nk lineage commitment nkps progress toan ink stage which is characterized by the acquisition ofnk11 and natural cytotoxicity receptor ncr expressionhowever nk cells at the ink stage are not yet functionallymature and their maturation continues in the bone marrowand the periphery the acquisition of dx5 and ly49 expressionmarks the maturation of nk cells to acquire optimizedcapacity nk cells continue a late maturation program whichis accompanied by an increase in their eï¬ector function andchanges in the expression of phenotype markers such asupregulation of cd11b cd43 and klrg1 or downregulation ofcd27 therefore based on the expression levels of cd27cd11bnk cell maturation can be divided into cd27cd11bˆ’cd27cd11b and cd27ˆ’cd11b stages nk cells maturefrom the cd27cd11bˆ’ink or m1 stage and then progressto the cd27cd11b transitional nk cell tnk or m2 stageand finally to the cd27ˆ’cd11b terminally mnk or m3stage during maturation nk cells gradually increase theircytotoxic capacity but decrease their potential for homeostaticexpansion nk cell maturation in humanshuman nk cells have developmental trajectories similar to thoseof mice figure hscs diï¬erentiate into clps and then nkps in humans the expression of cd122 on nkps is critical fornk cell lineage commitment the appearance of cd56 representsthe final step in the diï¬erentiation of nkps into nk cells thematuration of human nk cells can be divided into a cd56brightstage and a cd56dim stage based on the expression levels ofcd56 cd56bright nk cells are thought to be immature andcan diï¬erentiate into cd56dim nk cells with the acquisition ofcd16 while two subsets produce ‚ammatory cytokinescd56dim nk cells have more potent cytolytic activity cd56dimnk cells can further progress into latematuration stages withchanges in their surface markers and function the terminalmaturation of cd56dim nk cells with highest cytolytic activitycan be defined by the expression of cd57 approximately “ of all cd56dim nk cells in healthy adults express cd57on their surface interestingly highdimensional singlecell analysis can identify the high similarity between mousecd27ˆ’cd11b nk cells and human cd56dim nk cells andbetween mouse cd27cd11bˆ’ nk cells and human cd56brightnk cells additionally fu has showed that cd27 andcd11b can reflect distinct populations of human nk cells fromdiï¬erent tissues functionally similar with their counterparts inmice similarinnatelymphocytes the maturation of nk cells includes multiplephysiological processes to attain an optimal nk cell populationsize the maturation process usually requires the optimal egressof nk cells from the bone marrow and a finely tuned balancebetween survival proliferation and apoptosis at the steadystatemeanwhile optimal nk cell functional status at the singlecelllevel requires a dedicated transcriptional program dictated by anoptimal level of transcriptional factor activityto the diï¬erentiation process of othermodels used for investigation of nk cellmaturationbased on the above parameters several systems are availableto investigate the factors involved in the regulation of nk cellmaturationtool knockout mouse models provide a powerfultodetermine the eï¬ects of a geneofinterest on nk cellmaturation importantly an increasing number of studieshave employed nk cellspecific conditional knockout mousemodels in which cre recombinationdirected gene deletionoccurs soon after the acquisition of nkp46 “ thismodel allows gene deletion that is restricted to nk cells andgroup innate lymphoid cells ilc1s importantly italso allows the dissection of stagedependent eï¬ects elicitedby the geneofinterest on nk cell maturation adoptive transfer of nk cells into immunedeficient egil2rgˆ’ˆ’rag2ˆ’ˆ’ mice or reconstitution ofthe bonemarrow in lethally irradiated mice can recapitulate thematuration of nk cells under physiological conditions diï¬erent groups of nk cells can be monitored in acompetitive manner to assess cellintrinsic eï¬ects throughthe use of congenic markers such as cd4512 in vitro nk cell diï¬erentiation assays using op9 stromalcells provide an in vitro model to mimic cytokinedrivenphysiological nk cell diï¬erentiation from nk precursors this model also allows the determination of cellspecificeï¬ects associated with a geneofinterestseveral factors and pathways that play a role in nk cellmaturation have been identified using the abovementionedapproaches the results have demonstrated that nk cellmaturation is dependent on several critical signaling pathwaysand is triggered by a balance between extracellular signalscytokines and dictated by an optimal coordination oftranscription factor activity although nk cell maturation hasbeen extensively studied in mice knowledge about the factors thatcontrol human nk cell maturation remains limited neverthelessfrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationfigure dynamics of nk cell development and maturation in mice and human hematopoietic stem cells hscs differentiate into common lymphoid progenitorsclps and then differentiate into nk cell progenitors nkps the acquisition of cd122 marks nk cell lineage commitment from hscs inks generated from nkpscontinue to mature in the bone marrow and periphery for fully functional acquisition in mice cd27cd11b divides nk cell maturation into three stages in humancd56cd57 divides nk cell maturation into three stagesadvances in gene editing humanized mice models singlecell sequencing mass cytometry and genomewide associationstudies have led to a deeper understanding of how nk cellmaturation is regulated in humanscytokines that regulate nk cellmaturationincreasing evidence suggests that multiple cytokines are involvedin nk cell development table for instance il7 scf andflt3l are critical for cd122 nkp generation from hscswhile il15 is essential for nk cell lineage commitment andmaturation from cd122 nkps to mnk cells additionallymultiple cytokines have been found to be involved in nk cellmaturation by modulating il15 signalingil7 scf and flt3lstromafree culture has shown that preculture in il7 scf andflt3l is necessary for nk cell development by inducing il responsiveness in progenitors clps normally exhibithigh levels of il7 receptor scf receptor and flt3l receptorhowever the expression of these receptors is gradually lost duringthe process of nk cell maturation nevertheless these cytokineshave distinct roles in nk cell development for instance nk cellmaturation is almost normal in il7rα or il7deficient mice whereas the homeostasis of thymusderived cd127nk cells is exclusively dependent on il7 showing characteristicssimilar to those of human cd56highcd16ˆ’ nk cells il7promotes the survival of cd56bright nk cells by increasing bcl2expression although it does not increase nk cell cytotoxicityinterferongamma ifnÎ production or the expression ofactivation markers il7 alone is not sufficient to supporthuman nk cell development as evidenced by the findings inhuman il7 knockin nod scid gamma nsg mice scfpromotes the survival of peripheral ckit nk cells and theabsence of ckit signaling reduces the generation of nk cells fromfetal liver precursors additionally a marked deficiency ofnk cells has been observed in the spleen of mice lacking flt3l flt3l not only potently induces il15 responsivenessin progenitors but can also significantly expand nk cells byincreasing the number of il15expressing cd11chi dendriticcells dcs indicating that flt3l is important for the earlydiï¬erentiation of nk cells and nk cell expansionlymphotoxinlymphotoxins alpha ltα and beta ltβ belong to thetumor necrosis factor tnf ligand superfamily ltα boundto the ltβ receptor ltβr constitutes a membraneboundltα1β2 heterotrimer that is essential for secondary lymphoidfrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationtable factors involved in nk cell maturationfactorseffect stages affectednotereferences clp nkpˆ’ nkp ink mnk nkp ink mnk nkp ink mnk nkp ink mnk ink mnk ink mnk ink mnk ink mnk ink mnk ink mnk ink mnk± inkrequired for il15 responsivenessessential for development but limits maturationinduce expression of tbet gata3 blimp1 and id2downstream of il15 receptorsuppress ebox genes suppress socs3promote s1pr5 ifng repress eomesrepress tbetpromotes tbet expressioninduced by tbetinduced by tbet zeb2 ko phenocopy tbet kodispensable for early nk cell developmenttgfβ independentpromote autophagy or suppress tbet ˆ’transcription factorse4bp4tcf1ets1stat5id2tbeteomestox12prdm1zeb2gata3smad4foxo1cytokinesil7 scf flt3llymphotoxinil15il17tgfβthis table lists transcriptional factors or cytokines that either promote œ or suppress œˆ’ nk cell maturation the stages affected clp nkp ink or mnk as well asthe functions or features of these moleculescritical for nkp generation from hscessential for earlystage nk developmentpromote nk development maturation activation survival and homeostasisinduce socs3cell cycle arrest before mature stage nkp nkp nkp ink mnkˆ’ ink mnkˆ’ nkp ink mnk“ “““““ ““ tissue anogenesis as evidenced by defective lymph nodedevelopment and altered splenic microarchitecture in ltaˆ’ˆ’ltbˆ’ˆ’ and ltbrˆ’ˆ’ mice ltα and ltβ are expressed inactivated lymphocytes including t b and nk cells whereasltβr is expressed exclusively in nonlymphoid tissues includingin bone marrow stromal cells the ablation of ltα or ltβrleads to impaired earlystage development of nk cells “ bone marrow stromal cells from ltaˆ’ˆ’ or ltbrˆ’ˆ’ micecannot efficiently support early nk cell progenitors il15can overcome the arrest of ltaˆ’ˆ’nk cell development in vitro however nk cell progenitors from wildtype mice generatedmore nk11 cells than nk cell progenitors from ltaˆ’ˆ’ micein the presence of il15 these findings suggest that theinteraction between ltα on nk precursors and ltβr on stromalcells creates a permissive microenvironment that is essential forthe earlystage development of nk cellsil15il15 belongs to the Îc family of cytokines sharing a Îcchain with il2 and il15 signals through il15rβÎc encoded by il2rg heterodimers either alone withintermediate affinity or as a complex with il15rα with highaffinity a phenotype with a nearcomplete loss of nkcells is observed in the absence of either il15rα il15rβ Îcchain or il15 “ in contrast mice deficient for il2 or have normal nk cell development and maturationduring nk cell development il15 acts from cd122nkpsuntil the terminal maturation of nk cells indeed the ablationof il15 does not aï¬ect the production of prenkps or nkpsalthough il15 is widely expressed in various tissues and bymany cell types irf1dependent il15 production in the bonemarrow microenvironment is critical for nk cell generation importantly the eï¬ects of il15 on nk cell development andmaturation require transpresentation of il15 by il15α onbone marrowderived dcs “ in an il15αdosedependentmanner in addition il15 transgenic mice or mice thatoverexpress il15 have a dramatic increase in the number ofmnk cells il15 is also needed to support mnk cell survivaland promote mnk cell expansion in the steadystate similarwith the observations in mice il15 determines human nk cellmaturation and homeostasis which is supported by evidencefrom patients with Îc mutation and humanized mice as well asby experiments involving nk cell ex vivo developmentil17il17a is a pro‚ammatory cytokine that also plays animmunosuppressive role in some settings such as in tumorsby recruiting myeloidderived suppressor cells promotingangiogenesis or suppressing cd8 t cells in addition ourgroup recently found that il17 signaling negatively regulatesnk cell maturation and function ifnÎ production and thecytolytic activity of nk cells as well as nk cell antitumor andantiviral immune activity are enhanced in il17aˆ’ˆ’mice although total nk cell numbers are comparable with thosein wildtype mice increased percentages of terminal maturecd27ˆ’cd11b nk cells were observed in il17aˆ’ˆ’ il17f ˆ’ˆ’il17aˆ’ˆ’il17f ˆ’ˆ’ and il17raˆ’ˆ’ mice overexpression of il17a in vivo reduces cd27ˆ’cd11b nk cell subsets mechanisticallyil17a signaling suppresses il15inducedphosphorylation of stat5 via the upregulation of socs3 in nkcells leading to inhibition of nk cell terminal maturation on the other hand il17a exerts contextdependent eï¬ects onnk cells il17 signaling is required for ifnÎ production andthe cytolytic activity of nk cells derived from lipopolysaccharidefrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationlpsprimed mice as well as for optimal production ofgranulocytemacrophage colonystimulating factor gmcsfby nk cells in fungal infection which plays a nonredundant rolein activating neutrophils for fungal control tgfβisgrowth factor betatgfβtransforminga majorimmunosuppressive cytokine many cell types express tgfβand nearly alllymphocyte populations express the tgfβreceptor tgfβr deletion of the tgfβ receptor subunit tgfβrii enhances mtor activity and the cytotoxic activity of nkcells in response to il15 in the steadystate nkspecificdeletion of tgfβrii in ncr1cre tgfbr2flfl mice has a minimaleï¬ect on conventional nk cell maturation and homeostasis in contrast constitutive tgfβ signaling arrests nk cellmaturation moreover tgfβ blocks il15induced mtoractivation thereby suppressing the activation and functionsof nk cells consistent with the observations in micetreatment with tgfβ in vitro inhibits cytokinestimulatedmetabolism of human nk cells via canonical tgfβ signaling in contrasttransgenic mice expressing a dominantnegative form of tgfβrii under the control of the cd11cpromoter cd11cdntgf β rii exhibit an increase in the numberof mature nk cells in the periphery in an in vitro assayfor the diï¬erentiation of nk cell progenitors to less maturecd122nk11 and more mature nk11dx5 nk cells in thepresence of il15 and op9 stromal cells the addition of tgfβmarkedly blocked the derivation of mature nk cells from wildtype precursors whereas precursors from the cd11cdntgf β riimice were not aï¬ected mechanistically tgfβ was foundto mediate nk cell immaturity during ontogeny by arresting thecell cycle of nk cells at the least mature cd11bˆ’cd43ˆ’ andintermediate cd11bcd43ˆ’ stages as well as by limiting nkcell transition at the terminally mature cd11bcd43 stage as a result of resistance to tgfβ infant cd11cdntgf β riimice are protected against murine cytomegalovirus mcmvinfection in addition smad3 has been found to suppresse4bp4mediated nk cell development and eï¬ector functions asevidenced by the increased number of mnk cells in smad3ˆ’ˆ’mice and the increased levels of granzyme b il2 and ifnÎ insmad3ˆ’ˆ’nk cells meanwhile the silencing of smad3 inthe human nk92 cell line allows for the upregulation of e4bp4which subsequently promotes ifnÎ production thediï¬erences in phenotypes between cd11c cells and nkp46cells following blockade of tgfβr signaling might be due to theabrogation of the tgfβr signal at diï¬erent stages of nk celldevelopmentmaturation further studies are required to revealthe temporal regulation of nk cell maturation by tgfβtranscription factors thatregulate nk cell maturationnatural killer cell development is also regulated by sequential andcoordinated transcription factor activity figure for instancetranscription factors such as e4bp4 tox and id2 are requiredfor nk cell lineage commitment while id2 tbet eomes andfigure transcriptional regulation of nk cell development and maturationmultiple transcriptional factors mediate regulation at distinct stages during nkcell development and maturation e4bp4 tcf1 and id2 are essential for nkcell lineage commitment tbet eomes tox prdm1 gata3 zeb2 andsmad4 are critical for nk cell maturation the discrepancy of foxo1 in nkcell maturation needs further confirmationzeb2 are required for nk cell maturation transcription factorsthat mediate nk cell maturation usually promote the expressionof genes coding for eï¬ector molecules receptors responsible foregress and cellsurface maturation markerse4bp4e4bp4 is expressed in clps e4bp4ˆ’ˆ’ mice specificallylack nk cells indeed the numbers of prenkps rnkpsink cells and mnk cells but not those of clps are reducedin the bone marrow of e4bp4ˆ’ˆ’ mice ectopic expression ofe4bp4 in e4bp4ˆ’ˆ’clps can rescue nk cell production these findings indicate that e4bp4 acts at the clp stage and isrequired for nk cell lineage commitment prenkps and rnkpsare not aï¬ected in the absence of il15 signaling however theabsence of e4bp4 results in the failure of il15responsive nkpproduction specifically il15 cannot rescue nk cell productionin e4bp4ˆ’ˆ’ bone marrow in contrast ectopic expressionof e4bp4 enables limited nk cell production even in the absenceof il15 signaling however ectopic expression of eomesid2 or tbet cannot rescue nk cell production in the absenceof e4bp4 furthermore e4bp4 was found to directly regulatethe expression of eomes and id2 while the histone h2adeubiquitinase mysm1 has been found to be critical for therecruitment of e4bp4 to the id2 locus and be required fornk cell maturation but not nk lineage commitment inaddition notch1 has been identified as an e4bp4 target genein nk cells the abrogation of notch signaling blockednk cell production similar to that observed in e4bp4ˆ’ˆ’miceexposure to notch peptide ligands at an early stage rescuedthe defective nk cell development from e4bp4ˆ’ˆ’ progenitors moreover sumoylation and phosphorylation can regulatee4bp4 activity and then ‚uence nk cell development the ablation of either sumoylation or phosphorylation sitessignificantly increased the transcriptional activity of e4bp4 andpromoted the production of nk cells therefore not only thefrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationexpression level but also the activity of e4bp4 is critical fornk cell development however the regulation of e4bp4 activityby posttranslational modifications needs further investigationfirth reported that specific ablation of e4bp4 in eitherink or mnk cells had no eï¬ect on nk cell lineage maintenanceand homeostasis therefore these findings confirm thate4bp4 is required for nk lineage commitment but not for thematuration from the ink to the mnk stage or the survival ormaintenance of mnk cells although it has been reported that theupregulation of e4bp4 can enhance ifnÎ production in nk92cells the roles of e4bp4 in human nk lineage commitmentand development are not well definedtcf1tcf1 encoded by tcf7 gene is a member ofthe highmobility group hmg of proteins and is important for t celldevelopment and function tcf1 was initially found to bind tothe ly49a promoter and be required for the acquisition of ly49aexpression during nk cell development loss of tcf1 resultsin a greater than reduction in the levels of ly49d on nkcells subsequently studies on a tcf1 reporter mouse straindemonstrated that tcf1 was expressed by prenkps nkps inkcells and mnk cells but not clps in the bone marrow prenkps nkps and mnk cell numbers were reduced in tcf7ˆ’ˆ’mice moreover detailed analysis in splenic nk cells showed thattcf1 expression was high in cd27cd11bˆ’ nk cells declinedwith nk cell maturation and disappeared in cd27ˆ’cd11b nkcells surprisingly the number of terminally mature nk cells intcf7ˆ’ˆ’ mice is significantly increased these findings indicatethat tcf1 is essential for nk cell development but limits nkcell terminal maturation in humans tcf1 has been found tobe uniquely expressed in circulating cd56bright nk cells andnot in cd56dim nk cells the eï¬ects of tcf1 on humannk cell development and maturation as well as the underlyingmechanisms need further investigationets1ets1 is the founding member of the ets family of wingedhelixturnhelix transcription factors and is highly evolutionarilyconserved ets1 has been shown to function from the prenkpstage and is required for the expression of various transcriptionfactors including tbet and id2 activating nkrs includingnkp46 ly49d and ly49h and signaling molecules thelevels of mnk cells are significantly decreased in the bonemarrow and spleen of ets1ˆ’ˆ’mice moreover ets1ˆ’ˆ’mnk cells display impaired eï¬ector function and decreasedexpression of activating nkrs but increased expression ofinhibitory nkrs meanwhilein human nk cellstaveirne revealed that ets1 induced the expression oftranscription factors such as tbet gata3 and blimp1 thatdetermine nk cell development suggesting that ets1 mightplay a similar role as in humans moreover il2 and il can increase ets1 expression through erk12 signaling inhuman nk cells these observations indicate that ets1is essentialthe stagespecific requirement of ets1 for nk cell maturation requiresfurther investigationfor nk cell development howeverstat5downstream of il15 receptor jak1 associates with il2rβleading to stat3 phosphorylation while jak3 associates withil2rÎc resulting in stat5 phosphorylation the jak“stat5 pathway has been identified as being essential fornk cell maturation germline deletion of jak1 or stat5 andconditional deletion of jak1 or stat5 in nkp46 cells both lead toa marked reduction in nk cell levels as well as inhibition of nkcell maturation in the bone marrow and the periphery consistent with the reduced levels of mnk cells jak1floxfloxncr1icre tg or stat5floxfloxncr1icre tg mice showed defective nkcelldependent tumor surveillance moreover stat5 tetramersare required for the maturation of nk cells from the ink tothe mnk state in bone marrow and spleen as evidenced by thesubstantial decrease in mnk cell numbers but normal levelsof nkps and ink cells in stat5astat5b tetramerdeficientdouble knockin mice stat5b mutation also leads todefective human nk cell maturation and impaired lytic function these observations indicate that jak13stat5 is criticalfor nk cell maturationid2id2 is a member of the inhibitor of dnabinding id proteinfamily which acts by preventing eproteins such as e2a e2 and heb from binding ebox canntgcontaining targetgenes through heterodimerization id2 is expressed at high levelsafter nk cell lineage commitment and in all subsequent stages id2 was first reported to play an essential role in thegeneration of peripheral lymphoid ans and nk cells throughthe use of id2ˆ’ˆ’ mice although id2 deficiency does notreduce linˆ’cd122 nk cell progenitors in the bone marrow itdoes lead to reduced nk cell numbers and impaired maturationof nk cells as evidenced by the decreased expression of cd43and cd11b maturation markers as well as granzyme expression a more recent study showed that the hematopoietic deletionand acute deletion of id2 or id2 deletion in nkp46 cellsresults in the loss of most nk cells this indicates that id2 isrequired for nk cell maintenance at all stages of developmentid2 suppresses ebox genes required for nk cell maturationsuppresses socs3 expression and is required for normal il15receptor signaling strong il15 receptor stimulation by il2 preligated to an antiil2 antibody or socs3 deficiency partiallyrestores homeostasis in id2deficient nk cellsin support of the role of id2 in nk cell maturation deficiencyof mysm1 which mediates the recruitment of e4bp4 to the id2locus leads to defects in nk cell maturation but not nk lineagespecification or commitment mysm1 also plays a role inmaintaining an open chromatin structure at the id2 locustbet and eomestbet tbox expressed in t cells encoded by tbx21 is amember of the tbox family of transcription factors that areprimarily expressed by t cells and nk cells in tbetˆ’ˆ’ mice thenumber of nk cells is significantly decreased in the spleen liverand peripheral blood but modestly increased in bone marrownk cells these defects result from the impaired egress offrontiers in immunology wwwfrontiersinaugust volume 0cbi and wangmolecular regulation of nk cell maturationnk cells from the bone marrow tbet directly regulates s1pr5by binding to its locus and loss of tbet leads to thedecreased expression of s1pr5 in addition tbetdeficientnk cells express lower levels of the maturation markers cd43cd11b and dx5 and higher levels of ckit and integrin alphavwhich reflects the immaturity of nk cells therefore tbet isessential for nk cell maturationanother tbox transcription factor eomes eomesoderminalso plays nonredundant roles in nk cell maturation in micemost spleen nk cells express eomes expression of eomes marksnk cell maturation and eomes nk cells are characterized bythe expression of dx5 and the absence of trail expression eomes is required for nk cell maturation hematopoieticdeletion of eomes in eomesfloxfloxvavcre mice results in asubstantial reduction in the number of nk cells in the spleenand blood and to a lesser extent in the liver lymph nodes andbone marrow loss of eomes also renders nk cells phenotypicallysimilar to eomesˆ’ nk cells derived from wildtype mice withexpression of trail but lacking dx5 as well as decreased levelsof ly49 receptors and cd11b in addition trail inks requireeomes for conversion to dx5 mature nk cells following theirtransfer into il2rgˆ’ˆ’rag2ˆ’ˆ’ mice eomes is also required tomaintain nk cell maturity as temporal deletion of eomes inmature nk cells results in the loss of the maturation marker dx5and upregulation of traildespite the critical roles of eomes and tbet in nk cellmaturation loss of either transcription factor does not markedlyimpair the eï¬ector functions of nk cells at the singlecell levelin vitro further confirming that these factors have roles in nkcell maturation but not in nk cell identitytoxthe tox thymocyte selectionassociated hmg box proteinbelongs to a family of evolutionarily conserved dnabindingproteins besides tox family members include tox2 tox3and tox4 the tox protein is highly expressed in ink andmnk cells in the bone marrow the loss of tox significantlyreduces the frequency and number of mnk cells in the peripheryand bone marrow with a severe block in the transition from theink to mnk stage and to a lesser extent from the nkp to theink stage id2 expression is significantly reduced in the nk cellsthat remain in toxˆ’ˆ’mice however id2 expression does notrescue the defective maturation of toxˆ’ˆ’nk cells in additionyun demonstrated that tox enhances the maturationof human nk cells and aï¬ects though not directly regulatestbet expression during nk cell maturation this indicatesthat tox is required for nk cell maturation both in humansand in mice however the regulatory circuit downstream oftox during nk cell maturation requires further investigationsbesides tox tox2 has been found to play a critical rolein human nk cell maturation vong found that tox2is preferentially expressed in human nk cells among severalimmune cell populations and is upregulated during human nkcell maturation tox2 is essential for human nk cell maturationand cytotoxicity tox2 directly upregulates tbet expressionand tbet overexpression can rescue the tox2 knockdownmediated nk cell maturation defects these results implythat tox2 functions upstream of id2 in human nk cellshowever the role of tox2 in murine nk cell maturationremains to be elucidated combined these results show tox andtox2 play a crucial role in nk cell maturationprdm1the expression of the transcriptional repressor b lymphocyteinduced maturation protein blimp1 also known as prdm1can be induced by il15 stimulation and its expressionis tbetdependent blimp1 is constitutively expressed bynk cells and is upregulated during nk cell maturation in bothhumans and mice loss of blimp1 aï¬ects nk cell homeostasisleading to increased nk cell numbers in the bone marrow andlymph nodes and reduced nk cell numbers in the liver and thelungs loss of blimp1 also leads to impaired nk cell maturationas shown by the decreased levels of the cd27ˆ’cd11b nkcell subset and expression of the maturation markers cd43and klrg1 however blimp1 is dispensable for most eï¬ectorfunctions of murine nk cells and even suppresses the productionof ifnÎ tnf and ltαin human nk cells by directly bindingto multiple conserved regulatory regions an impairedresponse to il15 in the absence of blimp1 might be the causeof the aberrant maturation of blimp1ˆ’ˆ’ nk cellszeb2tb so synergizes with zeb2 to promote nk cell maturationtbet is necessary and sufficient to induce zeb2 expression andzeb2 expression positively correlates with nk cell maturation zeb2 is required for normal nk cell homeostasis deletionof zeb2 in nk cells results in the retention of nk cells in thebone marrow and reduced nk cell numbers in the peripheryzeb2 overexpression leads to a reduced number of nk cellsin the bone marrow moreover mice with nkspecific zeb2deletion lack mature cd27ˆ’ nk cells while zeb2 overexpressionincreases the numbers of mature cd27ˆ’ nk cells the reducednumbers of mature nk cells might be due to poor survivaland response to il15 as well as the downregulation of s1pr5expression in zeb2deficient nk cells additionally the levels ofthe maturation markers klrg1 and cd146 are also proportionalto the levels of zeb2 expression this implies that zeb2 promotesnk cell maturation interestingly although the absence of zeb2in nk cells compromises the overall nk cellmediated immuneresponse as evidenced by the increased susceptibility of nkzeb2ˆ’ï¿

"Chinese women. International Journal of Cancer40: 604“609 37 Wu-WilliamsAH DaiXD BlotW XuZY SunXW et al (1990) Lung cancer among women in north-east China. Br J Cancer62: 982“9872257230 38 ZhongL GoldbergMS GaoYT JinF (1999) Lung cancer and indoor air pollution arising from Chinese-style cooking among nonsmoking women living in Shanghai China. Epidemiology (Cambridge Mass)10: 488“494 PLoS One one 1932-6203 Public Library of Science San Francisco USA 24586842 3934888 PONE-D-13-30257 .0089518 Research Biology Genetics Genetic mutation Mutation types Cancer genetics Medicine Clinical research design Retrospective studies Diagnostic medicine Pathology Clinical pathology Test evaluation Oncology Cancer detection and diagnosis Cancer screening Cancer treatment Chemotherapy and drug treatment Clinical trials (cancer treatment) Cancers and neoplasms Lung and intrathoracic tumors Non-small cell lung cancer Oncology agents Clinical Validation of a PCR Assay for the Detection of EGFR Mutations in Non“Small-Cell Lung Cancer: Retrospective Testing of Specimens from the EURTAC Trial EGFR Mutation Testing in NSCLC in EURTAC Trial Benlloch Susana 1 * Botero Maria Luisa 1 Beltran-Alamillo Jordi 1 Mayo Clara 1 Gimenez-Capit¡n Ana 1 de Aguirre Itziar 2 Queralt Cristina 2 Ramirez Jose Luis 2 Cajal Santiago Ramn y. 1 6 Klughammer Barbara 3 Schlegel Mariette 3 Bordogna Walter 3 Chen David 4 Zhang Guili 5 Kovach Barbara 5 7 Shieh Felice 5 7 Palma John F. 5 Wu Lin 5 Lawrence H. Jeffrey 5 7 Taron Miquel 1 2 1 Pangaea Biotech SL Barcelona Spain 2 Medical Oncology Service-ICO Hospital Germans Trias i Pujol Badalona Spain 3 F. Hoffmann-La Roche Basel Switzerland 4 Genentech South San Francisco California United States of America 5 Roche Molecular Systems Pleasanton California United States of America 6 Pathology Department Vall d'Hebron University Hospital Universidad Autnoma de Barcelona Barcelona Spain 7 Roche Molecular Systems Pleasanton California United States of America Minna John D. Editor Univesity of Texas Southwestern Medical Center at Dallas United States of America * E-mail: sbenllochpangaeabiotech.com Competing Interests: BK MS WB DC GZ JFP LW are all current employees of Roche. BK DC JFP and LW have stock holdings in Roche. BK and HJL are former Roche employees and HJL has stock in Roche and has served as a paid consultant. FS was a paid consultant to Roche. SB JBA CM AGC are current employees of Pangaea Biotech. MLB is a former Pangaea employee. MT and SRyC have stock holdings in Pangaea Biotech. IdA CQ JLR are current employees of Medical Oncology Service-ICO Hospital Germans Trias i Pujol. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Conceived and designed the experiments: MT HJL. Performed the experiments: CM AGC JBA IdA CQ JLR. Analyzed the data: SB DC GZ CM AGC JBA IdA CQ JLR FS LW JFP HJL MT. Contributed reagents/materials/analysis tools: SB MLB JBA CM AGC IdA CQ JLR SRyC BK MS WB DC GZ BK FS LW JFP HJL MT. Wrote the paper: SB MLB JBA CM AGC IdA CQ JLR SRyC BK MS WB DC GZ BK FS LW JFP HJL MT. 2014 25 2 2014 9 2 e89518 23 7 2013 21 1 2014 2014 Benlloch et al This is an open-access distributed under the terms of the Creative Commons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original author and source are credited. The EURTAC trial demonstrated that the tyrosine kinase inhibitor (TKI) erlotinib was superior to chemotherapy as first-line therapy for advanced non-small cell lung cancers (NSCLC) that harbor EGFR activating mutations in a predominantly Caucasian population. Based on EURTAC and several Asian trials anti-EGFR TKIs are standard of care for EGFR mutation-positive NSCLC. We sought to validate a rapid multiplex EGFR mutation assay as a companion diagnostic assay to select patients for this therapy. Samples from the EURTAC trial were prospectively screened for EGFR mutations using a combination of laboratory-developed tests (LDTs) and tested retrospectively with the cobas EGFR mutation test (EGFR PCR test). The EGFR PCR test results were compared to the original LDT results and to Sanger sequencing using a subset of specimens from patients screened for the trial. Residual tissue was available from 487 (47%) of the 1044 patients screened for the trial. The EGFR PCR test showed high concordance with LDT results with a 96.3% overall agreement. The clinical outcome of patients who were EGFR-mutation detected by the EGFR PCR test was very similar to the entire EURTAC cohort. The concordance between the EGFR PCR test and Sanger sequencing was 90.6%. In 78.9% of the discordant samples the EGFR PCR test result was confirmed by a sensitive deep sequencing assay. This retrospective study demonstrates the clinical utility of the EGFR PCR test in the accurate selection of patients for anti-EGFR TKI therapy. The EGFR PCR test demonstrated improved performance relative to Sanger sequencing. This study was funded by Roche. The funders contributed to the study design data collection analysis decision to publish and preparation of the manuscript. Introduction The efficacy of many novel targeted cancer therapies can be predicted by the detection of specific biomarkers in the tumor. The FDA has indicated that if the identification of a specific biomarker is required for the safe and efficacious administration of a drug a well-validated FDA approved companion diagnostic assay is required for that drug. The optimal approval path for a new targeted therapy and its companion diagnostic is a parallel clinical development process that involves clinical trials for the investigational agent where the investigational diagnostic test is used to either select patients for the trials or to predict response to treatment and ends ideally with simultaneous health authority approval of the drug and the companion diagnostic. Successful examples of this include the co-development (and co-approval) of the BRAF inhibitor vemurafenib and its companion diagnostic BRAF V600E mutation assay for BRAF-mutant metastatic melanoma[1] and the ALK inhibitor crizotinib and its companion diagnostic ALK fusion gene test in advanced ALK-fusion positive non-small cell lung cancer (NSCLC) patients.[2] [3] [4] However in some cases predictive biomarkers for a targeted therapy are not recognized until after the drug is first approved. As an example the anti-EGFR antibody cetuximab was first approved in the US for the treatment of metastatic colorectal cancer in 2004. Numerous retrospective and prospective trials subsequently revealed that tumors harboring KRAS mutations were very unlikely to respond to cetuximab. In July 2009 FDA required labeling changes for cetuximab and another anti-EGFR antibody panitumumab requiring that the indications and usage state there was no treatment benefit with the drugs for patients whose tumors had KRAS mutations in codon 12 or 13 at a time when there were no FDA-approved diagnostic assays for KRAS mutations.[5] Only later in July 2012 did a KRAS mutation assay receive FDA approval based on the results of a prospective randomized trial highlighting the challenges of retrospectively validating a companion diagnostic assay after the pivotal drug trials have been completed.[6] The anti-EGFR TKI erlotinib was initially approved for all patients with advanced NSCLC who had progressed on first-line chemotherapy. A number of subsequent studies determined that patients with EGFR-mutant NSCLC had a high likelihood of responding to these TKI leading to trials in the first-line setting for EGFR-mutant cancer.[7] [8] [9] [10] [11] [12] [13] Four prospective randomized clinical trials studied in Asian populations demonstrated that erlotinib and gefitinib resulted in improved progression-free survival compared to chemotherapy for first line therapy in NSCLC patients with EGFR mutations.[7] [8] [9] [13] Other clinical studies in mixed ethnicity cohorts have concluded with similar results.[10][12] The EURTAC trial was a randomized phase 3 trial to assess the safety and efficacy of erlotinib compared with standard platinum-based chemotherapy for first-line treatment of a patient population with advanced EGFR-mutation detected NSCLC in a largely Caucasian population of European patients. Erlotinib-treated patients experienced significant improvements in median PFS (9.7 months vs. 5.2 months) compared to chemotherapy. Patients on the erlotinib arm also had a considerably higher percentage of responses (58% vs. 15%) in the intent-to-treat population.[11] This trial has been submitted for first line indication of erlotinib in EGFR mutated NSCLC patients. The majority of activating EGFR mutations are located in exons 19 (45%) and 21 (40“45%).[14] [15] [16] [17] [18] [19] [20] Guidelines from organizations such as ASCO CAP/AMP and NCCN recommend the use of anti-EGFR TKIs as first-line therapy in patients with EGFR-mutant advanced NSCLC based on the results of these pivotal clinical trials. [21] [22] [23] Recent recommendations by CAP/IASLC/AMP advise the identification of EGFR mutations present at >1% of which exon 19 deletions and an exon 21 mutation (L858R) account for greater than 90% of all mutations.[24] None of the guidelines specify the testing method to be used however the cobas EGFR Mutation test is CE-IVD approved and is recently FDA approved.[25] Here we present the retrospective analysis of a clinical validation study of the EGFR PCR test on a subset of lung cancer specimens from patients screened for the EURTAC trial. The EGFR PCR test demonstrated improved sample workflow relative to the LDTs used in the EURTAC trial enabling EGFR mutation screening in a single assay with a one-day turn-around time. The EGFR PCR test showed superior sensitivity and specificity compared with conventional Sanger sequencing. Methods The major study objectives were 1) to correlate the clinical outcomes (PFS BORR) from the subgroup of available samples tested by the EGFR PCR test to the results from the entire EURTAC population and 2) to compare the analytic performance of the EGFR PCR test to that of the original LDT and Sanger sequencing using massively parallel pyrosequencing (MPP) to resolve discrepancies observed between the other 3 testing methods. In the EURTAC trial1044 patients from hospitals in France Italy and Spain were screened using the LDT. For this study all samples were retrospectively analyzed under IRB approval from Copernicus IRB (00001313). Site specific IRB approval from each clinical site and written consent from all patients was obtained prior to the study conduct phase of NCT00446225.[11] [26] In 487 cases residual specimens were available for retesting with the EGFR PCR test (Figure 1). A single 5 µm section with at least 10% tumor content from each of the 487 specimens was used for the EGFR PCR test. Genomic DNA from existing eluate or extracted from additional sections was tested on Sanger sequencing and MPP. Table 1 lists the demographics of the patients screened for the EURTAC trial by the LDT sub-categorized by patients tested or not tested by the EGFR PCR test. Patients enrolled in the EURTAC trial were selected using a laboratory-developed test validated by the Laboratory of Oncology (ICO-Hospital Germans Trias i Pujol Badalona Spain) consisting of three methodologies.[26] In this study a single PCR-based assay for detecting EGFR mutations was used. Details of the analytical performance of this assay have been described previously.[27] .0089518.g001 Figure 1 Flow of samples through the study."

"Although there has limited evidence to confirm the rest genes to be molecular targets for targeted therapy these genes provide useful clues for targeted therapy. By gene ontology analysis the biomarkers inferred in gene-subtype lower logic relationships were significantly enriched in biological processes of ˜cell adhesion™ (GO: 0007155) and ˜epidermis development™ (GO: 0008544). The identified biological processes had nonrandom probability values and enrichment scores and they were also significant biological processes which were important for tumorigenesis of NSCLC. The discovered biomarkers in the biological processes ˜cell adhesion™ and ˜epidermis development™ (i.e. DST CLCA2 DSG3 PKP1 FAT2 DSC3 PVRL1 KRT5 GJB5 BNC1) account for more than a half of all discovered biomarkers. The expression of these genes were all sufficient and necessary conditions of the presence of SCC as well as the absence of AC. It indicates that genes annotated to epidermis development and cell adhesion may be differently regulated between AC and SCC. In previous research several genes involved in ˜cell adhesion™ as well as ˜epidermis development™ were significantly up-regulated in SCC compared to normal and AC [26] which is in accordance with our results. The majority of cell adhesion genes (predominantly desmosomal genes) and epidermis development genes have been found to be significantly up-regulated in SCC compared to normal tissue and the AC subtype. For example desmosomal genes (DSC3 and DSG3) and epidermis development genes (KRT5) were increased in SCC compared to the AC subtype. Our results strengthen the importance of ˜cell adhesion™ and ˜epidermis development™ in distinguishing AC from SCC. It indicates that cell adhesion genes and epidermis development genes play central roles in the drug delivery and are promising targets for novel therapies. In biomarkers identified in this paper could be used to classify patients for the treatment of NSCLC. A classification based on the discovered biomarkers could help to supply potential information in clinical decision making. The identified gene-subtype logic relationships and GO terms may extend perception to disease mechanisms for NSCLC. In addition the targeted therapy agents may also be designed to interfere with the discovered biomarkers. However several biomarkers and GO terms have been less well understood yet which needs further experimental research. Materials and Methods Data source and data processing We use the specimens of GSE10245 (a Gene Expression Omnibus accession number for microarray data) GSE37745 GSE18842 and GSE28571 to form a microarray expression data which are available from National Center for Biotechnology Information (NCBI http://www.ncbi.nlm.nih.gov/). Each specimen is annotated with a phenotype property (AC SCC and Normal) (). The microarray expression data (see Appendix S2) contains the expression data of probes in specimens. The microarray expression data is converted into a binary probe data using the Microarray Suite 5 (Mas5) algorithm [42]. The Mas5 algorithm generates a p-value which assesses the reliability of the expression level for each probe and a detection call which is a three-valued discrete data of a p-value. Specifically if a p-value is less than then the detection call is ˜Present™; if a p-value is greater than and less than then the detection call is ˜Marginal™; if a p-value is greater than then the detection call is ˜Absent™. Probes are flagged ˜Marginal™ or ˜Absent™ when the detection of probes is not considered to be significantly reliable. Hence it is reasonable to consider that the probes with flag ˜Marginal™ or ˜Absent™ are not significantly detected. In this work we turn ˜Marginal™ and ˜Absent™ flags to ˜™s and turn ˜Present™ flags to ˜™s. A ˜0™ in the th row and th column of the binary probe data mean the th probe is not detected in the th specimen while a ˜1™ indicates the probe is detected. Once converted the binary probe data is supplemented with an additional phenotype profile data. The phenotype profile data has three rows and columns. The st nd and rd rows correspond to AC SCC and Normal specimens respectively (Appendix S2). The phenotype profile data represents the properties of phenotypes where a ˜1™ in the th row and th column of the phenotype profile data means the th specimen belongs to the th phenotype while a ˜0™ means not. The probes are associated to genes according to the information of GPL570 (a microarray chip)(see Table S7). According to the number of genes that a probe detects probes can be classified into three categories: probes detecting a single gene probes detecting more than one gene and probes detecting no genes. In Table S7 there are probes associated to a single gene probes associated to more than one gene and probes associated to no genes. We are focused on the probes associated to a single gene. The binary probe data contains rows describing the detection patterns of probes. Current relationship-inference method Calculating uncertainty coefficient The vector describes the vector via either Type or Type lower logic function (see ) i.e. and constitute a logic pair. A logic combination of the vectors and describes the vector via one of the eight higher logic functions (see ) i.e. and compose a logic triplet. Uncertainty coefficient for a vector pair or a vector triplet is a measure to describe to what extent a vector or a combination of two vectors predicts another vector [22]. .0094644.t003 Lower logic function of vector . Type Symbol Lower logic function Logic statement The value of is iff the value of is The value of is iff the value of is ˜™ denotes the function symbol of type of lower logic relationships where and represents the sign for the lower logic relationships. .0094644.t004 Higher logic function of vectors and . Type Symbol Higher logic function Logic statement The value of is iff the values of both and are The value of is iff the value of is or that of is The value of is iff the value of or that of is The value of is iff the values of both and are The value of is iff the value of is and that of is The value of is iff the value of is and that of is The value of is iff the value of is or that of is The value of is iff the value of is or that of is The value of is iff either the value of or that of is The value of is iff the values of both and are or ˜™ denotes function symbol of type of higher logic relationships where and represents the sign for the higher logic relationships. The value of represents how well for the vector is described by the vector under a lower logic function where and is the symbol for lower logic functions. The value of is calculated as follows (Matlab codes available in Appendix S3):(1)where is the entropy of and where is the probability of occurrence of and is either or . is the entropy of the vector . is the joint entropy of and and where is the probability of occurrence of . The uncertainty coefficient for given which is denoted by is the maximum of and . Referring from and we got . The value of ranges from to where means that is independent of and means that is completely determined by . We calculate the degree to which the logic combination of the vectors and (e.g. ) describes a third vector as follows (Matlab codes available in Appendix S3):(2)where ; and are the entropy of and respectively; is the symbol for higher logic functions; is the joint entropy of and . As similar with we have . The uncertainty coefficient for predicted by a logic combination of and is denoted by . is equal to the maximum of the following five values: . The value of ranges from to . A well known measure the confidence is used to select the greatest possible rules by which probes related with phenotypes from the set of all possible rules [43]. Here the set of all possible rules are lower/higher logic functions corresponding to the maximum lower/higher uncertainty coefficients. Suppose the vectors and follow the lower logic function where . The confidence of is calculated as: where and refer to the joint probability of occurrence of and for the vectors and respectively. Suppose vectors and follow the rule where then the confidence of the rule () is also the ratio of to and and refer to the joint probability of occurrence of and for the vector and vector . We calculate the confidence for two lower (or higher) logic functions with the same value of (or ). The higher the confidence of a logic function the higher the probability that vectors follow the logic type corresponding to the logic function. The value of measures how well approximates a sufficient condition for and the value of measures how well the combination of and approximates a sufficient condition for . We improve the logic analysis by taking the reverse uncertainty coefficients into consideration. That is given the and to be the final lower and higher logic functions respectively we calculate the value of and as follows (Matlab codes available in Appendix S3):(3)where is either or and and are the same as those in e.q (1).(4)where ; and are the entropy of vector and respectively; is the joint entropy of vector and . Calculating random uncertainty coefficient Let be the histogram of the vector . Suppose is the set of distinct elements of . For each is the number of times appears in where and is the number of elements in [44]. Given the vectors and the random uncertainty coefficient and is calculated using the following steps: Generate random vectors and . and maintain the same distribution of the vectors and (i.e. ). Compute where is the uncertainty coefficient for given in a trial. Compute where is the uncertainty coefficient for given in a trial. The calculation of and involves the following three steps: Generate random vectors and maintaining the individual distribution and pairwise distribution. The vector retains the position of its elements unchangeably. Note that could determine and . We generate and maintaining and . Compute where is the uncertainty coefficient for given the combination of and in a trial. Compute where is the uncertainty coefficient for the combination of and given in a trial. Identification of probe-phenotype lower and higher logic relationships Thresholds are defined here to separate lower or higher logic relationships from logic pairwise or triplet combinations. Let and be the thresholds of lower and higher logic relationships respectively. We calculate the random uncertainty coefficients of all probe-phenotype pairwise (i.e. a probe and a phenotype) and triplet combinations (i.e. a probe pair and a phenotype). is the maximum uncertainty coefficient of all probe-phenotype pairwise combinations and is the maximum uncertainty coefficient of all probe-phenotype triplet combinations. If the values of both and are higher than then approximates a necessary and sufficient condition for . There exists a lower logic relationship between and . If the values of both and are higher than and there are no lower logic relationships between either or and then the logic combination of vector and approximates a necessary and sufficient condition for . There is a higher logic relationship between the combination of and and . Statistical analysis A p-value is defined as a measure to reflect how well vectors related in the form of discovered logic pairwise or triplet combinations compared to chance relations. Here an actual uncertainty coefficient is compared to the random one in a random trial. The p-value of a discovered logic pairwise or triplet combination is equal to the number of random trials in which either one of the two random uncertainty coefficients of pairwise or triplet combination of random vectors in both directions is higher than the actual one divided by the total number of random trails. Specifically we compare and to the distribution of and where and are the random vectors of and . For each pair of and we calculate the random uncertainty coefficients and in random trails. We have the p-value of the discovered logic pairwise combination: where means the number of random trials in which either one of the following two items and is tenable. Similarly the p-value of the discovered logic triplet combination is where means the number of random trials in which either or is tenable. False discovery rate In order to evaluate a global significance value of the actual discovered logic pairwise or triplet combinations we measure a false discovery rate (FDR) [45]. Given the threshold of lower logic relationships we estimate the number of discovered random logic pairwise combinations with the mean uncertainty coefficients larger than the threshold by chance. We generate random independent data and extract discovered random logic pairwise combinations from each random data. The estimated number of false discovered logic pairwise combinations (denoted as ) is calculated as the mean number of discovered random logic pairwise combinations obtained from these random independent data. If is the number of actual discovered logic pairwise combinations then is a simple estimated positive FDR for the given threshold. We can scan all probe-phenotype pairwise combinations but it take too much time to scan all triplet combinations. Therefore we randomly select a fixed number of triplet combinations (e.g. of all possible triplet combinations) and extract higher logic relationships with respect to actual and random (denoted as and ) respectively. This process is repeated for times and the FDR is the mean value of . The Matlab codes are available in Appendix S3. Cross validation In a random trial a fixed number of columns corresponding to each phenotype are selected from the original probe binary data and phenotype data to form the random probe binary data and random phenotype data. We check whether a logic relationship could be obtained in the random trial. The above processes are repeated for times where represents the number of all random trials."

RANK are expressed in different cell types and tissues throughout thebody They were originally described for their essential roles in bone remodeling and theimmune system but have subsequently been shown to provide essential signals fromregulating mammary gland homeostasis during pregnancy to modulating tumorigenesisThe success of RANKLRANK research serves as a paragon for translational researchfrom the laboratory to the bedside The case in point has been the development ofDenosumab a RANKLblocking monoclonal antibody which has already helped millionsof patients suffering from postmenopausal osteoporosis and skeletal related events incancer Here we will provide an overview of the pathway from its origins to its clinicalrelevance in disease with a special focus on emerging evidence demonstrating thetherapeutic value of targeting the RANKLRANKOPG axis not only in breast cancer butalso as an addition to the cancer immunotherapy arsenalKeywords osteoimmunology RANKLRANKOPG malignant tumor targeted therapy DenosumabEdited byLinda ConnellyCalifornia University of Science andMedicine United StatesReviewed byDhivya R SudhanINTRODUCTIONUniversity of Texas SouthwesternMedical Center United StatesSophia HL GeeUniversity of Miami United StatesCorrespondenceJosef M PenningerjosefpenningerubccaSpecialty sectionThis was submitted toWomen™s Cancera section of the journalFrontiers in OncologyReceived February Accepted June Published August CitationMing J Cronin SJF and Penninger JM Targeting theRANKLRANKOPG Axis for CancerTherapy Front Oncol 103389fonc202001283In the œseed and soil theory was first proposed by Stephen Paget for tumor metastasesto distant ans When tumor cells œseeds leave their primary site of origin and spreador metastasize the microenvironment œsoil of the target an is usually favorable for tumorcell anchoring and expansion of metastatic cells Bone is not only the site for primary bonetumors such as giant cell tumors and osteosarcoma but is also one of the most common distantmetastatic sites for solid tumors such as multiple myeloma MM breast cancer prostate cancerand nonsmall cell lung cancer NSCLC suggesting that the bone environment can serve asœsoil for tumor development and might also serve as a œseed for further metastatic spread Recentresearch on the bone microenvironment and its involvement in cancer biology has focused on thefield of osteoimmunology which includes the crosstalk between bone stromal cells osteoblastsand osteoclasts and immune cells Identifying key players regulating bone homeostasis couldpave the way for potential therapeutic cancer targets in particular to break the vicious circle ofmetastasis to the bonesThe receptor activator of the nuclear factor kappaB ligand RANKL also known as TNFSF11together with its receptor RANK TNFRSF11A the decoy receptor osteoprotegerin OPGTNFRSF11B and the recently identified receptor Leucinerich repeatcontaining Gproteincoupled receptor LGR4 has been shown to play critical bottleneck functions not only inregulating bone metabolism but also in immunity and tumorigenesis In this review we will brieflyintroduce the key functions of the RANKLRANKOPG axis in maintaining bone homeostasisand regulating immunity Furthermore we will discuss the role of this pathway from primaryFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertumorigenesis to cancer metastasis with particular attention tobreast cancer and the hormonal control of this pathway We willalso discuss recent data pointing to the RANKLRANK axis as anovel therapeutic target in BRCAmutated breast cancers and asa novel promising cancer immunotherapy agentRANKLRANKOPG AND BONEHOMEOSTASISBone provides strength and structure protects vital ans storesminerals such as calcium and is essential in the productionof hematopoietic cells Bone homeostasis is maintained by thebalance between mainly two types of cells osteoblasts derivedfrom mesenchymal cells which build bone and osteoclastsderived from bone marrow hematopoietic precursor cells whichresorb bone Figure Osteoblasts act as both mechanicalsensors together with osteocytes and coordinators for the boneremodeling process which is controlled by local growth factorsand systemic factors for example calcitonin or sex hormonessuch as estrogen The pathological imbalance between boneformation and resorption leads to the development of local orsystemic bone diseases such as osteopetrosis and osteoporosis The interaction and communication between osteoclasts andosteoblasts is intricately regulated in feedback loops to maintainbone homeostasis and this constant remodeling process of thebone matrix is critical for healthy bone strength and efficienthematopoiesis RANK TNFRSF11A OFE ODFR TRANCER ODARCD265 and RANKL TNFSF11 TRANCE ODF andOPGL “ are a receptorligand pair of the TNF receptorsuperfamily discovered at the end of the last millennium and wereidentified as key regulators of osteoclast development and bonemetabolism Figure Factors that can induce boneresorption such as the sex hormone progesterone vitamin D3PTHrP IL1 IL11 IL17 or TNFα “ act on osteoblaststo induce RANKL expression which then binds to its receptorRANK on the surface of osteoclast progenitor cells inducing preosteoclast diï¬erentiation into multinucleated fullyfunctionalosteoclasts RANKL also plays an important role in the continuedsurvival and function of osteoclasts “ Figure RANKLis produced as a membranebound protein which can alsobe shed as a soluble trimeric protein SheddaseresistantRANKL mice have been generated in which soluble RANKLis undetectable in the circulation bone mass or boneFIGURE Role of RANKLRANKOPG axis on bone homeostasis and immune system RANKL is secreted by osteoblasts and osteocytes when stimulated byparathyroid hormone PTH vitamin D andor prostaglandin PGE2 RANKL binds to RANK on the membrane of osteoclast progenitors preosteoclasts whichresults in bone resorption by mature osteoclasts Osteoprotegerin OPG binds to RANKL thus inhibiting RANK signaling and bone resorption RANKRANKL alsoplays a role in immune cell regulation and the crosstalk between both systems termed osteoimmunology T cells can also express RANKL which can both act onpreosteoclasts but can also act on dendritic cells DCs to promote their survival and to prolong T“DC interactions DCs can exhibit modulating effects onRANKmediated osteoclastogenesis through the secretion of OPG HSC hematopoietic stem cell MSC mesenchymal stem cellFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerstructure was not aï¬ected during development in these mice butadult mice displayed reduced osteoclast numbers and increasedcancellous bone mass Importantly the bone loss caused byestrogen deficiency was unaï¬ected by the lack of soluble RANKLThus these data show that it is the membranebound formof RANKL which is largely responsible for the physiologicalfunctions of RANKL although the soluble form can contributeto bone remodeling in adult mice Osteoprotegerin OPG TNFRSF11B acts as a decoy receptorfor RANKL and is induced by estrogen IL4 or transforminggrowth factor beta TGF OPG competitively binds toRANKL thereby interfering with RANKL“RANK interactionsand blocking bone resorption “ The relative levels ofOPG and RANKL are precisely controlled to ensure healthybone During pathological conditions such as menopauserelatedosteoporosis decreased estrogen levels result in decreased OPGand subsequently increased RANKL resulting in enhancedosteoclast activation and bone loss Recently leucinerichrepeat G proteincoupled receptor LGR4 was identifiedas an additional receptor for RANKL Similar to RANKLGR4 is expressed on osteoclasts but unlike RANK LGR4 is anegative regulator for osteoclast diï¬erentiation Therefore bothOPG and LGR4 are endogenous inhibitors of RANKLRANKsignaling A recent study has shown that RANKL reversesignaling from osteoclasts to osteoblasts couples bone resorptionto bone formation processes This is achieved through thesecretion of small extracellular vesicles from osteoclasts thatcontain RANK The authors showed that these RANK vesiclesbind membranebound RANKL on the osteoblasts and therebypromote bone formation by triggering RANKL reverse signalingvia activation of Runtrelated transcription factor Runx2Targeting RANKL reverse signaling represents a novel strategyto avoid the reduced bone production associated with inhibitionof osteoclastogenesis As RANKL is an important regulator of bone loss in bonemetastases associated with cancers such as multiple myelomaand in postmenopausal osteoporosis a specific fully human IgG2monoclonal RANKL antibody mAb has been developed whichneutralizes the activity of RANKL which has been designated asDenosumab The efficacy of Denosumab has been confirmed inmultiple clinical trials and Denosumab therapy is now approvedand widely used for the treatment of various boneassociateddiseases “RANKLRANKOPG IN THE IMMUNESYSTEMApart from bone homeostasis the RANKLRANKOPG axis isalso involved in various physiological immune processes RANKwas originally discovered on dendritic cells DCs and RANKLmediates the survival of DCs The interaction betweenactivated T cellderived RANKL and RANK expressed on DCsincreases the antigenpresenting capabilities of the latter thusaugmenting the number and cell cycle of antigenspecific Tcells as well as enhancing the immune response of memory Tcells Interestingly phenotyping of rankl and rankdeficient micerevealed a complete absence of peripheral lymph nodes but intactspleen and Peyer™s plaque structures “ Subsequent studieshave found that during embryogenesis RANKL is expressedby hematopoietic lymphoid tissue inducing LTi cells andmesenchymallymphoid tissue anizer LTo cells “RANKL has been demonstrated to stimulate lymphotoxin LTexpression and regulate LTi cell accumulation FurthermoreRANKL also triggers the proliferation of adult lymph nodestroma indicating that RANKL may directly activate LTo cells“ In the thymus the RANKLRANK pathway is criticalfor CD80 AIRE medullary thymic epithelial cell mTECmaturation involved in central immune tolerance RANKdeficient mice display mild autoimmunity at an advancedage RANKLRANK activation in lymphatic endothelialcells LECs is important for the tissueresident macrophagesnamely sinusoidal macrophage maturation not only duringembryogenesis but also after inflammationinduced loss of thesecells Moreover group innate lymphoid cells ILC3s inthe intestine use RANKLRANK interactions to control theirown abundance and intestinal homeostasis Genetic ablation ofRANKL specifically in IL3C cells leads to an increased number ofthese cells with enhanced levels of proinflammatory cytokinessuch as interleukin17A IL17A and IL22 during intestinalinfection Human patients carry RANK mutations and mice lackingRANKL or RANK exhibit a defect in B cell developmentresulting in a significant reduction in B cell numbers however these eï¬ects might be indirect because in themousetissuespecific deletion of Rank in B cells showedno diï¬erence in function nor development of B cells andblocking RANKRANKL with Denosumab does not apparentlyaï¬ect B cell physiology in osteoporosis patients In addition reports using Bcellspecific rankldeficient micehave shown that B cellderived RANKL increases osteoclastnumbers and bone loss brought on by estrogen deficiency Overexpression of RANKL in keratinocytes results in functionalalterations of epidermal dendritic cells and systemic increases inregulatory CD4CD25 T cells Tregs numbers Thereforeenvironmental stimuli can rewire the local and systemic immunesystems via RANKL The RANKLRANK system is alsoinvolved in M microfoldcell development a specific antigensampling cellular subtype found in the intestine as mesenchymalcells produce RANKL that can directly interact with intestinalepithelial cells to regulate M cell diï¬erentiation “Inhibition of mesenchymal RANKL impairs M celldependentantigen sampling and B celldendritic cell interaction in thesubepithelial dome SED resulting in decreased IgA productionand microbial diversity In addition B cells are absentin cryptopatches CPs and isolated lymphoid follicle ILFsformation was abrogated in rankl null mice Whether B cells or T cells are essential for bone loss isstill controversial Ovariectomy has been shown to enhance Tcelldependent TNFalpha production in a bone loss mousemodel because of the enhanced macrophage colonystimulatingfactor MCSF and RANKL In contrast anotherstudy suggested T cells are not involved in ovariectomyinducedFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancertrabecular bone loss Nevertheless it has been reportedin postmenopausal women that increased T cell activity andincreased RANKL production by T cells are associated withosteoporosis Furthermore studies in conditionalknockout mice to specifically eliminate RANKL in B cells or Tcells have shown that RANKL produced by B cells but not T cellsleads to bone loss by the induction of osteoclastogenesis Thelack of mature B cells does not prevent bone loss suggestingthat RANKL is derived from immature B cells Moreover it hasbeen reported that deletion of rankl in T cells does not change thenumber of T cells but results in impaired mature B cell numbersin the bone marrow suggesting that RANKL might promote Bcell maturation via paracrine signaling RANKLRANKLOPG IN MAMMARYGLAND PHYSIOLOGY AND BREASTCANCERBreast cancer is the most prevalent female malignancy Studies based on large populations have shown that womenwho receive estrogen plus progesterone hormone replacementtherapy called combined HRT are more vulnerable to breastcancer compared to women who receive estrogen only “furthermore progesterone levels have been demonstrated to bean independent risk factor for increased breast cancer incidence In rankl knockout mice our group was the first to reportthat during pregnancy RANKL deficiency results in a totalblock in the development oflobuloalveolar milksecretingstructures Whereas estrogen triggers the expansion ofthe mammary epithelium in puberty progesterone drives theproliferation of mammary epithelial cells in the estrous cycleand in pregnancy induces the growth and diï¬erentiation of themammary epithelium into ultimately milksecreting acini Figure Mechanistically progesterone induces progesteronereceptor PRpositive mammary epithelial cells to expressRANKL resulting in the proliferation of neighboring RANKmammary epithelial progenitor cells in an autocrine and alsoparacrine fashion “ Moreover RANKL can induce theproliferation of RANKpositive ductal epithelial cells through theFIGURE RANKRANKL pathway in mammary gland physiology and breast cancer A RANK is constitutively expressed on the membrane of luminal and basalepithelial cells including mammary stem cells MaSCs Stimulation with progesterone induces RANKL expression and secretion in progesterone receptor PRpositiveluminal epithelial cells RANKL binds in an autocrine fashion to RANK on luminal epithelial cells which stimulates further RANKL expression and in a paracrine fashionto RANK on basal epithelial cells resulting in enhanced RANK expression on basal mammary epithelial cells and the activation of the IKKαNFκB“cyclin D1 signalingaxis to induce a variety of physiological responses necessary for mammary gland development B Heterozygous BRCA1 mutationcarrying women canspontaneously lose the remaining wildtype BRCA1 gene from somatic mutation or epigenetic silencing Subsequently loss of BRCA1 protein can result in increasedgenomic instability DNA damage and genetic mutations eg TP53 Progesterone as well as synthetic progestins upregulate RANKL expression in PR luminalbreast epithelial cells which stimulates RANKmediated cell proliferation of adjacent progenitor cells as discussed in A Altogether the genotoxic stress and amplifiedproliferation cues culminate in uncontrolled proliferation and the development of breast cancerFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerinduction of Rspondin Therefore RANK and RANKL linksex hormones to mammary progenitor cell proliferation duringthe estrous cycle and in pregnancy Figure Clinically an increase in serum progesterone and RANKLlevels is associated with an increase in breast cancer risk inpostmenopausal women Higher concentrations of solubleRANKL are positively correlated with an increased risk ofestrogen receptorpositive but not estrogen receptornegativebreast cancer indicating that the RANKRANKLOPG axis maybe involved in the tumorigenesis of ER breast cancer Indeed in a hormoneinduced spontaneous mouse breast cancermodel RANKL is critical for the development of sex hormonedriven breast cancer Deletion of RANK and Ikkα akey downstream regulator of the RANK signaling pathway inmammary epithelial cells also significantly delayed progestinMPA and DNAmutation DMBAinduced mammary tumorformation further indicating that the RANKRANKL pathwaydrives breast cancer Furthermore the selective inhibitionof RANKL by RANKFc not only attenuated breast tumorprogression in a hormone and carcinogendriven mouse breastcancer model but also decreased the progression of breast cancerin a transgenic spontaneous tumor model BRCA1 and BRCA2 mutations are the most prevalent geneticdrivers for hereditary breast cancer in humans Interestinglywomen with germline BRCA12 mutations usually exhibithigher progesterone and estrogen levels during the gestationalphase ofthe estrous cycle compared to women withoutthese mutations Inversely decreased serum OPG levelsare associated with increased breast cancer incidence Moreover high levels of RANK expression were observed inbreast cancer samples from premalignant lesions and patientswith BRCA1 mutations SNP data analysis from theCooperative Tumor GeneEnvironmental Research iCOGSincluding approximately BRCA1 and BRCA2mutation carriers identified SNPs which were significantlyassociated with breast cancer risk at the TNFRSF11A locusencoding RANK Altogether these human data stronglysupport the idea that the RANKLRANKOPG axis is intimatelyinvolved in the tumorigenesis of BRCA12 mutationdrivenbreast cancerSubsequent animal studies provided direct evidence thatRANKL and RANK are critically involved in the oncogenesis ofBRCA1 mutationdriven hereditary breast cancer Figure Genetically engineered mice carrying Brca1 and Tp53 mutationsshowed hyperproliferation and malignancy in their mammaryglands at months of age the inactivation of the RANKLRANKpathway in these mice largely prevented the occurrence ofmalignanttumors and resulted in significantly prolongedsurvival Additionally the pharmacological blockade of RANKLusing RANKFc completely abolished the development ofprecancerousin the Brca1Tp53 doublemutatedbreast cancer model Amplification of RANKexpressingmammary duct progenitor cells can be found in the nontumorbreast tissue of BRCA1 mutant carriers and these cells havesimilar molecular characteristics as basallike breast cancercells RANKL inhibition also significantly suppressedthe proliferation oftumor anoids derived from BRCA1mutant human breast biopsy specimens and RANKLRANKlesionspathway blockade strongly reduced tumorigenesis in patientderived xenograft PDX breasttumor mouse model Thus independent work among diï¬erent laboratories usingdiï¬erent mouse models as well as studies using humanbreast epithelial progenitor assays hasled to the sameconclusion RANKLRANK aï¬ect mammaryprogenitorcells and are critically involved in the BRCA1mutation drivenmammary tumorigenesisTherefore we and others have proposed that the monoclonalantibody Denosumab which specifically inhibits RANKRANKLinteractions could potentially be used for the prophylactictreatment of breast cancer in BRCA12 carriers Indeed weposit that healthy women with BRCA1 mutation will benefit notexcluding an eï¬ect on other TNBCs In a pilot clinical studytermed BRCAD the proliferation marker Ki67 was significantlydownregulated in the breast biopsy of BRCA1 mutation carrierswho received shortterm treatment with Denosumab suggestingthat RANKL inhibition may be a feasible method for the chemoprevention of breast cancer in women with BRCA1 mutationsThis study requires additional patient data which is currentlyongoing Another clinical study DBEYOND which aimedto investigate whether neoadjuvant RANKL inhibition therapycan reduce tumor proliferation in premenopausal early breastcancer patients found no significant change in Ki67positivetumor cells in the breast cancer tissues treated with Denosumabbut the density of tumorinfiltrating lymphocytes TILs wasincreased in the stroma and tumor tissues upon Denosumabtreatment In addition to the now experimentally well validated role ofRANKLRANKOPG in the sex hormone and BRCA1 mutationdriven mammary cancer tumorigenesis it has also been reportedthat this pathway can induce epithelialmesenchymal transitionEMT in breast cancer cells as well as in prostate andendometrial cancers “ suggesting that RANKLRANKsupports tumorigenesis in various epithelial cancers Moreoverour group has recently reported on the role of RANKL andRANK in lung cancer We demonstrated that the inactivation ofrank in lung epithelial cells disrupts mitochondrial bioenergeticsand significantly reduceslung cancer development bothculminating in increased survival This genetic modeling inthe mouse supports findings in human clinical trials in whichRANK inhibition with the monoclonal antibody Denosumabresulted in prolonged survival especially in patients withnonsmall celllung cancer NSCLC adenocarcinomas andsquamous tumors Notably this Denosumabdependent survivaladvantage occurred in lung cancer patients irrespective of visceralmetastasis hinting that the underlying eï¬ects of RANKLRANKblockade in addition to those targeting the bone are involved Epidemiological reports have also uncovered genderdiï¬erences particularly in lung cancer with respect to etiologyprogression and treatment response believed to be due tosexrelated hormonal factors “ though the underlyingmolecular mechanisms are poorly understood We have recentlyshown in our experimental lung cancer model that by ablatingthe sex hormones in female mice we could eï¬ectively eliminatethe survival advantages brought about by loss of rank in the lungtumors Furthermore synthetic progesterone MPAdependentenhanced lung cancer initiation required RANK expressionFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in CancerTogether these data suggest that the sex hormone regulation ofRANKLRANK could also explain the gender diï¬erences seen inhuman lung cancerRANKRANKL AS REGULATORS OFMETASTASISStudies have now shown that the RANKLRANKOPG axis playsa role in the progression of malignant tumors by promotingtumor cell migration stimulating tumor neovascularizationand promoting distant metastasis of tumor cells Disseminated tumor cells are responsible for the earlymetastasis of tumors which frequently can be detected inthe bone marrow of patients with malignant tumors Thisœmicrometastases niche forms a favorable microenvironmentfor the development of metastatic spread protecting cancer cellsfrom various antitumor treatments and modulating anticancerimmune responses thereby allowing the tumor cells to escapeimmune surveillance The tumor microenvironment is acomplex milieu composed of distinct factors such as cytokinesextracellular matrix components and various cell types suchas fibroblasts endothelial cells and immune cells all ofwhich participate in cancer development progression andmetastasis In bone tissue the tumor microenvironmentincludes immune and tumor cells as well as osteoblasts andosteoclasts all of which participate in a œvicious cycle thataccelerates osteolysis and cancer cell proliferation through inpart the RANKRANKLOPG axis For instance cancercells can increase the expression of RANKL in osteoclastsby secreting parathyroid hormonerelated peptide PTHrP Tumor cells can also directly express RANKLand secrete cytokines such as interleukin IL1α TNFα macrophage colonystimulating factor MCSF orprostaglandin E2 PGE2 all of which promote osteoclastdiï¬erentiation and survival resulting in local osteolysis whichsupports metastatic growth “ Subsequently growthfactors released by the bone matrix such as insulinlike growthfactors IGFs fibroblast growth factor FGFs plateletderivedgrowth factor PDGF or bone morphogenetic proteins BMPspromote cancer cell proliferation “ In addition tocytotoxic drugs and endocrine disruptive drugstherapiestargeting the RANKRANKLOPG axis exhibit direct andorindirect antitumor eï¬ects by blocking the vicious cycle betweenbone and cancer cells “In a murine model of melanoma metastasis it was foundthat for malignant tumors with RANK expression RANKLproduced by osteoblasts and bone marrow stromal cells couldact as a chemical attractant and promote the migration andmetastasis of malignant tumors to these sites Similareï¬ects were also found in malignant tumors such as breastcancer “ prostate cancer “ and lung cancer The activation of phospholipase C PLC proteinkinase C PKC ERK and phosphatidylinositol3OH kinasePI3K pathways were involved in RANKinduced tumor cellmigration “ RANK engagement by RANKL inducestrimerization of the RANK receptor which then stimulates therecruitment and activation of the adapter protein TRAF6 viaTRAF6binding sites in the Cterminus of RANK™s cytoplasmictail TRAF6 in turn complexes with many other downstreamadapters and kinases to activate the aforementioned pathwaysMoreoverthe RANKLRANK pathway was also shown topromote the formation of new blood vessels and regulate thetumor microenvironment at the primary tumor site to promotethe migration of tumor cells into the bloodstream and formetastasis to distant ans “In breast cancer RANKL is also produced by Foxp3expressing Tregs and tumorassociated macrophages TAMsthat can aï¬ect tumor growth tumor cell dissemination andmetastasis RANKL expression on tumorinfiltratingregulatory T cells may also be involved in cancer metastasis TAMs are either M1 or M2 macrophages with M1 being antitumor and M2 TAMs promoting tumorigenesis ImportantlyM2 macrophages express RANK and are attracted by RANKLproduced by the tumor microenvironment The RANKLRANKpathway in M2 macrophages can regulate the production ofchemokines and promote the proliferation of Treg lymphocyteswhich supports the immunosuppressive milieu within the tumormicroenvironment Recently it has been reported that estrogenrelatedreceptoralpha ERRα an important factor of cancer cell invasivenesspromotes breast cancer cell dissemination from primarymammary tumors to the bone Intriguingly RANK hasbeen shown to be a target for ERRα Furthermore the metaexpression analysis of breast cancer patients has uncovereda positive association between metastases and ERRαRANKexpression as well as a positive correlation between ERRαand BRCA1 mutation carriers revealing a novel pathwaywhereby ERRα in primary breast cancer could promote earlydissemination of cancer cells to bone Moreover it wasrecently shown that RANKL serum levels are significantlyincreased in breast cancer patients who developed bonemetastases p and patients within the highest quartileof RANKL had a significantly increased risk of developing bonemetastases compared to those in the lowest HR 95CI“ p This study further suggests a role ofRANKL in breast cancer metastasis TARGETING RANKLRANK IN HUMANCANCERIn light of the diï¬erent roles of the RANKLRANK pathwayin bone metabolism and immune system functions therapytargeting this axis may not only control primary tumordevelopment such as in the case of breast cancer and reducebone metastasis which has been demonstrated in clinical trials but also exert a direct antitumor eï¬ect via regulatinglocal tumorassociated immune responses as observed in studiesusing the monoclonal RANKL antibody inhibitor Denosumab In randomized clinical trials Denosumab has shown rapideï¬ectiveness by directly impairing osteoclast activity andinducing osteoclast apoptosis Moreover Denosumabwassignificantly more eï¬ective in reducing urinary Nterminal peptides a biochemical marker for bone turnoverFrontiers in Oncology wwwfrontiersinAugust Volume 0cMing et alRANKLRANK in Cancerand more eï¬ective in delaying skeletalrelated events SREssuch as pathologic fractures spinal cord compression andhypercalcemia which greatly aï¬ect quality of life in patients withbreast cancer and castrationresistant prostate cancer CRPCbone metastases However the eï¬ect of Denosumab to delaySREs in patients with NSCLC and multiple myeloma MMpatients with bone metastases is comparable to bisphosphonatedrugs “ Moreover the benefit of Denosumab andbisphosphonates is not only restricted to osteolytic cancers suchas breast myeloma and NSCLC but also evident in osteoblasticcancers Recently it was demonstrated in osteoblastic cancerssuch as prostate cancer that Denosumab or bisphosphonate canaï¬ect the osteoclastosteoblast balance in the œvicious cycle ofbone destruction induced by metastasized cancer cells which highlights the potential rationale in treating osteoblasticcancer patients with Denosumab or bisphosphonatesIn a randomized phase III clinical trial comparing Denosumaband bisphosphonate zoledronic acid ZA in patients with solidtumors breast cancer prostate cancer multiple myeloma andbone metastases the results showed that Denosumab was similarto ZA in preventing or delaying the onset of primary SREs However in nonsmallcell lung carcinoma NSCLCn treatment with Denosumab showed a significantimprovement in overall survival In these patients nostatistically significant SRE delay was observed in Denosumabtreated patients suggesting that this survival advantage maybe independent of the bone system The result of arandomized phase III trial of multiple myeloma MM patientsn also demonstrated the eï¬ectivity of Denosumab toreduce the occurrence of primary SRE events moreover the useof Denosumab significantly improved progressionfree survivalPFS Whether this survival benefit is due to the decreasein the incidence of bone metastasis or whether Denosumab hasother antitumor eï¬ects requires further researchIn the randomized placebocontrolled phase III ABCSG18trial which enrolled postmenopausal female patients withearly hormone receptorpositive breast cancer the first clinicalfracture of the Denosumabtreated group was compared with theplacebo group and a significant protection of bone breaks wasdemonstrated hazard ratio [HR] · [ CI ·“·] p A median followup of months showeda significant improvement in the diseasefree survival DFS inthe Denosumabtreated group HR CI “Cox p These data suggest that adjuvant Denosumabcan significantly improve the DFS rate of HR postmenopausalbreast cancer patients However in another randomizedphase III clinical trial of breast cancer DCARE recent reportshave shown that adjuvant Denosumab does not reduce therisk of breast cancer recurrence or death in earlystage breastcancer patients receiving standard adjuvant therapy Theseinconsistencies which could be explained by diï¬erent cohortsfor patient stratifications eg more advanced early cases ofbreast cancer were included in the DCARE trials as comparedto the ABCSG18 study need to be further evaluated with largercohorts of patients and multiplecenter analysis Importantlya recent followup study of the ABCSG18 trial confirmed thethat blocking RANKL in an adjuvant breast cancer therapysetting not only markedly reduces the risk of breaking bones butalso significantly reduces the reoccurrence of the breast tumors It should be also noted that although there was nodiï¬erence in bonemetastasesfreesurvival in the DCARE trialDenosumab treatment significantly reduced the time to bonemetastasis at the site of first occurrence DENOSUMAB AS A NOVEL CANCERIMMUNOTHERAPYThe field of cancer immunotherapy has paved the way for a newparadigm to combat cancer by coaxing the body™s own immunesystem to seek out specifically target and destroy cancer cellsAmong the various approaches immunecheckpoint inhibitorsthattarget CTLA4 as

" tumor associated macrophages tam constitute the most abundant immune cells in the tumor stroma initiating pro inflammatory m1 or immunosuppressive m2 responses depending on their polarization status advances in tumor immunotherapy call for a detailed understanding of potential immunogenic mechanisms of irradiation routinely applied in rectal cancer patientsmethods to test the effects of radiotherapy on tam we ex vivo irradiated tissue samples of human rectal cancer and assessed the phenotype by flow cytometry we furthermore evaluated the distribution of leucocyte subsets in tissue sections of patients after short course radiotherapy and compared findings to non pretreated rectal cancer using an immunostaining approach anotypic assays ota consisting of macrophages cancer associated fibroblast and cancer cell lines were used to dissect the immunological consequences of irradiation in macrophagesresults we demonstrate that short course neoadjuvant radiotherapy in rectal cancer patients is associated with a shift in the polarization of tam towards an m1 like pro inflammatory phenotype in addition ex vivo irradiation caused an increase in the phagocytic activity and enhanced expression of markers associated with stimulatory signals necessary for t cell activation in ota we observed that this alteration in macrophage polarization could be mediated by extracellular vesicles ev derived from irradiated tumor cells we identified high mobility group box in ev from irradiated tumor cells as a potential effector signal in that crosstalks our findings highlight macrophages as potential effector cells upon irradiation in rectal cancer by diminishing their immunosuppressive phenotype and activate pro inflammation our data indicate that clinically applied short term radiotherapy for rectal cancer may be exploited to stimulate immunogenic macrophages and suggest to target the polarization status of macrophages to enhance future immunotherapeutic strategiesintroductionsince the introduction of immune checkpoint blockade immunotherapy has become an attractive therapeutic option in cancer1“ irradiation used as standard therapy in a number of solid malignancies induces immunogenic cell death icd by the release of damage associated pattern damp4 studies based on murine models indicated that irradiation induced dna damage of tumor cells elicited an antitumor immune response5 it was shown that alterations of the infiltrating immune cells by irradiation might be augmented when combined with immune modulating drugs6“ a detailed understanding of the impact of radiotherapy on the immune system in humans should allow application of radiotherapy as a part of novel immunotherapeutic concepts however little is known about the detailed regulation of the immunogenic effect of irradiation in clinical settingsmacrophages are one of the most abundant immune cell subsets in tumor tissue9 and play a key role in the cancer microenvironment11 tumor associated macrophages tam are functionally diverse13 and display high plasticity upon immunological stimuli14 the concept of m1 and m2 macrophages was introduced to describe the heterogeneity of this cell subset16 m1 like macrophages possess the capacity to clear infections in support of a t helper type th1 immune response17 whereas m2 like macrophages respond in general to th2 cytokines and are strongly enriched in the tumor microenvironment18 the concept of m1m2 has been challenged and is seen as an oversimplified approach to the phenotype of macrophages but can be viewed as a linear scale on which m1 and m2 present two extremes19 tam recognize damp and respond by producing a variety of cytokines and growth factors to promote innate and stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access adaptive immunity9 in response to these signals macrophages are able to undergo reprogramming with enhanced antitumor features making them an attractive target for anticancer therapies22 there are conflicting results with respect to the effect of irradiation on the macrophage phenotype klug et al demonstrated in a murine model of breast cancer and human pancreatic cancer that low dose irradiation gy induced repolarization of m2 like macrophages to m1 like macrophages via induction of nitric oxide synthase inos24 moreover expression of inos correlated with vessel normalization and an influx of cd8 t cells suggesting a tumor ablative as well as pro inflammatory effect of repolarized macrophages upregulation of inos was also observed in murine prostate cancer which has been irradiated with up to gy25 similarly agonists of the toll like receptor further stimulated the m1 phenotype of macrophages and enhanced the antitumor effects of irradiation in a murine model of breast cancer26 other studies suggested that irradiation of tumors was associated with a more immunosuppressive phenotype of tam27“ jones found that the depletion of macrophages by an anti csf antibody greatly increased tumor ablation upon irradiation gy in murine tumors generated from a colorectal and a pancreatic cell line obviously the effect of irradiation appears to depend on the model irradiation dose tissue as well as on the investigated time point after irradiation however despite the controversial reports an important role for tam in response to radiotherapy seems evidentmore recently extracellular vesicles ev have attracted attention in mediating signals to immune cells ev are rich in molecular cargos and are emerging as critical messengers in the cell to cell crosstalk they contain a variety of small signaling molecules which can be transferred to other cell types to modulate cell functions30“ thus we hypothesized that ev might be involved in macrophages regulation in the tumor microenvironment in irradiated cells using a clinically relevant approach we show that short course irradiation increased the proportion of m1 like tam in human rectal cancer tissue primary short term cultures and anotypic tumor assays ota consisting of tumor cell lines macrophages and cancer associated fibroblasts allowed to further dissect irradiation induced changes in colorectal tam using irradiated tumor cell lines we demonstrate that ev are able to mediate irradiation induced repolarization of macrophagesmethodspatients and tissue materialpatients with clinical t3 rectal cancer patients characteristics online supplementary table received neoadjuvant hyperfractionated radiotherapy over the course of days within the frame of a controlled clinical study34 as a control group we used historical surgical tumor specimen of non pretreated rectal cancer lesions of patients from the same institution with no history of irradiation therapy or cytoablative treatment for immunofluorescence and immunohistochemical stainings surgical paraffin embedded specimen of the cancer lesions were cut in µm sections and mounted on slides for ex vivo cultures and subsequent flow cytometric stainings rectal cancer tissue was obtained from patients with histologically verified rectal cancer with no history of irradiation therapy or cytoablative treatment studies involving patient material were performed according to the declaration of helsinki and approved by the local ethics committee of the medical university of vienna for ota hct116 ccl147 and dld1 ccl221 were purchased from atcc peripheral blood mononuclear cells for macrophage differentiation were isolated from healthy blood donorsprimary ex vivo cultures of leucocytes and flow cytometrytissue samples of non irradiated rectal cancer were minced resuspended in rpmi medium with fetal bovine serum and plated in a mm petri dish irradiation protocol was applied as indicated below after hour of incubation °c co2 a single cell suspension from cancer tissue was prepared briefly tissue was digested with collagenase iv unitsml and dnase i mgml for hour min in rpmi supplemented with fetal bovine serum and hepes buffer solution at °c afterwards cell suspension was rinsed through a µm mesh and leucocytes were isolated by density gradient centrifugation using ficoll gradients for flow cytometry analysis cells were stained with fluorescence antibodies listed in the online supplementary table livedead discrimination was performed with fixable viability dye biolegend samples were acquired on a facsaria iii bd and analyzed with flowjo software v1061irradiation protocola theratron mds nordion radiotherapy unit with a cobalt60 source was used for γirradiation of ota ex vivo cultures of rectal cancer tissue monocyte derived macrophages and cancer cell lines tissue samples of rectal cancer were minced and cultivated petri dishes with cancer tissue were irradiated with × gy after hours of incubation a single cell suspension was prepared ota and cancer cell lines hct116 and dld1 were irradiated with × gy or × gy and subsequently cultivated over the course of hours monocyte derived macrophages were irradiated with gy or gy a negative control gy was always transported to the radiotherapy unit but was not exposed to γirradiationex vivo phagocytosis assaymononuclear cells isolated of rectal cancer lesions were spun down at g for min at °c for ex vivo escherichia coli e coli phagocytosis assay irradiated and non irradiated leucocytes were resuspended in µl phrodo red e coli bioparticles thermo fisher scientific as per the manufacturer™s instruction a same set of cells were resuspended in phosphate buffered saline pbs as controls after hours incubation at °c stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessleucocytes were washed and stained for flow cytometry tam cd11b cd14 hla dr viable cd45 cells that were pe high were considered to be phagocytosing for ex vivo tumor cell phagocytosis assay tam were isolated via fluorescence activated cell sorting facs cd11b cd14 hla dr viable cd45 cells from healthy colon mucosa or colorectal cancer lesions and incubated overnight with ev isolated from irradiated × gy and non irradiated gy dld1 cells evtam after washing dld1 were labeled with carboxyfluorescein diacetate succinimidyl ester cfse thermo fisher µm and cocultured with tam at an effector to target cell ratio of the proportion of cfse tam was assessed by flow cytometry after hours of incubation at °cat the air liquid interface in dmem supplemented with egm for seeding of tumor cells nylon discs with collagen cell cylinders were transferred into well plates five hundred microlitres of a tumor cell suspension with × cellsml hct116 or dld1 were added to each well after hours the tumor cells attached to the surface of the collagen gels and the nylon meshes with the tumor cell colonized collagen cell cylinders were put back onto the metal grids and incubated for days with media changes every days prior to further experiments for further assessment ota were embedded in optimal cutting temperature media snap frozen in liquid nitrogen and stored at “°c until processing or fixed and embedded in paraffinimmunofluorescence and immunohistochemical stainingdirectly and indirectly labeled monoclonal antibodies as listed in online supplementary table and dapi as a nuclear marker were used an isotype was used as negative control in brief after incubation with the primary antibodies overnight at °c an appropriate secondary fluorescence labeled antibody was applied for min at room temperature followed by staining of dapi nuclear marker for immunohistochemical staining antibodies were mixed with bovine serum albumin in pbs and applied overnight to sections in a humid chamber at °c for visualization of the cells aec was used as chromogen sections were counter stained with hematoxylin for evaluation of staining results images of whole tissue sections one section per patient were acquired using a z1 axio observer microscope equipped with a ld plan neofluar × objective zeiss for leucocyte evaluation the tumor normal interface mm on tumor and normal zone was defined as regions of interests roi for γh2ax staining only tumor tissue was selected as roi roi were automatically quantified using tissuefaxstissuequest image analysis software tissuegnostics gmbhpreparation of otaota were set up as previously described35 briefly cancer associated fibroblasts caf were isolated and cultured from fresh samples of colon adenocarcinomas for the preparation of macrophages monocytes were isolated using the easysep direct human monocyte isolation kit stemcell technologies according to the manufacturer™s instructions × caf and monocytes per ota were mixed and pelleted by centrifugation at g for min for the collagen gel preparation all steps were performed on ice collagen solutions were prepared by mixing mgml of collagen i rat tail mgml becton dickinson and the fibroblastmonocyte suspension in dulbecco's modified eagle medium dmem supplemented with endothelial cell growth medium mv2 egm promocell a total of µl each of the collagen cell suspension were transferred into silicone gel casting devices after polymerization of the collagen solution the gels were lifted up on nylon mesh discs and transferred onto metal grids in well plates and cultured macrophage differentiationpbmc were obtained by ficoll plaque density gradient centrifugation pbmc were seeded at a concentration of ×106well well plates in rpmi medium monocytes were isolated using the ability of monocytes to adhere to non tissue culture treated plastic culture dishes attached cells were cultivated in rpmi medium with glutamax thermo fisher scientific supplemented with ngml macrophage colony stimulating factor m csf thermo fisher scientific fetal bovine serum uml penicillin uml streptomycin and µgml fungizone in a humidified atmosphere at °c cells were cultivated for days with two medium changes to obtain m1 lpsifnγ polarized macrophages cells were stimulated with ngml lipopolysaccharide lps sigma aldrich and ngml interferonγ ifnγ thermo fisher scientific for hours m2 il4il13 polarized macrophages were generated using ngml interleukin il4 strathmann and ngml il13 biolegendev preparation and characterizationev were isolated by differential centrifugation hct116 and dld1 cells were cultured in mccoy™s 5a medium supplemented with exosome depleted fetal calf serum briefly cancer cell line culture medium was centrifuged at g for min to pellet cells supernatant was transferred to new falcon tubes and then centrifuged at g for min to pellet dead cells and apoptotic bodies rotanta 460rc hettich supernatant was transferred to the high speed centrifugation tubes and large ev were removed by ultracentrifugation at g for min sorvall evolution rc thermo fisher scientific after filtration of the supernatant through a µm syringe filter small ev were pelleted by ultracentrifugation at g for min rotor t1250 suspended in ice cold pbs and collected after another ultracentrifugation at g for min rotor type sorvall wx ultra thermo fisher scientific the ev pellet was resuspended in cold pbs for further use ev count was determined using a nanoparticle tracking analyzer zetaview particlemetrix results were analyzed with zetaview software ev were added to macrophages at a stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access concentration of ×× cells we have submitted all relevant data of our experiments to the ev track knowledgebase ev track id ev20006537western blotcells were scraped from cell culture plates washed in ice cold pbs and lysed in × ripa buffer mm tris“hcl ph mm nacl np40 sodium deoxycholate sds containing × halt protease inhibitor cocktail thermo fisher scientific ev suspensions were mixed with × ripa buffer containing × halt protease inhibitor cocktail to obtain × concentration protein extracts were incubated at °c for min and centrifuged at g for min clear protein extract supernatants were quantified with the bca protein assay kit thermo fisher scientific one to five micrograms of protein were denatured with laemmli buffer loaded into each well of a acrylamide bisacrylamide gel containing sds and electrophoresed with magel for vhours proteins were transferred to a polyvinylidene fluoride membrane using the transblot turbo and the rta ready to assemble transfer kit bio rad after several hours of blocking pvdf membranes were incubated with primary antibodies online supplementary table at °c overnight after washing with tris buffered saline tween detergent membranes were incubated with the secondary antibody anti rabbit immunoglobulin g igg hrp linked antibody cell signaling technology or anti mouse igg hrp linked antibody cell signaling technology at room temperature for hour detection was done with clarity or clarity max western ecl substrate bio rad protein band intensities were quantified with imagequant tl ge healthcareelectron microscopyafter mounting cu mesh r22 holy carbon grids quantifoil with a tweezer into a leica gp leica microsystems grid plunger µl of ev sample solution were applied to grid™s front side and blotted for “ s grids were plunge frozen into liquid ethane for instant vitrification and transferred to a glacios cryo transmission microscope thermo fisher scientific equipped with a x feg images were recorded in low dose mode using the serialem software mastronarde with a falcon3 direct electron detector at magnifications of and with pixel sizes of and respectivelystatistical analysisstatistical analysis was performed using graphpad prism graphpad software statistical significance was determined by student's t test when comparing two groups the two way analysis of variance followed by tukey's multiple comparison test was used when comparing three or more groups significance was set at a p value of less than resultstam in rectal cancer polarize towards m1like phenotype upon irradiationwe first investigated the effect of irradiation on human tam in a primary ex vivo culture of rectal cancer specimen for this purpose minced tumor tissue was irradiated with gy and subsequently cultivated for hours before leucocyte isolation figure 1a the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer is shown in figure 1b tam as defined by cd14cd11bcd68hla dr accounted for over of all viable cd45 leucocytes in rectal cancer figure 1c with over cells per gram of tissue figure 1d there was no significant difference in percentage or absolute numbers of tam in non irradiated versus corresponding ex vivo irradiated tissue samples figure 1cd we then assessed the phenotype of treatment naïve and ex vivo irradiated tam using flow cytometry in naïve rectal cancer lesions tam were characterized by high expression of cd206 cd163 and cd64 and low levels of the chemokine receptor ccr7 indicating that the m2 like macrophage phenotype was present in untreated rectal cancer figure 1e this was also reflected in their cytokine pattern as more tam in untreated rectal cancer samples produced il10 il13 and il4 while few interferonγ ifnγ and tumor necrosis factor alpha tnfα producing tam were found in untreated samples in contrast ex vivo application of gy γirradiation to primary rectal cancer leucocytes resulted in a reduced presence of cd163 tam this phenotype correlated with enhanced levels of tnfα and inos tam as well as diminished detection of il10 and il13 tam hence low dose irradiation of rectal cancer tissue can polarize tam towards a pro inflammatory m1 like phenotype and may therefore directly contribute to antitumor activity by tnfα and inos productionlowdose irradiation reduces pd1 expression and enhances phagocytosis in tam to e colito further assess the distinct functional phenotype of tam on irradiation we investigated the ability of tam to phagocyte as an aspect of direct effector function and activation to model the phagocytic behavior of irradiated tam we incubated leucocytes derived from irradiated ex vivo tumor culture with ph sensitive pe labeled e coli particles in the phagosome the fluorescence of e coli particles increases as demonstrated in a representative example figure 2a phagocytosis by tam was significantly elevated upon irradiation with gy figure 2b demonstrating that the acquired shift towards m1 polarization due to irradiation was also functionally relevant as pd1 was previously shown to inhibit phagocytosis38 we next determined pd1 expression on irradiated ex vivo tumor culture derived tam we found variable pd1 expression on tam in non irradiated rectal cancer and observed a significant decrease of pd1 on tam derived from irradiated cultures figure 2c these stary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0cchanges induced by irradiation can be instrumental for phagocytic activity of tamirradiation promotes antigen presentation and cytokines associated with th1 responseone of the hallmarks of m1 polarization of macrophages is the acquisition of antigen presenting features leading to efficient th1 response17 as a first step to determine whether irradiation may result in improved capability of tam to initiate immune responses we analyzed the expression of cd86 on tam derived from irradiated ex vivo tumor cultures irradiation correlated with a significant increase of cd86 on the cell surface of tam as compared with tam derived from non irradiated naïve cultures figure 2d we next investigated hla dr on non irradiated tam versus tam irradiated with gy we observed hla dr low expressing tam and high expressing tam clearly dividing them into two groups figure 2e in non irradiated samples more than of tam expressed high levels of hla dr while irradiation significantly increased the fraction of hla drhigh tam to over figure 2f we next investigated whether irradiation can induce the expression of il12 p70 and il23 p19 as markers for the initiation of adaptive immune responses whereas il23 p19 showed a tendency to be produced by tam on irradiation il12 p70 was significantly induced in irradiated tumor tissue compared with open accesstreatment naïve samples figure 2g together these results suggest that low dose irradiation influences tam polarization in rectal cancers by equipping the cells with an hla drhiil12hi il23hi cytokine profile this observation emphasizes that irradiated tam have a higher probability to participate in antitumor immune responses directly by phagocytosis and cytokine secretion as well as indirectly by exhibiting a broad armamentarium that potentially activates t cells as main players of antitumor adaptive immunityshortcourse irradiation of patients with rectal cancer increases the m1m2 ratio of tamto corroborate our ex vivo data with the in vivo situation on short course irradiation we examined tam polarization and function in rectal cancer patients treated by a routinely applied radiotherapy protocol we made use of a cohort of patients in clinical stage t3 rectal cancer who received neoadjuvant hyperfractionated short course radiotherapy two times gy per day over the course of days figure 3a surgery was performed “ days after radiotherapy in these patients as a control we used surgical specimen from treatment naïve clinical t3 rectal cancer patients importantly the indication for radiotherapy was not correlated with a more severe tumor progression compared with the treatment naïve cohort figure ex vivo γirradiation induces polarization of tumor associated macrophages a tissue samples of naïve rectal cancer were minced and irradiated after incubation a single cell suspension of cancer tissue was prepared b representative example of the gating strategy for macrophages isolated from viable cd45 mononuclear cells of human rectal cancer c percentage of macrophages compared to total of viable cd45 cells in non irradiated and ex vivo irradiated rectal cancer lesions assessed by flow cytometry and presented as mean±sd n10 d mean numbers of macrophages in rectal cancer per gram in non irradiated and ex vivo irradiated rectal cancer presented as mean±sd n10 e expression of intracellular and extracellular markers in macrophages assessed by flow cytometry bars presented as mean percentage of indicated markers±sd of non irradiated and ex vivo irradiated macrophages n10 p005 p0001 p00001 by two tailed paired student™s t test gy gray tam tumor associated macrophages ifnγ interferonγ tnfα tumor necrosis factorα il interleukin inos inducible nitric oxide synthasestary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access irradiation increases phagocytosis and marker associated with antigen presentation and th1 response a figure representative histogram and flow cytometry plots indicating difference in pe fluorescence of non irradiated blue versus irradiated red tam in ex vivo phagocytosis assay total phagocytosis was analyzed by first gating on tam and then gating on pe cells b analysis of tam phagocytosis of phrodo e coli particles data presented as percentage of phagocytosis of non irradiated tam and corresponding irradiated tam gy n5 c percentage of tam positive for pd1 non irradiated versus ex vivo irradiated and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 d percentage of expression of cd86 in non irradiated versus ex vivo irradiated tam and representative histogram non irradiatedblue vs irradiatedred data presented as mean±sd n10 e representative plots histogram and f expression of hla dr on tam of non irradiated and ex vivo irradiated rectal cancer data given as mean±sd n10 g expression of il12 p70 and il23 p19 data presented as mean percentage±sd of total tam n10 representative example of il12 p70 and il23 p19 expression on tam p005 p0001 by two tailed paired student™s t test th1 t helper type gy gray tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen accessfigure short course irradiation in patients modulates the immune infiltrate with induction of macrophage polarization a irradiation protocol of patients with neoadjuvant hyperfractionated × gy per day over the course of five days radiotherapy for clinical t3 rectal cancer surgery was performed the following week b quantitative in situ assessment of infiltrating macrophages cd68 t cells cd3cd56ˆ’ nk cells cd56cd3ˆ’ and nkt cells cd56cd3 in non irradiated n25 and irradiated rectal cancer n45 assessed by multicolor immunofluorescence staining data are given as absolute numbers of positive cells per mm2±sd c analysis of immunohistochemical staining for γh2ax as irradiation response in non irradiated n25 and irradiated tissue sections data are given as mean±sd d representative examples of immunohistochemical staining for γh2ax images below are magnified × e representative immunohistochemical staining of cd68 cells in non irradiated n25 and irradiated tissue sections n45 images below are magnified × f representative example of immunofluorescence multicolor staining of cd68 macrophages pe m1inos fitc m2 cd163 apc g quantitative in situ analysis of immunofluorescence staining of ratio of inos m1 to cd163 m2 tams cd68 macrophages presented as m1m2 in non irradiated n25 compared to irradiated sections n45 data are given as mean±sd h percentage of il10 cells to total cd68 cells data are presented as mean±sd i representative image of multicolor immunofluorescence staining of il10 in macrophages cd68 peil10 fitc of irradiated rectal cancer p0001 p00001 by two tailed unpaired student™s t test gy gray inos inducible nitric oxide synthase tam tumor associated macrophages il interleukinstary a0v et a0al j immunother cancer 20208e000667 101136jitc2020000667 0copen access since the stage in the tnm classification of malignant tumors and cancer differentiation of the two cohorts were comparable online supplementary table to assess the composition of the leucocytic infiltrate and the impact of radiotherapy we evaluated the distribution of macrophages t cells nk cells and nkt cells via immunofluorescence staining and automated image analysis irradiated tumor sections showed significantly less infiltration of cd68 cells which correlated with a tendency for a decrease of cd3 t cells while the amount of nk cells cd56 cd3ˆ’ or nkt cd56 cd3 cells did not show significant differences figure 3b immunostaining of phosphorylated γh2ax was used to confirm radiotherapy induced dna damage in analyzed tumor specimen irradiated tumors revealed a significantly higher amount of γh2ax positive cells than non irradiated tissue figure 3c γh2ax foci were primarily located in the nuclei of tumor cells rather than in the cells of the tumor infiltrating microenvironment figure 3d in both patient cohorts cd68 cells accumulated in regions surrounding tumor cell clusters figure 3eto evaluate the in vivo effect of irradiation on macrophage polarization and to correlate results with ex vivo irradiated tumor specimens we determined the ratio of m1 like tam to m2 like tam by staining for inos and cd163 in cd68 cells irradiation was associated with higher numbers of inos expressing tam while cd163 tam were drastically reduced resulting in a significant increase in the m1m2 ratio figure 3fg similar to the ex vivo cultures we observed a sixfold decrease of il10 tam in tumors from irradiated patients as compared with treatment naive tumors figure 3hi these data are corroborating hallmarks of our ex vivo irradiation protocols with macrophages becoming active players within the tumor microenvironment upon γirradiationirradiation of anotypic cultures promote macrophage activation towards an m1like phenotypeto be able to further dissect the irradiation induced findings in patients tissue sections and ex vivo cultures we used anotypic cultures which were established with colorectal cancer cell lines hct116 or dld1 primary caf and peripheral blood derived macrophages online supplementary figure a in collagen i gels in contrast to primary cultures ota allowed culture periods of up to days to mimic the clinical protocol of short course irradiation ota containing either hct116 or dld1 were exposed to irradiation with two times gy or two times gy over the course of hours compared with non irradiated controls figure 4ab frozen tissue sections of ota were assessed for macrophage marker using immunostaining and automated image analysis the number of macrophages in ota was comparable and not significantly different among the distinct conditions online supplementary figure b non irradiated gy ota harbored macrophages resembling an m2 like phenotype of cd11bcd68 macrophages as indicated by high expression of cd163 figure 4cd low expression of ccr7 figure 4e and modest expression of il10 figure 4fg upon irradiation of ota cd163 and il10 were reduced whereas the expression of the pro inflammatory marker ccr7 was elevated thus the marker profile of ota correlated with those of primary ex vivo tumor cultures and surgical specimen following neoadjuvant irradiation these observa

" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [“] an rln countof ‰¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ‰¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ‰¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ‰¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1“t3“tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ˆ’ ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes ˆ’ positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd student™s t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor ci““p“reference“““age ‰¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ‰¥ glasa ‰¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3“rlnslymph node metastasislnr lodds ˆ’ ptnm stage ‰¥ iiilauren subtypeintestinaldiffusemixedunknown ci““““““““““reference““““reference““““““““reference“““p 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range “ and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were × ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ‰¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ‰¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ‰¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ‰¥ characteristicsunivariateormultivariateor cipreference““ ““age ‰¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ‰¥ glasa ‰¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3“lymph node metastasisptnm stage ‰¥ iiilauren subtypeintestinaldiffusemixedunknown ci““““““““““reference““““reference“““““reference“““p bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ‰¥ deng proposed an optimal rln count of ‰¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[“] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [“] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors™ contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ™ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol “son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer “li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol “ aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol “koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet “zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer “chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol “deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncol“amin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more œpersonalized approach tocancer staging ca cancer j clin “okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol “ deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res “kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer “ wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg “ dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg “ hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol “tóth d bíró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg “ maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer “liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg “sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer “ zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol “ alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg “ 0c"

the hypoxic tumour is a chaotic landscape of struggle and adaption against the adversity of oxygen starvationhypoxic cancer cells initiate a reprogramming of transcriptional activities allowing for survival metastasis andtreatment failure this makes hypoxia a crucial feature of aggressive tumours its importance to cancer and otherdiseases was recognised by the award of the nobel prize in physiology or medicine for research contributing toour understanding of the cellular response to oxygen deprivation for cancers with limited treatment options forexample those that rely heavily on radiotherapy the results of hypoxic adaption are particularly restrictive to treatmentsuccess a fundamental aspect of this hypoxic reprogramming with direct relevance to radioresistance is the alterationto the dna damage response a complex set of intermingling processes that guide the cell for good or for badtowards dna repair or cell death these alterations compounded by the fact that oxygen is required to inducedamage to dna during radiotherapy means that hypoxia represents a persistent obstacle in the treatment of manysolid tumours considerable research has been done to reverse correct or diminish hypoxia™s power over successfultreatment though many clinical trials have been performed or are ongoing particularly in the context of imagingstudies and biomarker discovery this research has yet to inform clinical practice indeed the only hypoxia interventionincorporated into standard of care is the use of the hypoxiaactivated prodrug nimorazole for head and neck cancerpatients in denmark decades of research have allowed us to build a picture of the shift in the dna repair capabilitiesof hypoxic cancer cells a literature consensus tells us that key signal transducers of this response are upregulatedwhere repair proteins are downregulated however a complete understanding of how these alterations lead toradioresistance is yet to comefacts— hypoxia is present in almost every solid tumour— hypoxia is a major barrier to effective radiotherapyand is associated with radioresistance— the hypoxic tumour is highly heterogenous withregions of chronic and acute hypoxia altered ph andimmune ltration— differences in gene expression and protein functioncan occur between acute or chronic and mild orsevere hypoxiacorrespondence mahvash tavassoli mahvashtavassolikclacuk1head and neck oncology group centre for host microbiome interactionking™s college london hodgkin building london se1 1ul ukedited by i amelio— all dna damageresponsehomologousddr pathwaysincludingnonhomologous end joining missmatch repair andthe fanconi anaemia pathways have been shown tosuffer alterations in hypoxiarecombination— activation of ddr transducer protein atm is seenin severe hypoxia in the absence of classical atmactivatingstranddna breaksfeaturesdoublesuchas— atr is also activated most likely in response tohypoxiainduced replication stress— however downregulation of dna repair effectorproteins such as rad51 and brca12 is seen— results of ddr reprogramming include geneticinstability aberrant cell cycle and apoptotic control the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of open questions— precisely how do alterations to the ddr in hypoxialead to radioresistance for example when genomicinstability and generation of radioresistant clonestakes several cell divisions to set in how does adecrease in dna repair ability lead to increasedradioresistance— what aspects of the hypoxic response could betargeted to radiosensitise or more effectively treattumours particularly in the context of ddr forexampleupregulated ddrtransducers such as atm atr and dnapkcs orinduce synthetic lethality following downregulationof dna repair effectorscan wetarget— do different types of cancers have different patternsof ddr alteration within hypoxic tumours thisparticularly needs further research as tissues havebeen shown to have different oxygen pressureslevels of hypoxia and hypoxic heterogeneity— can we use the data on this subject to develop abiomarkerhypoxiainducedradioresistance as we have done using hypoxia as asingle parametersignatureof— how can we monitor hypoxic tumours during thecourse of a patient™s disease to help guide treatmentand— how can wereportingreliableensureinterpretation of in vitro and in vivo dataintroductionofprogramstranscriptionalhypoxia is present in almost every solid tumour aninevitability of cancer™s characteristic disanised andfunctionally inefficient vasculature rapid growth anddemanding metabolism1 the result is a comprehensiverewritingupdownregulating certain genes and proteins allowing cells toevade apoptosis and migrate to areas with better oxygenperfusion crucially this microenvironmentally inducedintracellular shift also results in genomic instability gialterations to dna repair and resistance to cell killing bycancer therapies after decades of research it has becomeclear that the relevance of hypoxia for both oncogenesisand treatment resistance is inescapablein the context of radiotherapy rt a link betweenresistance and low intratumoral oxygen pressure has beenknown since the publication of a study modelling oxygenflow in lung tumours years ago2 for some cancerssuch as head and neck squamous cell carcinomashnsccs hypoxia is a major contributing factor to localrt failure3“ advancesin developing technologiesallowing for more precise delivery of rtimaging oftumours and sensitisation to treatment while protectingnormal tissues have led to improved locoregional controland quality ofsincelife for patients78 howeverofficial of the cell death differentiation associationtreatment for many cancers like hnsccs which incidentally are also some of the most hypoxic depends onrt the hypoxic problem remains particularly pertinent9in recent years efforts have been made to correct orreverse hypoxia including administering hyperbaric oxygen therapy to patients1011 reducing cellular oxygenconsumption12 and increasing blood vesselfunctionality1314 to more precisely tackle resistance induced byhypoxia researchers have also soughtthethreshold of treatability of hypoxic tumours by use ofsensitizers15“ as a third arm in our battle plan researchhas also gone into developing methods to detect hypoxiaincluding the use of specialised imaging techniques petct scanning combined with hypoxiadetecting radionuclides often studied in tandem with genetic signaturesseeking to genotypically define these tumours1819to lowerhowever very few of these advancements have allowedus to overcome hypoxiainduced radioresistance rrthough research activity in this area remains strong amore complete understanding of how the hypoxic environment contributes to rr particularly by modulation ofpotentially targetable dna damage response ddrpathways is warranted this review will outline our current knowledge of the molecular processes that underpinhypoxic rr particularly in the reprogramming of the ddrradiotherapy mechanism of action”therequirement of oxygenseminal work by gray and colleagues during the 1950sproved that the efficacy of rt was dependent on theavailability of oxygen within the tissue22021 radiationinduces damage through the direct and indirect generation of double stranded breaks dsbs in dna in thepresence of oxygen damage induced is “ times morelikely to end in cell death22 this effect is best explainedby the oxygen fixation hypothesis where radicals produced directly or indirectly by ionizing radiation ir areoxidised to dna in the presence of oxygen23 making thedamage irreversible24 thistothe hypothesis with the notion that these lesions cannotbe restored to an undamaged state as the damage isœï¬xed to dna by oxidisation25 thus without oxygendamage induced is transient and hypoxic cells experiencefar reduced radiationassociated damagelast pointis crucialthough crucial the requirement of oxygen to inducedamage is not where the story ends for rr as it does notfully explain the level of rr we observe this is evidencedby the fact that restoration of oxygen to tumours forexample through applying hyperbaric oxygen does notrestore radiosensitivity26 importantlyit also does notaccount for changes that occur with respect to dnarepair which have been shown to be crucial in impactingthe radiation response27 as these changes are retainedpast the point of radiotherapy administration 0cbegg and tavassoli cell death discovery page of the landscape of the hypoxic tumourin vivo the hypoxic region exists on a gradient ofoxygen pressures with oxygen levels throughout the tissue ranging between severe hypoxia “ mildhypoxia and anoxia with around consideredphysoxia see table for definitions tissue oxygenpressures are usually measured in mmhg however sincethe majority of research on hypoxia and the ddr hasbeen performed in vitro where oxygen levels are measured in percent for the purpose of this review o2 willbe referred to predominantly the difference betweenin vivo and in vitro measurements of oxygen is importantthough with tissue normoxia physoxia classified ataround o2 or mmhg and in vitro normoxia beingaround o2 fig the hypoxic tumour is a space of restricted proliferationparticularly when oxygen levels are o2 cell cyclearrest and decreased protein synthesis juxtaposed againstaccelerated aggressivity microenvironmental interactionsand altered ph28“ at the most oxygendepleted borderexists the barren land of necrosis with the highly proliferating and comparatively treatmentsensitive aerobiccells closest to the blood vessel fig tumour hypoxia does not develop in a linear fashionand is highly heterogenous and changeable the hypoxictumour is dynamic with fluctuating vessel functionalityand cycling oxygen levels creating regions of acute andchronic hypoxia31 where part of the tissue may sufferacute hypoxia after temporary occlusion of a blood vesselsocalled perfusion limited in which oxygendeprivationcycles last sometimes minutes sometimes hours beforesubsequent reoxygenation chronic hypoxia is diffusionlimited where oxygen levels become a factor of distancefrom the blood vessel31 compounded with this is thediffering rates of oxygen consumption and responsivenessto oxygen availability in cells within the tissue to be ableto fully understand and ultimately treat the hypoxictumour it must be remembered that the changeability ofoxygen concentrations in the hypoxic tumour also predictably ‚uences its behaviour and response to treatment in the context of radiotherapy cells with o2 levels is where we see most resistance so called radiobiological hypoxia24 whether this is acute and thereforefollowed by reoxygenation or chronic oxygen deprivedfor more than h can have marked differences on theensuing genomic and proteomic changes that ultimatelyallow for hypoxic survival32 thus within one tumourdifferent regions are likely to have a completely differentresponse to the same dose of radiotherapyitaside from the oxygen status the involvement of otherenvironmentalfeatures affected by hypoxia must beconsidered an additional outcome of hypoxic adaption isthe concomitant phenotypic shift of the microenvironmentis now accepted that hypoxia can induce‚ammation33 demonstrated even by patients whodevelop mountain sickness after prolonged periods athigh altitude34 hypoxic tumours are known to havehigher ltration of protumour immune cells such asm2 macrophages35 a feature known to be involved inrr3637 the same could also be said with respect to thehypoxicinduction of highly rt resistant cancer stemcells38 though this subject needs further research thelikelihood of an interplay between intracellular geneticreprogramming as a result of hypoxic adaption and themicroenvironment in mediating radioresponse is stronggenetic reprogramming”the hifswithin this chaotic showground of heterogeneity andat the core of cellular adaption to hypoxia are the alteredgenetic pathways that push for survival against adversitycommonly dysregulated genes include glut1 involvedin altered glucose metabolism vegf involved intable glossary of terms multiple classifications of the terms used to describe hypoxia exist throughout the literaturethis represents a general consensus and what is used in this reviewtermhypoxianormoxiaphysoxiaanoxiasevere hypoxiamild hypoxiaacute hypoxiachronic hypoxiadefinitionreduced oxygen levels usually ‰¤ o2 mmhg in in vitro studiesnormal atmospheric oxygen used in in vitro studies o2 mghgphysiological levels of oxygen in tissues between mmhg tissue specific see fig complete absence of oxygen o2 o2“ o2incubation in hypoxic conditions “ hincubation in hypoxic conditions hradiobiological hypoxiaoxygen levels where the efficacy of radiotherapy is half maximal mmhg04 o2official of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of fig approximate oxygen levels reported in different tissues in mmhg used in in vivo experiments and o2 used in in vitroexperiments note that normal tissue normoxia or physoxia is considerably less than the o2 used in vitro as normoxia adapted frommckeown139 liu140 and graham26fig the heterogeneity of the hypoxic tumour tumours suffer from reduced oxygen availability due to the disanised nature of thevasculature where occlusion of a blood vessel bv occurs tumours are said to be under perfusion limited hypoxia pl hypoxia where lack ofoxygen is a function of distance from the vessel cells experience diffusion limited dl hypoxia when these states are temporary h it is said tobe acute or chronic when h within hypoxia tumour cells undergo considerable genetic reprogramming contributing to therapy resistance andmetastatic behaviourofficial of the cell death differentiation association 0cbegg and tavassoli cell death discovery page of neoangiogenesis and lox involved in remodelling ofthe extracellular matrix7in the nobel prize in physiology or medicine wasawarded to three scientists gregg semenza williamkaelin and sir peter ratcliffe for their contributions toour understanding of cellular oxygensensing mechanisms39 this included the discovery of a group of transcription factors regulated by hypoxia that allow forcellular adaption40 these hypoxiainducible factor hifproteins hif13 are transcription factors composed oftwo subunits the α subunits reside in the cytoplasm andto rapid degradation “ min41 underare subjectnormal circumstances this degradation is mediated bythe actions of prolylhydroxylasesphd14 whichhydroxylate hifα at the oxygendependentdegradationdomains oddd of note phd2 and phd3 are themselves transcriptional targets of hif alluding to possiblenegative feedback systems in place though conflictingresults suggest this system doesn™t always function effectively to constrain cancer growth42“ subsequentlyhydroxylation by phds recruits the vonhippellandauvhl protein45 this alongside other proteins forms ane3 ubiquitin ligase complex ubiquitinating hifα forproteasomal degradationin hypoxia due to the lack of molecular oxygen neededfor hydroxylation4246 this degradation cascade does nottake place and hifα subunits translocate to the nucleusto associate with hifβ subunits47 the hif complex ininteraction with its coactivators p300 and crebbindingprotein cbp then binds to hypoxia response elementshresin dna to initiate transcription of hiftarget genesintheincludingcontrolledadditional layers of hif regulation also exist to keepthis pathway in check such as factor inhibiting hif fihwhich hydroxylates hif subunits at asparagine residuesblocking their association with p300cbp48 some evidence has shown that hifs also undergo other posttranslational modifications including phosphorylation andacetylation as a further method of regulation4247 however as with many such processes in cancer it can beaberrantlyhypoxiaindependent stabilisation of hifα by oncogenes such asegfr and mtor4950 and depletion of hifregulatoryfactors4251 the hif proteins themselves can interactwith a number of factors relevant in cancer such as p53mutants present in human papillomavirus hpvnegativehnsccs and nonsmall celllung cancers nsclcsresulting in transcriptional control of protumorigenicgenes52 since both hifs and p53 compete for binding ofp300cbp to enact transcriptional control the hifs havea unique relationship with this highly cancerrelevantprotein53 inactivation of p53™s transcriptional abilities hasbeen observed54 though again conflicting results exist forthis55official of the cell death differentiation associationmost of the work investigating hifdirected transcriptional changes in hypoxia has focussed on the actions ofthe bestknown hif hif1 however both hif2 andhif3 also play a role in hypoxic transcriptional control42interestingly relative expression of the hifs has beenshown to differ between hypoxic tissues demonstratingthat each may have specific functions56 in some casesthey may indeed work in concert as hif2 has beenshown to be induced when hif1 is depleted50hifs are master regulators of the hypoxic response andconcurrent with the notion that hypoxic tumours areradioresistant depletion of hif1α in tumour modelsradiosensitises cells41 one study showed that intermittenthypoxia showed less radiationinduced cell death bothin vitro and in mice via stabilisation of hif1α57 thisinvestigation also found that intermittent hypoxia had amore significant effect than chronic hypoxia hif1α hasalso been shown to function via the hif1α“myc pathway in which hif1α competes with the transcriptionactivator myc for sp1 binding in the target gene promoter to downregulate mismatch repair mmr genesmsh2 and msh6 in o2however how exactly hifs contribute to rr of thehypoxic tumour be itthrough their transcriptionalfunctions or interactions with other proteins is so farunresolved notably some radio and chemotherapiesthemselves upregulate or stabilise hifs41though genetic reprogramming in hypoxia can lead to anumber of alterations for the purpose of this review wewill focus on those associated with rr and ddr formore general reviews see schito60 and tsai61reprogramming of the ddrthe ddr is a complex process consisting of overlapping and interconnected pathways initiated by differentforms of dna damage arguably one ofthe mostimportant homeostatic processes it allows us to withstand constant and numerous dna damageinducinginsults the result of this protection ensures that onlyreliable genomes are passed on to the next cellular generation for cancer considering both the power ofmutagenesis in driving oncogenic potential and the factthat many cancer therapies function by inducing dnadamage the ddr has considerable relevance for therapyresistance and tumour progressionrepair of dna is a tale of three acts firstly the damagepropagates a signal that recruits sensors to the site ofdamage secondly the signal is amplified by transducersand thirdly response pathways are initiated by effectorsfor each part of the process welldefined though notexclusive sets of proteins act as sensors transducers andeffectors respectively28 shrouding these repair processesare signals to stall the cell cycle initiated by chk1 orchk2activated cdc25 and p21to allow time for 0cbegg and tavassoli cell death discovery page of clearance of this damage and initiation of apoptoticpathways for example as initiated by atm™s interactionwith p53 if the repair is unsuccessful6263for the repair of radiationinduced dsbs two primarypathways are put to use homologous recombination hrand nonhomologous end joining nhej the formerconsidered less errorprone uses sister chromatids torepair dna and as such can only take place during g2sphase of the cell cycle nhej predominates in g1 but canoccur at any stage of the cell cycle and often results in thegeneration of insertiondeletion mutations which havethe potential to lead to more oncogenic alterations hr ismediated primarily by the recruitment to sites of damageof master transducer of the dsb response atm ataxiatelangiectasia mutated a phosphoinositide3kinaserelated protein kinase pikk following detection by themrn complex composed of mre11rad50nbs1which initiates activity of effectors including rad51 andbrca1 nhej occurs following sensing of damage by theku proteins ku70 and ku80 and signal transduction ofku in complex with dnapkcs dnadependent proteinkinase catalytic subunit”together forming dnapk andsubsequent activity of effectors dna ligase iv ligivand xrcc464alteration of ddr pathways has been seen across manycancers compared to normal tissue perhaps the mostwellknown are the mutations in brca12 in aggressivehereditary breast and ovarian cancers65 understandablywhere hypoxia represents an exaggerated form ofaggressive tumours the ddr pathways in hypoxia operatedifferently to those in normoxia indeed this is true forevery aspect of the ddr process dna damage in theform of dsbs is reduced in conditions of hypoxia o2and hypoxia alone does not induce dsbs2466 researchhas shown that different members of the ddr pathwayscan be either activated or downregulated in conditions oflow oxygen see tables and for reported alterationsto hr nhej and mismatch repair mmr pathwayscrucially whether the cells are in acute or chronichypoxia or h and at what level of oxygen depletion may define the ensuing response despite this delineation there lacks within the literature proper reportingof experiments carried out in either acute or chronic mildor severe hypoxia with interchangeability in use of termssee table for a consensus of parameters used with thesedefinitionssensorsthe mre11rad50nbs1 mrn complex is responsible for sensing dna damage and initiating both the hrand nhej pathways by recruitment of transducers such asatm via nbs167 while the mrn complex is consideredthe main sensor responsible for recruiting and activatingatm followingdamage atrip atrinteractingofficial of the cell death differentiation associationprotein and ku7080 are sensorsresponsible forrecruitment of atr and dnapkcs respectively67 fig though there are many overlapping interactions atminatminteracting protein with roles in replication stressrs genome stability and the base excision repair berpathway has also been shown to recruit atm independent of dna damage68repression of the mrn machinery has been seen inchronic hypoxia days in a medulloblastoma modelwith transcriptomic downregulation of both mre11a andnbs1 resulting in downregulation of etoposideinducedatm and p5369 nbs1 has also been shown to stabilisehif1α particularly in response to ir70 while hif1α hasbeen shown to downregulate nbs1 the authors of onestudy where reduction of nbs1 was seen after h in o2 noted that this repression resulted in the induction ofγh2ax and 53bp1 foci in hypoxia suggesting the presence of dna breaks59 interestingly all components ofthe mrn were found to be downregulated both at themrna and protein level in nsclcs harbouring egfrmutations incubated in severe hypoxia o2 thisdownregulation in egfrmutated cells correlated withtheir increased sensitivity to egfrinhibiting drugs71sensing of damaged dna is a crucial step in theinitiation of repair and begins with changes to the chromatin72 γh2ax a phosphorylated variant of histoneh2ax is induced by mrn activation and accumulates atsites of damage in the chromatin preceding recruitmentand necessary for retention of key ddr signalling proteinsincluding mrn and atm73 studies also show thatγh2ax is crucial for retaining mediators such as 53bp1p53 binding protein mdc1 mediator of dnadamage checkpoint and brca1 at sites of damage66h2ax is primarily phosphorylated by atm but can alsobe phosphorylated by atr and dnapkcs63 indeed aswell as by radiation and chemotherapies γh2ax has alsobeen shown to be induced by hypoxia following replication fork stalling this phosphorylation has been shown inchronic severe hypoxia to occur in a hif atr or atmdependent manner74“ crucially some evidence hasshown the phosphorylation of h2ax present only inproliferating cells7778 the downstream effects of thisactivation have been linked to other consequences ofhypoxic regulation including angiongenesis79 via induction of vegf80 experimentally resolution of γh2ax fociafter irradiation is often used as a marker of dsb repair astheory dictates that the phosphorylation should disappearafter damage is repaired however in hypoxia this heavilyrelied upon protocol may necessitate further finetuningku70 and ku80 together forming a heterodimericcomplex tether damaged dna at breaks and are keysensors of dsbs responsible for recruitment of dnapkcs as part of the nhej pathway63 the ku complex hasbeen shown to be both upregulated and downregulated by 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the homologoursrecombination hr pathways in hypoxiaprotein role in ddrmechanism of alterationalteration conditions and consequencesreferencenbs1sensor of dsbs in hr activated atmas part of the mrn complexcid129 pasb domain of hif1αmre11sensor of dsbs in hr activated atmas part of the mrn complexcid129 atmtransducer of hr in dsb repaircid129 autophosphorylation atser1981atrtransducer of dna repair induced byreplication stresscid129 rad51effector of dsb repair in hrrad52effector of dsb repair in hrrad54 motor protein effector of dsbrepair in hrbrca1effector of dsb repair in hrcid129 e2f4p130cid129 lsd1cid129 ezh2cid129 mir210cid129 mir373cid129 mir210cid129 cid129 e2f4p130cid129 h3k4 demethylation via lsd1cid129 downregulated in chronic mild hypoxia days o2cid129 downregulation in acute mild h o2cid129 resulted in induction of γh2ax and 53bp1 focicid129 downregulated in chronic mild hypoxia days o2cid129 activated in acute hypoxia o2cid129 increased expression and activity o2“ hcid129 mediated by src and ampk signallingcid129 activated in acute o2cid129 resulted in phosphorylated p53 andaccumulation and growth arrestcid129 downregulation in chronic severe hypoxia o2 “ h and or “ hcid129 decreased radioresistancecid129 increased genomic instabilitycid129 downregulation in o2 hcid129 decreased mrna expression o2“ hcid129 downregulated o2 hcid129 decreased mrna expression o2“ hcid129 downregulation in chronic severe hypoxia o2 hcid129 decreased mrna expression o2“ hcid129 downregulation in o2 hcid129 decreased radioresistancecid129 decreased expression o2 “ hcowman69to59cowman69hashimoto88bencokova28hammond75meng119bindra oliveira82meng119crosby118meng119meng119lu117bindra120oliveira82meng119brca2effector of dsb repair in hrcid129 hypoxia in different studies81 one study found downregulation of ku80 after h in mild hypoxia o282another using severe hypoxia o2 found upregulation of ku70ku80 in a431 cells alongside many othermembers of the nhej pathway and proteins generallyinvolved in metastatic progression83 a study in humanand mouse hepatoma cells found upregulation of the kuheterodimer upon incubation in hypoxia o2 or withhypoxia mimics and downregulation associated with hif1βdeficient cells84 alternative subpathways of nhejalso exist possibly as insurance for when classical nhejmediators are inoperative howeverthe impact ofhypoxia on these pathways has not been extensivelystudiedofficial of the cell death differentiation associationtransducersatm and atr are two of the most important proteinsinvolved in transduction of the ddr as pikk familymembers they phosphorylate a number of proteinsinvolved in propagating the signal and repairing dna aspart of both the hr and nhej pathways as well asundergoingtheresponse until dna is repairedautophosphorylationto maintainbroadly speaking atm has been shown to be activatedparticularly in acute hypoxia as shown in the study bybencokova et al the pattern of activation in this contextdoes not match rtinduced atm activation which follows mrn recruitment to dsbs85 as atm phosphorylation does not correlate with the presence of dsbs 0cbegg and tavassoli cell death discovery page of table a nonexhaustive list showing alterations to sensors transducers and effectors of the nonhomologous endjoining nhej pathways in hypoxiaproteinrole in ddrmechanism of alterationalteration conditions and consequencesreferenceku70ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku80sensor in nhej pathwaysrecruits dnapkcsin complex with ku70cid129 cid129 dnapkcstransducer of nhej pathwaycid129 autophosphorylation atser2056dna ligiv effector of nhej repairxrcc4effector of nhej repaircid129 cid129 cid129 decreased mrna expression o2 “ hcid129 upregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 “ hcid129 upregulation o2 hcid129 downregulation o2 hcid129 downregulation in cervical tumour sectionscid129 upregulation o2 “ hcid129 decreased mrna expression o2 “ hcid129 increased expression and activity o2 “ hcid129 activated in mild hypoxia “ o2 led to positiveregulation of hif1 and upregulation of glut1cid129 decreased mrna expression o2 “ hcid129 decreased mrna expression o2 “ hmeng119ren83lara81um84oliveira82ren83lara81um84meng119hashimoto88bouquet103meng119meng119table a nonexhaustive list showing alterations to sensors transducers and effectors of the mismatch repair pathwayin hypoxiaprotein role in ddrmechanism of alterationmlh1dimerises to pms2 to form themutlα complex in mmrpms2dimerises to mlh1 to form themutlα complex in mmrmsh2dimerises with msh6 forms themutsα complex in mmrmsh6dimerises with msh6 forms themutsα complex in mmrcid129 mad1maxcid129 mntmaxcid129 dec12cid129 mir155cid129 lsd1cid129 hdaccid129 hypoacetylationhypermethylation on h3cid129 cid129 mycmaxcid129 hif1α via sp1cid129 mir155cid129 hcid129 p53cid129 hif1α via sp1cid129 mir155cid129 hdaccid129 p53cid129 hypoacetylationhypermethylation on h3official of the cell death differentiation associationalteration conditions andconsequencescid129 downregulation “ h o2cid129 downregulation in h o2cid129 increased expression “ h o2resulting in genomic instability instem cellscid129 downregulation at protein level“ h o2cid129 resulting in genomic instability instem cellscid129 downregulation “ h o2referencebindra121127mihaylova128nakamura129rodriguezjimenez145lu115mihaylova128rodriguezjimenez145bindra121127koshiji58cid129 downregulation “ h o2cid129 increased expression “ h o2resulting in genomic instability instem cellskoshiji58rodriguezjimenez145 0cbegg and tavassoli cell death discovery page of often reduced or absent in severe hypxoia28 the authorsof this study emphasized that atm activation was specificto the level of hypoxia only phosphorylated at o2and hifindependent since phosphorylation was maintained even in hifknockout cells activation of atmwas attributed to autophosphorylation the result of whichwas an activation of targets much like dna damageinduced atm activation but dependent on the activityof cell cycle regulator mdc128 this study did not analyserr but the results suggest that atm activation byhypoxia is likely enacted as a means of halting the cellcycle in order to allow for dna repairthe pattern of atm activation in hypoxia however isnot clearcut and may depend on cancer type atm canbe regulated by a number of factors as part of the hypoxicresponse including mrn or atmin but also by posttranslational and epigenetic factors86 one study foundthat atm was downregulated along with hif1α by amicrorna mir18 resulting in radiosensitivity87the study by hashimoto et al88 showed atm activation alongside activation of a number of other key ddr orcancerrelated proteins including dnapkcs akt andegfr and decreased expres

the molecular heterogeneity of renal cell carcinoma rcc complicates the therapeutic interventions for advancedmetastatic disease and thus its management remains a significant challenge this study investigates the role of thelncrna cdkn2bas1 and mir1413p interactions in the progression and metastasis of kidney cancer human renalcancer cell lines achn and caki1 normal rptec cells tissue cohorts and a series of in vitro assays and in vivo mousemodel were used for this study an overexpression of cdkn2bas1 was observed in rcc compared to normal samplesin tcga and our inhouse sfvamc tissue cohorts reciprocally we observed reduced expression of mir141 in rcccompared to normal in the same cohorts cdkn2bas1 shares regulatory mir141 binding sites with ccnd1 andccnd2 genes direct interactions of cdkn2bas1mir141cyclin d1“d2 were confirmed by rna immunoprecipitationand luciferase reporter assays indicating that cdkn2bas1mir141cyclin d1“d2 acts as a cerna network in rccfunctionally attenuation of cdkn2bas1 andor overexpression of mir141 inhibited proliferation clonogenicitymigrationinvasion induced apoptosis in vitro and suppressed tumor growth in xenograft mouse model furtheroverexpression of cdkn2bas1 is positively correlated with poor overall survival of rcc patients expression of mir141also robustly discriminated malignant from nonmalignant tissues and its inhibition in normal rptec cells induced procancerous characteristics cdkn2bas1 attenuation or mir141 overexpression decreased ccnd1ccnd2 expressionresulting in reduced rac1ppxn that are involved in migration invasion and epithelial“mesenchymal transition thisstudy for the first time deciphered the role of cdkn2bas1mir141cyclin d axis in rcc and highlights this networkas a promising therapeutic target for the regulation of emt driven metastasis in rccintroductionrenal cell carcinoma rcc is one of the most commoncancers in the usa accounting for nearly deathsand new cases in surgery is the first line oftreatment resulting in successful resection and longtermdiseasefree status with an overall survival rate of morecorrespondence rajvir dahiya rdahiyaucsfedu1department of urology veterans affairs medical center san francisco anduniversity of california san francisco san francisco ca usa2department of surgery university of miami miller school of medicine miamifl usafull list of author information is available at the end of the these authors contributed equally pritha dasgupta priyanka kulkarniedited by e candithan however in approximately of localizedrcc cases recurrence occurs with distant metastasis2the obstinate nature of rcc to current treatment regimens is a primary cause of poor prognosis in patients withmetastatic recurrence lack of sensitivity to both chemotherapytherapeuticoptions difficult3“ it is therefore of utmost importanceto improve our understanding of rcc pathogenesis byidentifying new biomarkers that lead to better predictionand therapeutic intervention of aggressive rcc6and immunotherapy makesemerging lines of evidence suggest that cancer aggressiveness is associated with epithelial“mesenchymal transition emt7 it is a wellorchestrated process involved in the authors open access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate ifchanges were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material ifmaterial is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this license visit httpcreativecommonslicensesby40official of the cell death differentiation association 0cdasgupta cell death and disease page of tumor invasion and metastasis comprising characteristicphenotypic changes through transition from polarizedimmotile epithelial cells to motile mesenchymal cells8emt changes in cellular morphology and migratoryproperties are governed by numerous factors9 increase inmesenchymal properties accompanied by augmentedexpression of mesenchymal markers like ncadherbronectinvimentin and matrix metalloproteinasemmps and decreased expression of epithelial markerslike ecadherin αecatenin claudin etc10“ are common emt phenomena often the progression of cancerthrough emt is significantly induced by the interaction ofcyclind with its binding partner cdk4 which act astranscriptional regulators controlling cell proliferationand migration14“ it is well known that cyclind regulates the ratelimiting step in cell cycle progression fromg1 to s phase accumulating evidence also suggest thatabnormal cyclindcdk4overexpression promotestumor growth and metastasis17 but how this correlateswith tumor metastasis or controls cell adherence andinvasion is poorly understoodreports show that noncoding rnas are involved in thefactors involved in emt18 micrornasregulation ofmirnas a naturally occurring class of small noncodingrna molecules of nucleotides long19 are known toregulate gene expression via both translational inhibitionand mrna degradation20 whereas long noncoding rnaslncrnas with more than nucleotides can also actas regulators for tumorsuppressive mirnas in differentcancers21“ recently a class oflncrnas have beencategorized as competing endogenous rna cernawhich involves crosstalk among lncrnas mrnas andtheir shared mirnas thus a novel regulatory mechanismis hypothesized suggesting that lncrnas and mrnascommunicate with each other by competing for commonmirna response elements24“in this context we describe the novel role of lncrnacdkn2bas1 and mir1413p mir141 in the regulation of cyclind to govern the metastatic progression ofrcc to our knowledge this is the first report to directlydemonstrate that cdkn2bas1mir141 interaction is acrucial component in rcc progression and metastasisthrough the cyclindrac paxillin pathwaymaterials and methodscell lines and cell culturethe normal rptec atcc number crl4031 andrenalcancer achn atcc number crl1611and caki1 atcc number htb46 cell lines were purchased from the atcc manassas va these humanderived celllines were authenticated by dna shorttandem repeat analysis by atcc cell line experimentswere performed within “ months of their procurementresuscitation achn cells were cultured in mem mediaofficial of the cell death differentiation associationcaki1 cells in and mccoy 5a medium and rptec cellsin dmemf12 medium atcc® „¢ all mediawere supplemented with fbs and 1x antibioticspenicillin and streptomycin cell lines were maintainedat °c and humidified atmosphere of co2mirnasirna transfectionsto induce overexpression or knockdown cells were transiently transfected with either mirvana mirna mimic nmoll or antimir mirna inhibitor nmollthermo fisher scientific and nmoll of sirna sigmaaldrich using lipofectamine rnai max thermo fisherscientific according to the manufacturers™ protocol toverify transfection efficiency mirvana mirna mimicnegative control mirna inhibitor control and sirnacontrol were used respectively in each transfection experiment at the same concentration all transfection experimentswere carried out for hclinical specimensformaldehydefixedparaffinembedded ffpe tissue specimens from patients undergoing radical nephrectomy wereobtained from the san francisco veterans affairs medicalcenter sfvamc written informed consent was obtainedfrom all patients and the study was in accordance withinstitutional guidelines irb approval no allpatient samples were pathologically confirmed for clear cellrcc ccrcc and slides were reviewed by a boardcertifiedpathologist for the identification of tumor foci and adjacentnormal tissue apart from sfvamc cohort tcgakirctcgakich tcgakirp icgc and geo cohorts forrcc from online databases were also used to check theexpression levelsrnamirna extraction and quantitative realtime pcrqrtpcrtotal rna was extracted from microdissected ffpetissues and cell lines using mirneasy ffpe and mirneasymini kits qiagen respectively in accordance to manufacturer™s instructions mature mirna and mrnas wereassayed by qrtpcr using quantstudio flexreal timepcr system applied biosystem using fast sybr®green master mixtaqman universal pcr master mixprobes and primers applied biosystems inc foster cityca usa following manufacturer™s protocol humangapdh and rnu48 were used as endogenous controlsand relative expression of rnamirna were calculatedusing comparative ct threshold cycle primer sequencesare provided in supplementary table t1dna methylation analysis insilico in cell lines and 5azacdr treatmentdna hypermethylation of the mir141 promoter innormal and rcc samples was first confirmed in the 0cdasgupta cell death and disease page of tcga database using wanderer software27 in order toconfirm the methylation status of the mir141 promoterin rcc cell lines we extracted dna from achn andcaki1 using dneasy tissue kit qiagen sodium bisulphite modification was done using ez dna methylationgold kit zymo research orange ca usa followingthe manufacturer™s protocol bisulfitetreated dna wasanalyzed by methylationspecific quantitative polymerasechain reaction msqpcr with primer pairs specific formethylated and unmethylated regions of the mir141promoter msqpcr was performed as described earlier28 for each sample the percent of methylation wascalculated by the difference of ct in methylated samplectm and ct in unmethylated sample ctu the primers sequences are mentioned in supplementary table achn and caki1 cells were treated daily with μmoll5azadeoxycytidine 5azacdrfor h29 and total rna was isolated using a mirneasy minikit qiagen to check mir141 expressionsigmaaldrichcell viability clonability migratory invasion andapoptosis assayscell viability was measured at and h using acelltiter aqueous solution cell proliferation assay kitpromega madison wi following the manufacturer™sinstructions for colony formation assay cells were seeded at a low density cellsplate after h oftransfection and were allowed to grow until visible colonies were formed plates were then stained with giemsafollowed by crystal violet and colonies were countedculture inserts of 8µm pore size transwell costar wereused for migration and invasion assay inserts were coatedwith matrigel bd biosciences µgwell for invasionbriefly h posttransfection cells were counted andplaced on inserts at × cellsml for migration and × for invasion in serumfree medium and wereallowed to migrateinvade for “ h at °c cellsmigrated or invaded through the pores were fixed stainedwith crystal violet crystal violet was solubilizedwith methanol and quantified at nm by a kineticmicroplate reader spectra max molecular devicesfacs analysis for apoptosis was done h posttransfection using annexin vfitc and 7aad kit beckmanin accordance with the manufacturer™scoulter incinstructions cold pbs washed cells were resuspended in1x binding buffer and stained with annexin vfitc7aad viability dye after min of incubation at roomtemperature in the dark stained cells were analyzed usingbd facsverse bd pharmingendualluciferase reporter assaythe wild type wt and offtarget ot luciferasereporter constructs were made by ligating annealed custom oligonucleotides containing putative target bindingofficial of the cell death differentiation associationsites and corresponding nontarget mutant sites into thepmirglo reporter vector luciferase constructs µgwere cotransfected into achn and caki1 cells along with nmoll mir141 mimic or controlmir using transfection reagent jetprime polyplustransfection illkirchfrance luciferase activities were measured using thedualluciferase assay promega madison wi h posttransfection relative luciferase activity was calculated bynormalizing firefly luciferase to renilla luminescencerna immunoprecipitation rip assaysimultaneous binding of mir141 to lncrna andmrna was confirmed by rip assay an imprint rip kitwas used following the manufacturer™s protocol sigmaaldrich st louis mo usa igg control and ago2antibodies were used forimmunoprecipitation theimmunoprecipitated rna fraction was reverse transcribed to cdna using high capacity cdna reversetranscription kit thermo fisher fold enrichment oflncrna and mrna to ago2 with respect to igg wascalculated using quantitative rtpcrwestern blot and immunofluorescence analysistotal protein extraction was performed as describedpreviously18 proteins were then separated by nupage“ bistris protein gels invitrogen and subsequentlytransferred onto nitrocellulose membraneresulting blots were blocked using odyssey blockingbuffer licor and subsequently probed with primaryand secondary antibodies blots were scanned using anodyssey infrared imaging system scan and quantificationwas carried out with the licor odyssey® scanner andsoftware licor biosciences the primary antibodiesused are listed in supplementary table for immunofluorescencetransfected achn andcaki1 cells were fixed in paraformaldehyde for minfollowed by blocking 1x pbs5 normal goat serum triton x100 for h at room temperature cellswere then incubated overnight in fold diluted primary antibody at °c cells were then reprobed with fold diluted secondary antibody for h and counterstained with µgml of ²6diamidino2phenylindoledapi for min cells were then mounted on a slideusing prolong gold antifade reagent images were captured using zeiss microscope model axio imagerd2transientlyrenal cancer xenograftswe studied the antitumorigenic effects of mir141 inestablished tumors using a renal cancer xenograft nudemouse model as previously described630 male nude mice“ weekold n charles river lab were subcutaneously injected with × caki1 cells oncepalpable tumors were formed mice were randomized intwo groups for the treatment and control groups five in 0cdasgupta cell death and disease page of each synthetic mirna mir141 mimicmircon of μg was complexed with μl siportamine transfection reagent ambion in μl pbs and deliveredintratumorally in 3day intervals tumor volume wascalculated according to the formula x2 y2 where x yx width y length experiments were terminated days after the last treatment day tumor measurements and statistical analysis were performed byresearchers who were blinded for the control and treatment groups all animal care was in accordance withinstitutional guidelines iacuc approval no statistical analysisall quantified data represents an average of at leastthree independent experiments or as indicated statisticalanalyses were performed using graphpad prism andmedcalc error bars represent ± standard error meansem the mann“whitney u test was used to assessthe difference between mirna expressions in tumornormal adjacent tissue significant differences betweenthe groups were determined using the student™s ttest alltests were performed either one tailed or two tailed andresults were considered statistically significant at p ‰¤ receiver operating curves roc were performed toevaluate the potential of mir141 to differentiate betweenmalignant and nonmalignant samples using medcalcsoftware showing area under curve auc and confidence interval kaplan“meier analyses for overall survival with respectto mir141 methylation levels intcgakirc cohort were generated using softwareœezrhttpsdoi101038bmt2012244 tumormeasurements and statistical analyses for all experimentswere performed blindly for the control and treatment groupsresultslncrna cdkn2bas1 is oncogenic and is a direct target ofmir141initially we found cdkn2bas1 is an oncogeniclncrna in rcc based on tcga fig 1a icgc andgeo databases fig s1 tcga cohort also revealed thathigh cdkn2bas1 expression increases from lower gradeand stage to higher grade and stage fig 1b moreoverhigher expression is significantly p correlated tooverall survival fig 1c in agreement with these datacohorts significantly higher cdkn2bas1 expression wasalso seen in rcc cell lines achn caki1 as compared tonormal rptec fig 1d and sfvamc cohort fig 1epatient and tumor characteristics are summarized insupplementary table t3 roc analysis shows an auc of p ci “ fig 1f suggesting the diagnostic potential of cdkn2bas1 to discriminate between normal and tumor tissues we usedcomputational algorithms and identified putative mir141binding sites in the cdkn2bas1 sequence fig 1g toofficial of the cell death differentiation associationexamine potential mir141cdkn2bas1 interactionexperimentally we performed luciferase reporter assayboth achn and caki1 cells cotransfected with mir141and cdkn2bas1 wild type binding site revealed a consistent reduction ofluciferase activity suggesting thatmir141 directly interacts and regulates cdkn2bas1fig 1h thus all these data suggest that clinicallyimportant cdkn2bas1 is an oncogenic lncrna in rccand is a novel target of mir141cdkn2bas1 inhibition by sirna suppressestumorigenicity in rcctransient transfection of achn and caki1 cells withcdkn2bas1 sirnas for h showed significant reduction in cdkn2bas1 expression fig 2a cdkn2bas1knockdown in both cell lines significantly inhibited cellproliferation figs 2b s2a and clonogenic survivalfigs 2c s2b with a significant increase in apoptosis fig2d e decreased cell migrationinvasion figs 2f s2c dwith simultaneous changes in emt markers such as anincrease in epithelial markers αecatenin and claudinand decrease in mesenchymal markers vimentin andfibronectin were also observed figs 2g s3expression of mir141 and its clinical importance in renalcarcinomaand samples lowersince our results confirmed cdkn2bas1 as a directtarget of mir141 we examined mir141 status andclinical importance in rcc expression of mir141 wassignificantly downregulated in rcc cell lines fig 3aand in tumor samples fig 3b“e compared to normal celllinesignificantlydecreased with increasing grade stages and in metastaticcompared to nonmetastatic tumors fig 3c“e patientand tumor characteristics are summarized in supplementary table t3 roc analysis showed an auc of p ci “ fig 3f suggesting thatmir141 can be used as a potential diagnostic parameterto discriminate between normal and tumor tissuesexpressionsepigenetic regulation of the mir141 locuswe identified a genomic site rich in cpg island locatedupstream of the mir141 in chromosome 12p13 in thetcga cohort we observed hypermethylation of mir141promoter in tumor tissues as compared to normalfigs 3g s4a which is significantly associated with poorpatient survival fig 3h similarly rcc cell lines achnand caki1 also showed hypermethylation compared tonormal rptec cells fig 3i further we treated achnand caki1 cell lines with demethylating agent 5azacdrand observed decrease in methylation fig s4b withconcomitant increase in mir141 expression fig 3jindicating possible epigenetic regulation a significantdecrease in the expression of methylation regulatory 0cdasgupta cell death and disease page of fig lncrna cdkn2bas1 is oncogenic in renal cancer and is a direct target of mir141 a expression levels of cdkn2bas1 among kircnormal tumor kich normal tumor and kirp normal tumor patient samples in tcga cohort using wanderersoftware pvalue calculated by mann“whitney twotailed test b expression of cdkn2bas1 in tcgakirc cohort among different grades normal grade “ grade “ and stages normal stage i“ii and stage iii“iv c overall survival in tcgakirc cohort as performedby kaplan“meier analysis using ualcan software d relative expression levels of lncrna cdkn2bas1 in rcc cell lines achn and caki1 e expressioncdkn2bas1 in matched pairs of rcc tissue samples from sfvamc cohort pvalue calculated by mann“whitney twotailed test f receiver operatingcurve roc analysis on sfvamc cohort showing ability of lncrna cdkn2bas1 to differentiate between malignant and nonmalignant samples sfvamccohort g predicted binding sites of mir141 in cdkn2bas1 sequence h luciferase assays showing decreased reporter activity after cotransfection witheither wildtype wt offtarget ot cdkn2bas1 or luciferase control constructs ev with mirconmir141 in achn and caki1 cellsgenes such as dnmtl dnmt3a and dnmt3b werealso noted after 5azacdr treatment compared to control dmso in both achn and caki1 cell lines31mir141 overexpression phenocopies functional effectsobtained with cdkn2bas1 inhibition in vitro andsuppresses tumorigenicity and in vivowe sought to determine if cdkn2bas1 causes itsantitumorigenic effects through mir141 we checkedthe effect of mir141 overexpression in rcc cellstransient transfection of mir141 mimic in achn andcaki1 cells for h led to over expression of mir141compared to control mircon fig 4a also overexpression of mir141 significantly reduced cdkn2bas1 expression fig 4b indicating a reciprocal correlation between mir141 and cdkn2bas1 a significant decrease in cell proliferation over time fig 4cand marked decreasefig 4din clonogenicityofficial of the cell death differentiation association 0cdasgupta cell death and disease page of fig knockdown of lncrna cdkn2bas1 reduces tumorigenicity in renal cancer a relative expression of cdkn2bas1 in achn and caki1 cellstransfected with cdkn2bas1 sirnas b cell proliferation assessed by mts assay after knockdown of cdkn2bas1 in both cell lines with sirna2 cgraphical representation showing knockdown of cdkn2bas1 with sirna2 significantly decreased colony formation in achn and caki1 cellsd e achn and caki1 cell lines showing significant induction of apoptosis early late compared to control after knockdown of cdkn2bas1f reduced migration and invasion in cdkn2bas1 sirna transfected cells compared to control treatment g changes in emt related proteins in bothachn and caki1after knockdown of cdkn2bas1compared to controls were also observed further westudied the therapeutic potential of mir141 in amouse xenograft model a significant decrease intumor growth was observed by intratumoral delivery ofmir141 mimic compared to control over the course ofexperiment average tumor volume in the controlgroup was mm3 compared to mm3 in micethat received mir141 mimic fig 4e in additionmir141 overexpression significantly induced apoptosis with a concomitant decrease in the viable population in both rcc celllines compared to controlfig 5a this proapoptotic role was supported by theinduction of cleaved caspase3 cleaved polyadpribose polymerase parp an increase in bax and adecrease in bcl2 at protein levels fig 5b a significantdecrease in migration fig 5c and invasion fig 5dwas also observed in both rcc cell lines with mir141overexpression we also examined emt markers aschange in migration and invasion are directly associated with emt our results showed an increase inepithelial markers αecatenin and claudin with concomitant decrease in mesenchymal markers fibronectinand vimentin at both protein fig 5e and mrnaofficial of the cell death differentiation associationfig s5 levels taken together overexpression of mir phenocopies the functional effects of cdkn2bas1 inhibition in vitro and tumor growth suppressioneffects in vivolike cdkn2bas1aremir141cdkn2bas1 interaction negatively regulatescyclind and its downstream effectors in rcccyclind1cyclind2alsooncogenic in rcc fig s6 and are direct targets of mir as discussed earlier lncrnas can act as cernas tocarry out their regulatory functions32“ we observedthat cdkn2bas1 shared regulatory mir141 bindingsites with cyclind1cyclind2 fig 6a and therebysponges mir141 allowing cyclind1d2 to be expressedin tumors to determine potential mir141cdkn2bas1cyclind interaction experimentally we performedrip assay both achn and caki1 cells over expressingmir141 revealed significant enrichment of cyclind1cyclind2 and cdkn2bas1 with ago2 as compared toigg control fig 6b moreover decreased luciferaseactivity also confirmed direct binding of mir141 tocyclind in mir141overexpressing achn andcaki1 cells compared to controls fig 6c we also found 0cdasgupta cell death and disease page of fig expression clinical significance and epigenetic regulation of mir141 in renal cancer a mir141 expression levels in achn caki1 andrptec cells b expression levels of mir141 in kirctcga cohort normal and tumor c expression levels of mir141 in normal n nonmetastatic n metastatic n kirc patient samples in tcga cohort d expression levels of mir141 in kirctcga cohort amongdifferent grades normal grade “ grade “ and stages normal stage i“ii and stage iii“iv e relative mir expression in rcc tissue vs matched adjacent normal regions n among different grades normal grade grade “ andin different stages normal stage i“ii stage iii“iv as assessed by qrtpcr sfvamc cohort f receiver operating curve roc analysisshowing ability of mir141 to differentiate between malignant and nonmalignant samples g methylation for kirc patient samples in tcgakirccohort for probe cg02624246 h overall survival with tcgakirc methylation as performed by kaplan“meier analysis i methylation status of mir141promoter in rcc cell lines compared to nonmalignant rptec as assessed by msqpcr j expression of mir141 in 5azacdr treated and untreatedachn and caki1 cell lines results are representative of three independent experiments p value calculated by student t test bar mean ± standarderror mean semthat overexpression of mir141 or inhibition of cdkn2bas1 significantly decreased cyclind1d2 expression atboth the mrna fig 6d e and protein levels figs 6f“hs8a b this effect was significantly attenuated by mir inhibitor fig s7 indicating that cyclind expressionis dependent on the interaction between mir141 andcdkn2bas1 we further observed a decrease in rac1 asmall gtpase and a reduction in the phosphorylation ofpaxillin a focal adhesion protein at mrna levelsfigs s3 s5 and protein levels figs 6g i“j s8c“f inboth mir141 overexpressed and cdkn2bas1 inhibitedrcc cell lines which in turn are involved in regulatingcellular migrationinvasion cumulatively these resultsindicate that suppression of cdkn2bas1 by mir141inhibits renal cell proliferation invasion and migration byinhibiting cyclind rac1 and phosphorylation of paxillinofficial of the cell death differentiation association 0cdasgupta cell death and disease page of fig mir141 overexpression mimics the knockdown effect of lncrna cdkn2bas1 in vitro and reduces tumorigenicity in vivo a relativeexpression of mir141 in achn and caki1 cells transfected with mir141 mimic and control b significant decrease in cdkn2bas1 expressioncompared to control in both cell lines overexpressed with mir141 c rcc cell proliferation after transfecting with mir141 mimic and control asassessed by mts assay d colony formation and its graphical representation in mir141 overexpressing achn and caki1 cells compared to controlse pictures of excised tumors are taken at the termination of experiment day graph represents tumor volume after intratumoral injection ofcontrol or mir141 mimic into established tumors injection was started at day and was followed for days each mouse in both groups mirconand mir1413pmimic received a total of eleven injections intermittently data represent the mean of each group and error bars are sem results arerepresentative of three independent experiments p value calculated by student t test bar mean ± standard error mean semattenuation of mir141 exerts tumorigenic attributes innormal rptec cellswe next determined whether attenuation of mir141induces tumorigenic characteristics in normal rpteccells by targeting cdkn2bas1 and cyclind transienttransfection of mir141 inhibitor indeed showed a significant decrease in mir141 expression fig 7a and anincrease in cdkn2bas1 expression fig 7b along withother procancerous phenotypes such as increased cellproliferation fig 7c colony formation fig 7d migration and invasion fig 7e as compared to controlsadditionally a significant increase in cyclind1 cyclind2 rac1 and paxillin pxn expressions were observed inmir141 inhibited rptec cells fig 7f a noticeableincrease in prometastatic fibronectin and vimentin with aconcomitant decrease in antimetastatic claudin andαecatenin genes were also observed in mir141 inhibited rptec cells compared to controls fig 7gdiscussionprior studies have shown the regulatory role of noncoding rnas in tumorigenesis especially in the emtofficial of the cell death differentiation associationpathway leading to cancer aggressiveness cdkn2bas1also known as anril is located at chromosome 9p21cdkn2bas1 is reported to be upregulated in tumortissues and function as an oncogenic lncrna in pancreatic ovarian and laryngeal squamous cell carcinoma36“ human mir141 is located at chromosome12p1331 and is transcribed from a mir200 familyclusterinterestingly expression of mir141 is controversial since it exhibits either oncogenic39“ or tumorsuppressive roles42“ in specific types of cancer theprime goal of the present study was to understand therole of cdkn2bas1mir141 interactions in regulatingrcc progression and metastasisin this study we identify cdkn2bas1 to be a crucialoncogenic lncrna that plays an important role in renalcarcinogenesis cdkn2bas1significantly overexpressed in rcc and the expression increases fromlower to higher grades and stages lncrna cdkn2bas1directly interacted with mir141 as it was found to be anovel target of mir141 in contrast to cdkn2bas1 weobserved significant attenuation of mir141 expression inrcc cell lines and tumor samples compared to normalis 0cdasgupta cell death and disease page of fig ectopic mir141 expression induces apoptosis and inhibits migrationinvasion in renal cancer cells a apoptosis assessed by flowcytometric analysis of annexinvfitc 7aad“stained achn and caki1 cells transfected with mirconmir141mimic graph represents totalapoptosis early late b immunoblots showing apoptotic proteins in mirconmir141mimic treated achn and caki1 cells with gapdh asendogenous control c migration assay and d invasion assay as seen in pictures and graphical representation of both achn and caki1 cells after mir overexpression compared to control e immunoblot assay showing emt related proteins in mirconmir141mimic treated achn andcaki1 cells with βactin as endogenous control graphs are average of three independent experiments p value calculated by student t testbar mean ± standard error mean semcell line or matched normal samples as it is known thatextensive dna hypermethylation of cpg islands is highlycorrelated to activation of cancerspecific genes45 wechecked the methylation status of mir141 in normal andrcc tissues interestingly insilico analysis showed thepresence of cpg island in the promoter region of mir and we also found hypermethylation of mir141 intcga samples as compared to normal this hypermethylation is also found to be significantly associatedwith poor survival of patients similar results were alsoobserved in rcc cell lines compared to a normal rpteccell line functionally inhibition of cdkn2bas1 andoroverexpression of mir141 significantly inhibits thetumorigenic characteristics such as cell proliferationclonogenicity migration and invasion whereas inducesanticancer apoptotic phenotype in rcc in vitro in vivodata show suppression of tumor growth by mir141overexpression conversely attenuation of mir141 innormal rptec cells induced precancerous characteristicsindicated by increased proliferation migration andinvasionfrom a clinical point of view noncoding rnas signatures are powerful tools for early cancer diagnosisofficial of the cell death differentiation associationmaking them attractive candidates as diagnostic andprognostic biomarkers184647 our results revealed thathigher expression of cdkn2bas1 is positively correlatedwith poor overall survival probability of rcc patientsindicating its prognostic capability cdkn2bas1 canalso discriminate normal from tumor samples showing itsdiagnostic potential similarly mir141 expression canalso robustly distinguish between cancerous from noncancerous samples and hence has potential to be a diagnostic biomarker for rcc collectivelythese resultshighlight the biomarker potential of cdkn2bas1mir expression in rcc although it needs to be confirmedin a larger independent sample cohortinterestingly we found tha

"Cardiac arrhythmias Atrial fibrillation Sudden cardiac death Long QT syndrome Torsade des pointes Ventricular tachycardia Ventricular fibrillation As the coronavirus COVID19 pandemic marches unrelentingly more patients with cardiac arrhyth mias are emerging due to the effects of the virus on the respiratory and cardiovascular CV systems and the systemic ‚ammation that it incurs and also as a result of the proarrhythmic effects of COVID19 pharmacotherapies and other drug interactions and the associated autonomic imbalance that enhance ar rhythmogenicity The most worrisome of all arrhythmogenic mechanisms is the QT prolonging effect of various antiCOVID pharmacotherapies that can lead to polymorphic ventricular tachycardia in the form of torsade des pointes and sudden cardiac death It is therefore imperative to monitor the QT interval dur ing treatment however conventional approaches to such monitoring increase the transmission risk for the staff and strain the health system Hence there is dire need for contactless monitoring and teleme try for inpatients especially those admitted to the intensive care unit as well as for outpatients needing continued management In this context recent technological advances have ushered in a new era in im plementing digital health monitoring tools that circumvent these obstacles All these issues are herein discussed and a large body of recent relevant data are reviewed Elsevier Inc All rights reserved Introduction The ongoing pandemic of coronavirus disease COVID19 has created a global tumult [] According to current data of patients with COVID19 infection are afflicted by acute my ocardial injury with an attendant higher mortality rate compared with those without cardiac injury commensurate with the degree of cardiac troponin cTn elevation [“] Furthermore of patients develop cardiac arrhythmias Table including malig nant ventricular arrhythmias VAs [ ] with a higher prevalence noted in patients admitted to the intensive care unit ICU [] Importantly clinically stable patients may have a low preva Abbreviations AAD antiarrhythmic drug AF atrial fibrillation APCs atrial pre mature complexes AZM azithromycin COVID19 coronavirus CQ chloro quine cTn cardiac troponin CV cardiovascular CYP cytochrome P450 ECG elec trocardiogram HCQ hydroxychloroquine ICU intensive care unit LQTS long QT syndrome NSVT nonsustained ventricular tachycardia OOHCA outofhospital cardiac arrest SCD sudden cardiac death TdP torsade des pointes VAs ventricular arrhythmias VF ventricular fibrillation VPCs ventricular premature complexes VT ventricular tachycardia ˆ—Corresponding author Email address asmotenetgr AS Manolis lence of arrhythmias [] however critically ill patients are at much higher risk for cardiac arrhythmias [] Cardiac arrhythmias including lifethreatening VAs may be the consequence of direct effects of COVID19 infection but also of the deleterious effects of systemic illness and the adverse reactions to drugs employed in the treatment of this pandemic Table Fig [ “ ] A recent study indicated that among patients with ± years men African Ameri COVID19 mean age can receiving care in the ICU there were cardiac arrests incident atrial fibrillation AF episodes bradyarrhythmias and nonsustained ventricular tachycardias NSVTs [] Arrhythmias occurring in patients admitted to the ICU included cardiac arrests all events of cardiac arrest occurred in this group AF odds ratioOR vs those not in the ICU and NSVT OR Car diac arrests were associated with acute inhospital mortality Among patients with confirmed COVID19 ex hibited myocardial injury as indicated by elevated cardiac troponin T cTnT levels [] During hospitalization patients de veloped ventricular tachycardia VTventricular fibrillation VF patients with elevated cTnT levels had more frequent VAs vs p compared with those with normal cTnT levels A recent singleday snapshot survey of stable patients with 101016jtcm202008002 Elsevier Inc All rights reserved Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Cardiac Arrhythmias Occurring in Patients with COVID19 Infection Sinus tachycardia Sinus bradycardia Conduction disturbances AVBBBB Atrial premature complexes Atrial fibrillation Supraventricular tachycardia Ventricular premature complexes Nonsustained ventricular tachycardia Polymorphic ventricular tachycardia Torsade des pointes Sustained ventricular tachycardia Ventricular fibrillation Pulseless electrical activity AVB atrioventricular block BBB bundle branch block Table Mechanisms of Arrhythmogenicity in Patients with COVID19 Infection Acute myocardial injury Myocarditis Hypoxia Systemic ‚ammation Autonomic imbalance SNS overactivity virusinduced vagal nerve injury Electrolyte abnormalities QT prolonging drugs antiCOVID pharmacotherapies AADs other agents Drugdrug interactions Cardiovascular comorbidities hypertension coronary artery disease cardiomyopathy AADs antiarrhythmic drugs SNS In this review we present current data about the whole spec trum of cardiac arrhythmias encountered in patients with COVID infection either attributable to the effect of the virus itself on the cardiovascular CV and the respiratory system andor to the effects of the treatments that these patients receive in combina tion with autonomic imbalance that is incurred by this unrelenting pandemic Acute myocardial injury and arrhythmias As mentioned in patients with evidence of acute myocardial injury the prevalence of cardiac arrhythmias is higher compared to patients without myocardial injury [] In a recent retrospec tive cohort study among patients with severe COVID19 had a cTnI level measured upon hospital admission of whom had positive results showing cardiac injury [] In patients with cardiac injury mortality was higher compared to patients without cardiac injury vs p Arrhythmias were found in of the patients with cardiac injury includ ing patients with VT or VF all of whom died [] A recent meta analysis of studies including COVID19 patients showed that patients with cardiac injury and newly occurring arrhythmias were at higher risk of developing severe disease or requiring ICU admission relative riskRR p [] sympathetic nervous system Sinus tachycardia Sinus tachycardia is the most common rhythm disturbance in patients with COVID19 infection due to multiple reasons such as fever respiratory insufficiencyhypoxemia hemodynamic compro mise fearanxiety pain and several other physical and emotional symptoms [] Bradycardiaconduction disturbances According to a retrospective series of patients transient si nus bradycardia lasting days is a possible manifestation of COVID19 hence another reason for close monitoring [] There may be many reasons for bradycardia but severe hypoxia ‚am matory injury of the sinus node by circulating cytokines and exag gerated response to medications are possible triggers Interestingly bradycardia has been suggested as a warning sign of the onset of a serious cytokine storm Fig The schema illustrates the various arrhythmias encountered in patients with COVID19 infection as a consequence of the virus infection affecting the heart and lung andor producing systemic ‚ammation the adverse proarrhythmic effects of COVID therapies and the drugdrug interactions that may occur see text for long QT discussion AF interval PEA pulseless electrical activity SB sud sinus tachycardia sympathetic nervous system STach den cardiac death SNS ventricular arrhythmias VF TdP ventricular fibril lation VT atrioventricular block LQT torsade des pointes VAs sinus bradycardia SCD atrial fibrillation AVB ventricular tachycardia COVID19 showed a incidence of arrhythmias limited to AF in and supraventricular tachycardia SVT in patients [] A Heart Rhythm Society HRS online survey of electrophysiology professionals n indicated that AF was the most commonly reported tachyarrhythmia whereas severe sinus bradycardia and complete heart block were the most common brad yarrhythmias in hospitalized patients with COVID19 [] Ven tricular tachycardiaVF arrest and pulseless electrical activity were reported by and of respondents respectively A meta analysis of retrospective cohort studies comprising pa tients with COVID19 showed that the pooled incidence was for cardiac arrhythmia for cardiac arrest [] p According to a retrospective cohort study of COVID19 pa tients multivariable logistic regression indicated that among other ECG abnormalities the presence of one or more atrial premature contractions APCs odds ratio OR a right bun dle branch block RBBB or intraventricular block IVB OR p increased the odds of death [] Another study analyzing the ECGs of COVID19 patients showed that abnormal PR interval behavior paradoxical prolonga tion or lack of shortening with increasing heart rate was associ and need ated with increased risk of death vs p for endotracheal intubation vs p compared to patients with PR interval shortening [] Atrial fibrillation According to a recent survey of electrophysiology professionals atrial fibrillation AF was the most commonly encountered car diac arrhythmia observed in patients with COVID19 infection [] Several mechanisms could be involved in the pathogenesis of AF in these patients virusinduced cardiac injury that could lead to perimyocarditis hypoxemia frequently occurring in these patients systemic infection common occurrence of the COVID19 infection Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx in older patients who are already susceptible to AF and sympa thetic nervous system overactivity could all account for such a high incidence of this arrhythmia in this particular population [ ] Guidance on acute management of AF In cases of AF associated hemodynamic compromise as done in all cases of hemodynamically unstable arrhythmias synchronized direct cur rent cardioversion should be used to restore sinus rhythm [] In all other cases one needs to initially proceed with a rate control strategy with use of a betablocker when there is no contraindi cation eg bronchospasm acute heart failure a calcium channel blocker in the absence of heart failure andor digoxin In cases of heart failure digoxin andor amiodarone may be used to achieve rate control For newonset AF within the last hours restoring sinus rhythm is the next target This can be achieved with use of class IA IC or III antiarrhythmic drugs AADs with the selection of the appropriate agent made as based on the presence where only amiodarone seems to be relatively safe or absence of under lying structural heart disease where all other options are available with the caveats being detailed in the discussion that follows be low also taking into account drug interactions with COVID phar macotherapies that are in use A major concern in using specific AADs relates to the baseline measurement of the QT interval and the coadministration of QTprolonging drugs see discussion be low Most importantly all AF patients should be receiving prophy lactic anticoagulation therapy with intravenous heparin Impact of national lockdown on newonset AF diagnosis An other aspect of the impact of national COVID19 lockdowns on the diagnosis of AF has been recently reported by a Danish study [] Using Danish registries the number of patients receiving a new onset AF diagnosis during the first months of and was compared A lower incidence of newonset AF during the weeks of lockdown was noted compared with the corresponding weeks in incidence rate ratios RRs for the weeks and There was a drop in total numbers vs [] Patients diagnosed during lockdown were younger and with a lower CHA2DS2VASc score while history of cancer heart fail ure and vascular disease were more prevalent During lockdown patients with newonset AF suffered an ischemic stroke and died compared with and pa tients during the corresponding period respectively The au thors concluded that following a national lockdown in Denmark a drop in registered newonset AF cases was observed indicat ing that the risk of undiagnosed AF patients developing complica tions could potentially translate into poorer outcomes in patients with AF during the COVID19 pandemic Ventricular arrhythmias In the setting of acute myocardial injury and acute myocarditis in patients with COVID19 infection various and serious ventricu lar arrhythmias VAs may occur [] Other important triggers in clude the severe respiratory insufficiency and the systemic ‚am mation incurred by COVID19 infection as well as the proarrhyth mic effects of COVID therapies and other drug interactions and also the autonomic imbalance superimposed in patients afflicted by the disease [ ] Furthermore hypoxemia which is common in these patients and electrolyte disturbances occurring for various reasons in this group of patients may aggravate arrhythmogenic ity Depending on preexisting or currently emerging CV disease various VAs may be encountered including ventricular premature complexes VPCs nonsustained VT NSVT and sustained VTVF Special attention is required for the development of polymorphic VT in the form of torsade des pointes TdP in the setting of QT prolongation either preexisting or acquired and induced by drugs especially when combination therapies are employed that are po tentially proarrhythmic [] Acute myocardial injury noted in of COVID19 patients can be the inciting factor for various VAs [ ] Among pa tients with confirmed COVID19 malignant VAs VTVF developed in patients during hospitalization patients with ele vated cTn levels had more frequent malignant arrhythmias vs [] A recent retrospective cohort study of patients with severe COVID19 indicated that among having a cTn level measured on admission with showing cardiac in jury arrhythmias developed in of the patients in cluding patients with VT or VF all of whom died [] Critically ill COVID19 patients often have comorbidities that can increase the risk for malignant VAs These include electrolyte abnormalities hypokalemia hypomagnesemia fever an ‚am matory state and most importantly COVID19 pharmacotherapies that are potentially proarrhythmic as they prolong the QT interval and may thus trigger TdP and sudden cardiac death SCD [] On the other hand the acute myocardial injury induced by the virus could also independently prolong the QT interval According to a recent report of a Kawasakilike syndrome temporally associated with COVID19 infection in children among whom myocardi tis was diagnosed in patients left ventricular ejection fractionLVEF range “ of these patients displayed im portant ECG changes that included QT interval prolongation and occasional VAs not attributable to any QTprolonging drug [] Inhospital cardiac arrest As mentioned among patients hospitalized with COVID19 infection all cardiac arrests occurred among patients admitted to the ICU [] In a retrospective cohort study inhospital VTVF occurred in of patients with cardiac injury all of whom died [] Outofhospital cardiac arrest OOHCA A recent Italian study compared all the consecutive outofhospital cardiac arrests OOHCA in the months following the first documented case of COVID19 in the region with those which occurred in the same time frame in [] The cumulative incidence of COVID19 from February to April in the study territory was COVID19100 inhabitants and the cumulative incidence of OOHCA was cases100 inhabitants with a increase as compared with OOHCAs in vs in p The authors concluded that the increase in OOHCAs in is significantly correlated to the COVID19 pandemic and is coupled with a reduction in shortterm outcome A French study comparing the OOHCAs of the pandemic period to the mean of the total over weeks in the non pandemic period indicated that the maximum weekly OOHCA in cidence increased from to per million inhabitants p before returning to normal in the final weeks of the pandemic period [] There was a higher rate of OOHCA at home less bystander cardiopulmonary resus vs p citation vs p and shockable rhythm vs and longer delays to intervention median p min vs min p The proportion of OOHCA patients ad mitted alive decreased from to p in the pan demic period After adjustment for confounders the pandemic pe riod remained significantly associated with lower survival rate at hospital admission odds ratio p COVID19 infection accounted for about one third of the increase in OOHCA incidence during the pandemic Druginduced prolongation of QTc interval and torsade des pointes Several agents employed for treating COVID19 infection may prolong the QT interval and lead to polymorphic VT in the form of TdP Table Chloroquinehydroxychloroquine and azithromycin which have been recently used for potential prophylaxis or treat ment for COVID19 infection are listed as definite causes of TdP Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table QTProlonging Drugs in COVID19 Infection Antibiotics Antiviral agents Anesthetics Antiemetics Antiarrhythmics Antipsychotics ChloroquineHydroxychloroquine Macrolides Azithromycin Quinolones LopinavirRitonavir Favipiravir Tocilizumab Fingolimod Propofol Domperidone Class IA Class III Haloperidol at crediblemeds [] According with the FDA azithromycin other macrolides and fluoroquinolones can cause lethal arrhyth mias as a potential consequence of QTinterval prolongation [] Chloroquine hydroxychloroquine Chloroquine CQ and hydroxychloroquine HCQ have been used for treatment and prophylaxis of malaria while they have also been employed for treatment of amebiasis that is occurring out side the gastrointestinal tract rheumatoid arthritis and lupus ery thematosus These agents were also found to have antiviral effects and have been proposed for the treatment of COVID19 infection [] However both these agents can be proarrhythmic by pro longing the QT interval and potentially initiating lifethreatening VAs including TdP they can also cause QRS widening Chloroquine interacts with multiple cardiac ion channels including the human etheragogorelated gene hERG potassium channel a reduction in hERG channel potassium current is the main cause of acquired druginduced long QT syndrome Recent experimental data indi cated that HCQ markedly increases the action potential dispersion and results in the development of repolarization alternans and ini tiates polymorphic VT [] Preliminary findings from a recent study suggested that the QTc prolonging effect of CQ is dosedependent [] Among pa tients enrolled with COVID19 infection were allocated to highdosage group ie mg CQ bid for days and to lowdosage group ie mg bid on day and qd for days Lethality until day was in the highdosage group of and in the lowdosage group of The highdosage group presented more instance of QTc interval ms compared with the lowdosage group Respiratory secre tion at day was negative in only of patients The authors suggested that the higher CQ dosage should not be rec ommended for critically ill patients with COVID19 because of its potential safety hazards especially when taken concurrently with azithromycin and oseltamivir A recent disproportionality analysis of HCQassociated CV ad verse reactions using the FDA adverse event reporting system FAERS database of datasets and patient records indicated that HCQ was associated with higher reporting odds ratios ROR of TdP ROR CI to complete atrioventricular AV block ROR CI to and QT prolongation ROR CI to [] QT prolongation and TdP are more frequent with high doses for a comparatively short period and represent the most common HCQassociated side effects A systematic review of data on COVID19 patients showed that of COVID19 patients treated with CQHCQ developed QT prolongation [] Ventricular arrhythmias developed in COVID patients from a group of treated with highdose CQ The authors suggest daily ECG monitoring and other risk mitigation strategies to be adopted in order to prevent possible arrhythmic sideeffects Macrolide antibiotics Azithromycin AZM also can cause modest QT interval pro longation but not through potent hERG channel blockade rather when used chronically through an increase in peak and late cardiac sodium current to cause potential loading of cardiomyocytes with sodium and calcium to produce calcium overload Advanced age and female gender are considered risk factors [] Azithromycin can also provoke nonpause“dependent polymorphic VT in the ab sence of QT prolongation [ ] After reviewing the data of AZM regarding risk of QT prolonga tion and associated TdP the FDA revised AZM product labels ad vising against its use in patients with known risk factors such as QTinterval prolongation hypokalemia hypomagnesemia bradycar dia or use of certain QTprolonging antiarrhythmic agents includ ing class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents [] Antiviral agents The combined antiviral regimen of ritonavirlopinavir approved for human immunodeficiency virus HIV infection was also con sidered to be able to suppress SARSCoV2 replication [ ] Lopinavir is metabolized by the hepatic cytochrome P450 sys tem CYP3A [] it also inhibits drug transporters such as Pglycoprotein Pgp [] Thus ritonavirlopinavir may increase plasma concentrations of drugs primarily metabolized by CYP3A or substrates of these drug transporters Ritonavirlopinavir may require dose reductions or avoidance of CYP3Amediated drugs such as rivaroxaban and apixaban Ritonavirlopinavir has also been shown to cause QT and PR interval prolongation or occasionally second or thirddegree AV block particularly in patients with un derlying structural heart disease and preexisting conduction sys tem abnormalities [] Due to its competitive inhibition of the RNAdependent RNA polymerase favipiravir is being evaluated in treating patients with COVID19 alone or in combination therapies the risk for QT inter val prolongation by favipiravir is considered to be low [] Other agents Fingolimod is an immunomodulator and immuno suppressant which reduces lymphocyte migration and is used in the treatment of multiple sclerosis [] it has been proposed as a potential adjuvant therapeutic agent against COVID19 [] Fin golimod has Ltype calcium channel blockade effect causing pro longation of PR RR and QT interval It also activates acetylcholine dependent potassium channels IKach in sinoatrial node causing dosedependent bradycardia [] Thus fingolimod increases the risk of bradycardia and heart block through Ltype calcium channel and IKach blockade [] Combined therapies Treatments employed for COVID19 may increase arrhythmia risk particularly the risk for VAs through drug interactions Drug combinations can lead to greater prolongation of cellular action potential duration analogous to QT prolongation compared with single drug therapies [] The combination effect can result from both pharmacokinetic and pharmacodynamic drug interactions Importantly females with preexisting CV disease seem to be more susceptible to druginduced arrhythmias compared to males with CV disease or healthy persons of either gender Please cite this as AS Manolis AA Manolis and TA Manolis COVID19 infection and cardiac arrhythmias Trends in Cardiovascular Medicine 101016jtcm202008002 0cJID TCM IN PRESS [m5G August ] AS Manolis AA Manolis and TA Manolis Trends in Cardiovascular Medicine xxx xxxx xxx Table Measures to Prevent Arrhythmias in Patients with COVID19 Infection ¢ Withhold QT prolonging drugs in patients with baseline QTc ¢ Withdraw QTprolonging drugs when QTc increases to ¢ Do not use chloroquinehydroxychloroquine azithromycin other macrolides fluoroquinolones lopinavirritonavir or favipiravir in patients with known risk factors such as prolonged QTc hypokalemia hypomagnesemia bradycardia or concomitant use of certain QTprolonging antiarrhythmic drugs including class IA eg quinidine and procainamide and class III eg dofetilide amiodarone and sotalol agents ¢ Maintain K ¢ Monitor QTc via ECG or preferably via telemetry monitor or smart phone measurements ms compared to baseline measurement ms or if QTc is prolonged by ms or with known LQTS mEqL and Mg level to level to mgdL An online survey of electrophysiology professionals revealed that of respondents reported having to discontinue therapy with HCQ AZM due to significant QTc prolongation and reported cases of TdP in patients on HCQCQ and AZM [] Amiodarone was the most common antiarrhythmic drug used for VA management Among COVID19 positive suspected patients stud ± years male received AZM HCQ ied age ± and received both drugs [] Baseline mean QTc was ± ms p with medications Sig ms and increased to ± ms vs nificant prolongation was observed only in men ± ms in women p of patients reached critical ms or QTc QTc prolongation maximum QTc ‰¥ ms Changes in ‰¥ ms if QRS QTc were highest with the combination compared to either drug ± with much greater prolongation with combination vs AZM vs ‰¥ ms or QTc increase of No patients manifested TdP ‰¥ ms if QRS ± ms p ± ms p ± ms vs Another recent cohort study of patients treated for COVID with CQHCQ reported that patients received CQ received HCQ and also received AZM [] Although the maximum QTc during treatment was signifi cantly longer in the combination group vs the monotherapy group TdP was not ob served in the entire population and there were no arrhythmogenic deaths reported A study of COVID patients receiving combined HCQAZM therapy indicated longer QTcinterval than before ther ‰¥ ms had apy vs ms p higher values of transaminases p compared with those with ms [] At h Holter ECG monitoring COVID19 QTc patient and no control had No patients showed R on T VPCs Analysis of h QTc dynamics revealed that COVID19 patients had higher QTc values than controls with no significant hourly variability Therapy with HCQ and AZM pro longs QTc interval in patients with COVID19 particularly in those with high levels of transaminases ‰¥ run of NSVT p patients with a QTc Interestingly in nonCOVID patients a retrospective cohort study identified only two SCDVA events among com bination users CQHCQ plus AZM [] However the doses were lower in this study compared to doses used in COVIDpatients drugs were not used acutely in a hospital setting as currently done for COVID patients fewer cardiac patients received the drugs all suggesting an attenuated risk for cardiac arrhythmias in this par ticular cohort Nevertheless when all measures and precautions are taken Table the incidence of QT prolongation and the TdPevent rate may remain low In a recent study of patients with COVID ± years male HCQAZM was infection mean age ‰¤ 480ms and potassium level initiated only if baseline QTc was mmolL [] Two patients were not eligible for drug ± ms initiation QTc ± ms after h of combined therapy The and increased to treatment had to be stopped because of significant QTc prolonga tion in patients No druginduced TdP nor death was ob served In this specific population HCQAZM could not be initiated or had to be interrupted in ‰¥ ms Baseline average QTc was of the cases QTc monitoring Congenital long QT syndrome LQTS with a prevalence of in the general population may often be asymptomatic and if an ECG has not been recorded it will remain unknown to the affected person and the first manifestation may be SCD usually triggered by a drug [] Furthermore silent genetic variants or œforme fruste  of congenital LQTS encountered in of people may render a person vulnerable to QT prolongation TdP and SCD [] Therefore a large number of healthy individuals will be at an increased risk of a druginduced LQTS Data suggest that in African Americans may be at a higher risk of druginduced TdP during the COVID19 pandemic due to clustering of intrinsic genetic susceptibility ie exclusive oc currence of the proarrhythmic ion channel variant pSer1103Tyr SCN5A acquired risk factors eg electrolyte disturbances and QTcprolonging drug use and COVID19“specific risk factors eg profound hypoxemia and cytokine storm [] A heart ratecorrected QT QTc interval is measured with use ˆšof various formulas among which the Bazett™s correction formula is most commonly used QTc QT RRsec QTc is defined as pro longed when it exceeds ms in males and ms in females as measured preferably in lead II or V on a standard 12lead ECG [] A prolonged QTc predisposes to polymorphic VT in the form of TdP that may degenerate into VF and SCD For the wideQRS adjusted QTc methods that have been suggested include the JT ad justment obtained as QTc“QRS [] or subtracting of the QRS duration from the measured QT [] For patients receiving QTprolonging drugs it is imperative to monitor the QTc interval during treatment Table Traditionally this can be accomplished by obtaining a 12lead ECG however in the era of the COVID19 pandemic this poses a certain risk and puts considerable strain on medical personnel and the health sys tem [] Many telemetry systems are equipped with features of real time QTc monitoring and could be used in hospitalized pa tients and those managed in the ICU setting In addition smart phone heart monitors are also capable of providing remote accu rate QTc measurements [] In this context AliveCor has recently received clearance from the FDA to market the KardiaMobile6L device a previously FDAapproved device for AF detection for QTc monitoring of COVID19 patients treated with QT prolonging drugs such as CQHCQ [] Similarly the Apple Watch ECG an F

ShortTerm Consequences ofPediatric Anticancer TreatmentRegarding Blood Pressure MotorPerformance Physical Activity andReintegration Into Sports StructuresTina Keiser Dominik Gaser Christiane Peters Renate OberhofferFritz Sabine Kesting   and Irene von Luettichau  Edited byKirsten K NessSt Jude Children™s Research Hospital Department of Sports Medicine and Exercise JustusLiebig University GieŸen GieŸen Germany Department of Sport andHealth Sciences Institute of Preventive Pediatrics Technical University of Munich Munich Germany Department ofPediatrics and Children™s Cancer Research Center Kinderklinik M¼nchen Schwabing TUM School of Medicine TechnicalUnited StatesReviewed bySeth E KarolSt Jude Children™s Research HospitalUnited StatesJacques GrillInstitut Gustave Roussy FranceCorrespondenceSabine KestingsabinekestingtumdeIrene von LuettichauIreneTeichertvonLuettichaumritumde These authors sharesenior authorshipSpecialty sectionThis was submitted toPediatric Oncologya section of the journalFrontiers in PediatricsReceived January Accepted July Published August CitationKeiser T Gaser D Peters COberhofferFritz R Kesting S and vonLuettichau I ShortTermConsequences of PediatricAnticancer Treatment RegardingBlood Pressure Motor PerformancePhysical Activity and ReintegrationInto Sports StructuresFront Pediatr 103389fped202000463University of Munich Munich GermanyBackground Cardiovascular diseases in childhood cancer survivors are knownlate sequelae following treatment Arterial stiffness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictors to assess the statusof cardiovascular health Frequent inpatient stays and reduced physical activity PAduring treatment can lead to noticeable impairments regarding motor skills and physicalperformance The present study examined parameters of cardiovascular health motorperformance and the status of integration into sports structures shortly after cessationof treatmentMethods A crosssectional monocentric study was conducted from April to June Participants “ yrs mixed cancer entities during maintenance therapy andfollowup care were recruited Peripheral and central systolicdiastolic blood pressurepSBP pDBP cSBP and PWV were assessed using the MobilOGraph® The MOONtest MOtor performance in pediatric ONcology was used to scale motor performancePA levels and status ofintegration into sports structures were assessed with aquestionnaire referring to the KiGGS study All measured data were compared topublished reference valuesResults Forty participants ± years female were recruited ± years posttreatment PSBP zscore ± p pDBP ± p and cSBP ‰¥ years ± p were significantly increasedcompared to reference values PWV was also elevated but not significantly Motorperformance was reduced in almost all motor abilities Thirtysix percent of the examinedgroup did not participate in physical education at school to the full extent Only reported hour of daily moderatetovigorous PA as recommended for children andadolescents by the World Health anization Half of the participants were active sportsclub members before treatment but one third did not resume their former membershipFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerConclusionIncreased cardiovascular parameters and impaired motor performanceshortly after cessation of treatment physical inactivity and low rates of integration intoregular sports programs highlight the support needed Young cancer patients shouldreceive early support in coping with physical limitations preferably soon after diagnosisMotor deficits could be reduced by applying targeted interventions Furthermorea regular sports therapy program during in and outpatient care could increaseengagement in PA to possibly counteract risk factors and improve cardiovascular healthKeywords childhood cancer cardiovascular health motor performance physical activity sports reintegrationblood pressure arterial stiffnessINTRODUCTIONExtensive research and optimized treatment regimens resulted inan increase of the 5year survival rate to in the USA and of the 15year survival rate to for patients under theage of in Germany However childhood cancer is a raredisease It contributes only around to all malignant diseasesin developed countries Worldwide children underthe age of and adolescents aged between and are diagnosed with cancer every year As a consequence ofthe success in the treatment of childhood cancer the importanceof survival quality and prevention of late sequelae have receivedmore attention during the last yearsKnown negativelongterm consequences ofintensivetreatment for childhood and adolescent cancer patients ofteninclude adverse eï¬ects on the cardiovascular system Cardiovascular diseases are the most frequently reported causesof death in childhood cancer survivors following secondarytumors Arterial stiï¬ness pulse wave velocity PWV andcentral systolic blood pressure cSBP are potential predictorsfor cardiovascular diseases frequently investigated in medicalresearch to evaluate the status of a patient™s cardiovascularhealth PWV describes the velocity of the pressure wave in the aortawhich spreads from the left ventricle through the arterial vascularsystem during systole Noninvasive investigation of the PWVvia ultrasound or oscillometric methods provides informationon the elasticity ofthe vascular system and enables earlyrecognition of damages in the vessels Thus in order to detectpotential structural modifications in the vascular system andindicators for arterial stiï¬ness at an early stage this subclinicalparameter should be surveyed continually Previous data indicatea positive correlation of PWV with arterial vascular stiï¬ness Moreover elevated PWV reflecting subclinical vasculardamage was shown in pediatric patients after hematopoieticstem cell transplantation On the contrary another studyinvestigated elevated blood pressure levels but no statisticallyAbbreviations PWV Pulse Wave Velocity WHO World Health anizationcSBP central systolic blood pressure pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure BMI Body mass index MOONMotor performance in pediatric oncology KiGGS German Health Interviews andExamination Survey for Children and Adolescentssignificant variation for PWV in pediatric cancer survivorscompared to healthy children and adolescents In addition to the abovementioned late sequelae severalproblems already arise during treatment and often persistthroughout survivorship For instancelongterminpatient stays and reduced physical activity during treatmentcan lead to noticeably reduced physical performance ofsurvivors and reintegration into sportschildhood cancerstructures mightberehabilitationprocess throughoutfrequentaï¬ectedIn healthy populations reduced physical activity leadsto negative consequencesfor cardiovascular health Additionally the necessary use of anthracyclines in almost ofapplied therapy regimens in childhood cancer increases the riskof cardiovascular morbidity and mortality eightfold comparedto agematched patients not receiving anthracyclines indicatingthe importance of reducing such longterm consequences Due to a poor state of health and impaired immune functionsports options such as physical education at school engagementin sports clubs or recreational sports are no longer feasibleduring therapy Consequently reintegration after cessation oftreatment is associated with even more barriers due to diseaseand treatmentrelated impairments Circumstances ofanticancer treatment can lead to inactivity resulting in deficitsof fine and gross motor skills reduced muscle strength andpoor physical fitness following treatment Especiallymotor performance in pediatric bone tumor patients oftenremains reduced until at least years after cessation of treatment Impairments of physical performance have been shown topersist throughout survivorship which may complicate thesurvivors™ reintegration into both social and sports structures aswell as the development of a longterm active lifestyle According to a questionnairebased study childhood cancersurvivors™ reintegration rate into physical education at school isvery low especially after treatment for bone tumors The lackof comprehensive oï¬ers of physical activity promotion and motordevelopment might exacerbate motor impairments and problemsof reintegration into sports structures Von Korn et al examined motor performance usingthe Fitnessgram Rcid13 as well as peripheral blood pressure centralblood pressure and PWV using the MobilOGraph Rcid13 in childrenafter treatment for childhood cancer n aged ± years ± years postdiagnosis Their results show reducedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood Cancermotor performance of childhood cancer survivors compared toreference values of healthy children However no correlationcould be drawn regarding cardiovascular parameters and motorperformance The present crosssectional study aimed atinvestigatingvarious parameters of cardiovascular health motor performanceand status of physical activity in children and adolescentsshortly after cessation of anticancer treatment or during ongoingoral maintenance therapy The collection of such data isof considerable importance for the early detection of healthimplications related to both disease and treatment Moreoverthe findings will help to support the development of preventivestrategies regarding the health of children and adolescents treatedfor cancer Appropriate strategies during primary and secondaryprevention and following cancer treatment tertiary preventionneed improvementsMATERIALS AND METHODSDesignThe crosssectional monocentric study was performed over aperiod of months April“June at our institution Theassessment of cardiovascular parameters using the MobilOGraph Rcid13 was followed by the MOON test MOtor performance inpediatric ONcology to evaluate motor performance Finally theparticipants completed a standardized questionnaire referring tothe KiGGS study German Health Interview and ExaminationSurvey for Children and Adolescents to collect data regardingtheir current level of physical activity and status of integrationinto sports structures The Ethics Committee of the School ofMedicine of the Technical University of Munich approved thestudy project number SSR Participation was voluntaryand informed written consent was signed by each participant aswell as by his or her legal guardian All data was collected encodedpseudonym and in accordance with privacy policy standardsParticipantsPrior to addressing the participants all eligible children andadolescents were screened using the electronic patient recordSAP Rcid13 ERP Patients were recruited during routine followupvisits The following inclusion criteria were applied childrenand adolescents during maintenance therapy and followupcare of a pediatric oncological disease and currently agedbetween and years No restriction was applied regardingthe period posttreatment Exclusion criteria were medicalcontraindications such as fever acute infection orthopedicrestrictions and mental retardation insufficient knowledgeof the German language and absence of written informedconsent The attending physician confirmed participation forall recruited children and adolescents Following these inclusioncriteria children and adolescents were initially found eligibleFigure Outcome MeasuresAnamnestic and Anthropometric DataAnamnestic and clinical data ie type of cancer treatmentregime end of therapy was obtained from the electronic patientrecord SAP Rcid13 ERP The nursing staï¬ assessed anthropometricdata height and weight during routine medical examinationseca electronic column scaleseca mechanicalmeasuring rod Body mass index BMI was calculated as aratio of body weight kg per square body height m2 By usingthe reference values of a healthy German cohort BMI wasconverted into percentiles and classified in underweight 10thpercentile normal weight 10thˆ’90th percentile and overweight90th percentile Cardiovascular ParametersPrior to the measurement the participants had to rest for atleast min in a supine position PWV central blood pressureand peripheral blood pressure were assessed using the MobilOGraph Rcid13 IEM GmbH Stolberg Germany and HMS ClientServer Version an oscillometric noninvasive methodMeasurements were performed on the left upper arm The cuï¬was inflated twice with a rest of s in between Cuï¬ sizewas chosen according to the circumference of the participant™sleft upper arm An ARCSolver Algorithm calculated the cSBPindirectly as well as the PWV from recorded brachial pulseRaw data was transformed into zscores and compared by usingzscores of a healthy reference cohort PSBP and pDBPwere compared to references from the national cohort of children and adolescents KiGGS study For the assessmentof PWV and cSBP values references from Elmenhorst et al of healthy children and young adults were used To evaluatethe results of the parameters cSBP and PWV the examinedparticipants were separated into two groups years and‰¥ years According to the age distribution of the referencevalues participants years were compared to heightmatchedreference values and participants ‰¥ years were compared toagematched referencesThe measurement using the MobilOGraph Rcid13 was previouslyused several times in pediatric patients “ and was alsosuccessfully applied in childhood cancer survivors Motor PerformanceTo quantify motor performancethe MOON test MOtorperformance in pediatric ONcology was applied Thetest assesses motor abilities coordination speed flexibility andstrength and consists of eight test items eyehand coordinationinserting pins static balance static stand upper extremitycoordination throwing at a targetspeed reaction testmuscular endurance sittostand flexibility stand and reachhand grip strength handheld dynamometry and muscularexplosive strength medicine ball shot The test lasts min onaverage Data of each item was compared to published age andsexmatched reference values of a healthy population within anage range of to years Data of participants older than years was compared to reference values of healthy 17yearoldssince reference values of healthy 18yearolds are not availableCalculation of a total score is not possible within this toolInstead each item was analyzed individually and the percentagedeviation to reference values was computedFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Flow chart of recruitment Out of participants eligible could not be addressed due to several medical examinations in different departments of thehospital and resulting in missing time slots Longer periods posttreatment are associated with fewer appointments for followups and more medical examinationstake place in day This does not necessarily mean that the children who could not be included in the study due to missing time slots are medically more complexTen participants refused participation Thus the sample included participantsPhysical Activity and Reintegration Into SportsStructures After Acute TreatmentPhysical activity levels and status of integration into sportsstructures were assessed with a standardized selfreportingreferring to the KiGGS study Thequestionnairequestionnaire wassupplemented by several disease andtreatmentrelated aspects in accordance with the study ofKesting et al to investigate potential barriers regardingreintegration and participation in sports activities eg barrierswith respect to exemption from physical education at school ornonparticipation in sports clubs sports therapy oï¬ers duringtreatment The KiGGS study oï¬ers the reference values ofhealthy children and adolescents n for comparison ofour dataData AnalysisCardiovascular parameters were analyzed and compared to thehealthy reference population with the one sample ttest Motorperformance was analyzed using the Wilcoxon signedranktest in comparison to age and sexmatched reference valuesPearson correlation was applied to calculate possible associationsbetween motor performance and cardiovascular parametersBMI and the period posttreatment The MannWhitneyUtestwas performed to evaluate anthracyclinemediated eï¬ects oncardiovascular health as well as diï¬erences within subgroupsregarding diï¬erent entities and levels of physical activity motorperformance physical education at school and achievement ofphysical activity recommendationsExplorative twosided statistical tests were conducted andp ‰¤ was considered statistically significant No adjustmentfor multiple comparison was conducted Correlations coefficientρ were classified according to Cohen Descriptive statistics were calculated with Microsoft Excelversion for demographic characteristics and medicaldata GraphPad Prism version was used to perform allfurther statistical analyses Data analysis was performed inconsultation with the Institute of Medical Informatics Statisticsand Epidemiology of the Technical University of MunichRESULTSParticipantsOut of eligible children and adolescents who met the inclusioncriteria a total of participants female with variouscancer entities were recruited and examined Table Performed AssessmentsFor various reasons not alltests could be realized withall participants In participants cardiovascularparameters could not be evaluated due to missing time slotsFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Anthropometric and medical characteristics of the participants n CharacteristicsN Mean ± SD MedianRangeAge at diagnosis years ± Age at assessment years ± ““ years‰¥ years Period postdiagnosis years ± Period posttreatment years ± ““ year“ years yearsLeukemiaLymphomaBone tumorBrain tumorOther solid tumors Body mass index¢ kgm2 ± “UnderweightNormal weightOverweightChemotherapyAnthracycline applicationCumulative dose mgm2RadiotherapyChestdirected radiation Anthracycline chest radiation Surgical tumor resectionRelapse ± “FIGURE Cardiovascular parameters shown in zscores and compared topublished reference values pSBP peripheral systolic blood pressurepDBP peripheral diastolic blood pressure PWV pulse wave velocity cSBPcentral systolic blood pressure Significant values p ‰¤ Results are given in Mean ± SD M median RangeOther solid tumors alveolar rhabdomyosarcoma n carcinoid tumor of the appendixn nephroblastoma n focal nodular hyperplasia liver n mature cysticteratoma ovary n thoracic ganglioneuroma n papillary thyroid carcinoman neuroblastoma n ¢BMI was converted into percentiles and classified in underweight 10th percentilenormal weight 10thˆ’90th percentile and overweight 90th percentile Thirteen participants received a combination of anthracyclines mainly the combination ofDoxorubicin and Daunorubicinduring routine appointment and a lack of willingness to prolongthe outpatient visit Six of the participants could not performthe MOON test due to medical limitations current orthopedicrestrictions n examinationrelated limitations ie a draintube in the crook of the arm for followup MRT n andabandonment due to lack of time n The central venousdevice has already been explanted in all participants prior toour studyOf the remaining participants not everyone performedevery test item Two participants could not perform the testitem speed due to a drain tube in the crook of the armFour participants could not accomplish the test item muscularexplosive strength due to orthopedic restrictions as well asexaminationrelated limitations Three participants did notperform the item muscular endurance of the legs n hadcrutches n severe muscular deficit in the legs n lackof time Two participants could not perform the test item handgrip strength with both hands due to lack of time n andinfusion needle in the crook of the arm n The test itemupper extremity coordination was measured in n participantsbecause reference values are provided for children between and years onlyCardiovascular HealthIn participants all parameters were assessed Based on theunderlying reference values for cSBP and PWV of Elmenhorstet al the group was separated into participants aged years heightmatched reference values and ‰¥ years agematched reference values PSBP zscore ± p pDBP zscore ± p as well as cSBP values‰¥ years zscore ± p were significantlyincreased compared to reference values of healthy children andadolescents Figure PWV was elevated but not significantly years zscore ± p ‰¥ years zscore ± p Comparison of cardiovascular parameters of participantswho received anthracyclines during intense therapy with acumulative dose of ± mgm2 and subjects whodid not receive cardiotoxic agents did not show statisticallysignificant diï¬erencesMotor PerformanceThe participants™ n motor performance wasreduced in almost all motor abilities compared to the referencevalues of healthy children and adolescents Table Significantimpairments became obvious in the following dimensionsmuscular explosive strength p upper extremitycoordination p muscular endurance of the legs p Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Results of the MOONtest compared to reference values n Motor abilityTest itemEyehand coordinationStatic balanceφSpeedInserting pins timeStatic stand contactsReaction test timeUpper extremity coordinationθFlexibilityThrowing at a target pointsStand and reach cmMuscular explosive strengthMedicine ball shot meterMuscular endurance legsSittostand secHand grip strengthHandheld dynamometry kgRightLeftNMean ± SD of differenceMedian pvalueto reference values ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’All results were compared to the reference values of each single test item Speed could only be measured in participants muscular explosive strength in participants andhand grip strength in right and left participants due to lack of time orthopedic restrictions or drain tube in the crook of the armFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesφ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsθ Although all participants completed this test item reference values are provided for children between and years onlyM median abs indicates absoluteBold numbers indicate significant values p ‰¤ TABLE Results of the MOONtest comparing participants treated for leukemialymphoma and participants treated for brain tumors compared to reference valuesLeukemiaLymphoma n Brain tumor n Motor abilityEyehand coordinationStatic balanceφSpeedFlexibilityMuscular explosive strengthMuscular endurance legsHand grip strength rightHand grip strength leftNMean ± SD Median ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’NMean ± SD Median pvalueˆ’ ± ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ ± ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ All results were compared to the reference values of each single test item φ Static balance was assessed counting the contacts with a foot to the ground while balancing on a rail in this context a negative difference to reference values represents fewercontacts and therefore better resultsFor the test items static balance and flexibility the absolute differences were used as the measured values would have fluctuated around zero and would have givenoversized percentagesDue to insufficient number of participants the comparison regarding the test item upper extremity coordination was disregarded reference values only provided for children aged and yearsM median abs indicates absoluteBold numbers indicate significant values p ‰¤ and hand grip strength on the right hand p The performance of eyehand coordination speed flexibility andhand grip strength on the left hand were also reduced but notsignificantly In the test item static stand the study participantsperformed slightly better compared to the reference populationFor upper extremity coordination throwing at a target onlyreference values from children aged “ years are availableTherefore comparison of the collected data was only possiblewith the same age group n Data for olderparticipants was not collectedComparing motor performance of participants diagnosedwith leukemialymphoma n and participantsn diagnosed with brain tumorsrevealedsome diï¬erences Table Children and adolescents treatedfor brain tumors performed significantly worse in eyehandcoordination than participants treated for leukemialymphomap Moreover the performances in all other testedmotoric dimensions of participants diagnosed with braintumor were deteriorated compared to participants treated forleukemialymphomas but not significantlyTo determine influencing factors on motor performance thecorrelation between motor performance results and BMI as wellas the period posttreatment was performed With increasingBMI values of static balance deteriorated significantly ρ Frontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerFIGURE Pearson correlation between static balance and BMI kgm2¢ leukemialymphoma —¦ bone tumor –³ brain tumor cid7 other solid tumorsHorizontal line indicates the reference values The absolute difference fromreference values is given number of contacts to the ground A negativedifference means fewer ground contacts and therefore better performanceThirty four participants performed the testp which corresponds to a moderate to high correlationFigure A negative diï¬erence to reference values means fewercontacts to the ground and therefore better performance in staticbalance Furthermore some nonsignificant correlations werefound Deteriorated eyehand coordination ρ ˆ’ p and flexibility ρ ˆ’ p were also associatedwith a higher BMI However superior values in upper extremitycoordination ρ p muscular explosive strengthρ p hand grip strength right ρ p left ρ p and muscle endurance ofthe legs ρ p were associated with increasedBMI The test item speed showed no association with BMIρ p A longer period posttreatment was significantly associatedwith decreased eyehand coordination ρ ˆ’ p corresponding to a moderate correlation Figure Especially participants treated for a brain tumor with a longerperiod posttreatment showed deteriorated values in eyehandcoordination Speed performance was deteriorated in participantswith longer posttreatment period ρ ˆ’ p Physical Activity and Reintegration IntoSports StructuresAccording to the selfreported questionnaire n did not participate in physical education at school to full extend n were not admitted to school sports activitiesand n were partly excluded Table Neitherof the two participants treated for bone tumor was takingpart in physical education at school n whereaschildren with other tumors participated to a notably higherrate Treatmentrelated muscular deficits n andosteonecrosis n were the most common reasons forparticipants not taking part in physical education at schoolFIGURE Pearson correlation between eyehand coordination and periodposttreatment months ¢ leukemialymphoma —¦ bone tumor –³ brain tumorcid7 other solid tumors Horizontal line indicates the reference values Thirtyfour participants performed the test itemTABLE Participation in physical education at school subdivided into entitiesn Participation Partial exemption Full exemptionN N N Entire group n LeukemiaLymphoma n Bone tumor n Brain tumor n Other solid tumors n Abs N number Four out of children were still attending kindergarten and could notanswer the question regarding participation in physical education at schoolOnly n reported moderatetovigorous physicalactivity for min daily as generally recommended by the WHOfor healthy children and adolescents Table This percentageis comparable to the achievements in the healthy referencepopulation n Every second participant questioned was an active memberin a sports club whereas n did not return to asports club following cancer treatment Almost onethird n has never been a sports club member Reasons fornot engaging in sports club activities of participants wereno interestfun n physical weakness n no time n anxiety n andphysicianbased prohibition due to clear medical reasons n Nearly all participants n were active inrecreational sportsFurther analyses pointed toward diï¬erences in physicalactivity and sports club participation especially betweenparticipants with brain tumors and leukemialymphomas Thenumber of participants in recreational sports was reported highin both groups leukemialymphoma and brain tumor In contrast a diï¬erence was found in sports club activitySixtysix percent of leukemialymphoma patients were membersFrontiers in Pediatrics wwwfrontiersinAugust Volume 0cKeiser et alPhysical Consequences After Childhood CancerTABLE Physical activity and engagement in sports club and recreationalsportsEntire groupReference populationn n Physical activity guidelinesWHO mindayDaily physical activityDaily walking distance kmDaily walking distance “ kmDaily walking distance kmSports club activity currentlyFormer membershipRecreational sports activityAbs N number Reference values derived from the national cohort of healthy childrenand adolescents in the KiGGS study German Health Interviews and Examination Surveyfor Children and Adolescents of a sports club whereas only of participants with a braintumor were active in a sports club On the other hand almosthalf of the children treated for brain tumor and of thechildren treated for leukemialymphoma were former membersConcerning possible correlations between motor abilities andphysical education at school participation in sports clubs orrecreational sports defined as activeinactive as well as meetingthe physical activity recommendations no significant associationscould be determined Likewise the comparison of motor abilitiesof participants receiving sports therapy during treatment didnot show any correlation Almost half of the participants n took part in a sports therapy programme duringtreatment which was mainly oï¬ered as care and varied greatlyin terms of training interventions without any standardizationDISCUSSIONtreatmentThe results of our study clearly present evidence for deterioratedcardiovascular function in children and adolescents shortlyafter cessation of cancerIncreased pSBP andincreased pDBP are risk factors for cardiovascular diseasesregarding guidelines for arterial hypertension Potentialcardiovascular consequences such as stroke sudden deathheart failure and peripheral artery disease due to elevatedblood pressure values are described in the aforementionedguidelines as well as in the literature Accordinglychildhood cancer survivors with elevated blood pressure are atrisk to experience such cardiovascular late eï¬ects Regarding10years survivors of childhood cancer a higher prevalenceof hypertension is assumed and cardiovascular diseaserelated deaths are eighttimes more likely in childhoodcancer survivors compared to the general population Our findings support previous study results which depictcomplications such as increased blood pressure prehypertensionand hypertension in children and adolescents treated for cancer This study aimed at investigating specific parameters thatcould serve as early predictors for potential damage to thecardiovascular system Recent evidence of cSBP as a suitableparameter to determine the elasticity of blood vessels suggeststhat cSBP is more closely related to cardiovascular events in thefuture than brachial blood pressure Increased cSBP inparticipants in our study may result from early changes in arterialwall stiï¬ness As a further parameter to detect early impairmentsin elasticity of the vascular system PWV was investigated Incontrast to prior studies no decisive change was observedin PWV While anthracycl

" inflammatory bowel disease ibd is the collective term for chronic immunemediated diseases ofunknown multifactorial etiology arising from the interplay between genetic and environmental factors andincluding two main disease manifestations ulcerative colitis uc and crohn™s disease in the last few decadesnaturally occurring alkaloids have gained interest because of their substantial antiinflammatory effects in severalanimal models of disease studies on mouse models of ibd have demonstrated the antiinflammatory action of themain tobacco alkaloid nicotine in addition anatabine a minor tobacco alkaloid also present in peppers tomatoand eggplant presents antiinflammatory properties in vivo and in vitro in this study we aimed to evaluate theantiinflammatory properties of nicotine and anatabine in a dextran sulfate sodium dss mouse model of ucresults oral administration of anatabine but not nicotine reduced the clinical symptoms of dssinduced colitisthe result of gene expression analysis suggested that anatabine had a restorative effect on global dssinducedgene expression profiles while nicotine only had limited effects accordingly map findings revealed that anatabinereduced the colonic abundance of dssassociated cytokines and increased il10 abundances our results support the amelioration of inflammatory effects by anatabine in the dss mouse modelof uc and suggest that anatabine constitutes a promising therapeutic agent for ibd treatmentkeywords plantderived alkaloid mouse model nicotine colitis correspondence pedroantonioruizcastropmicomblainephillipspmicom juliahoengpmicom pedro a ruiz castro ulrike kogel giuseppe lo sasso blaine w phillips andalain sewer contributed equally to this work1philip morris international rd philip morris products sa quai jeanrenaud neuch¢tel switzerland2philip morris international research laboratories pte ltd science parkroad the kendall science park ii singapore singapore the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cruiz castro of inflammation page of crohn™s disease cd and ulcerative colitis uc the mainclinical phenotypes of inflammatory bowel diseases ibdare chronic relapsing inflammatory disorders that affectthe gastrointestinal tract ibd are thought to result froman inappropriate and continuing inflammatory responseto commensal microbes in a genetically susceptible hostenvironmental triggers such as increased hygiene druguse stress smoking and diet influence the onset of thedisease over the past decades naturally occurring alkaloids from plant or medicinal herb sources have sparkedconsiderable interest because of their significant antiinflammatory and antioxidant properties [“]alkaloids”a class of natural bioactive compoundsderived from amino acids that contain one or moreheterocyclic nitrogen atoms”are produced by a widerange of living anisms such as bacteria fungi plantsand animals in plants alkaloids are produced assecondary metabolites in response to environmentalbiotic or abiotic interactions and they confer protectionthrough a range of insecticidal antimicrobial and pharmacological attributes the antiinflammatory activities ofplantderived alkaloids have been documented in severalanimal models of disease including respiratory distress spinal cord injury hepatic fibrosis cancer and ibd [ ] the protective effects of alkaloids havebeen attributed to amelioration of inflammatory responsesand colonic oxidative stress [ ] promotion of epithelial barrier function and positive regulation of gutmicrobiota pyridine alkaloids present in tobacco nicotiana tabacum have been the subject of intensive research nicotinethe major alkaloid in tobacco accounts for approximately of the total alkaloid content of tobacco while thestructurally related nornicotine and anatabine are themost abundant minor pyridine alkaloids accounting for to of total alkaloids other pyridine alkaloids intobacco such as anabasine anabaseine and cotinine arepresent in smaller amounts nicotine and all minortobacco alkaloids have been shown to be pharmacologically active upon binding to several nicotinic acetylcholinereceptors nachrs tobacco nachr agonists suchas nicotine anatabine anabasine anabaseine and cotininedisplay protective effects in animal models of several inflammatory conditions including sepsis parkinson™sdisease alzheimer™s disease and ibd several in vitro and in vivo studies have shown a clearnicotinedependent positive effect on inflammatory processes [ ] in a previous study oral nicotine administration reduced the severity of dssinduced colitis andreduced colonic tnfα synthesis while subcutaneous injection or minipump infusion had no effect highlightingthe crucial role of administration route for the protectiveeffects of nicotine in dss colitis nicotine has alsobeen shown to attenuate the severity of dss colitis andexpression of il6 in cd4t cells a recent studysuggested that nicotine ameliorates dssinduced inflammation through inhibition of signaltransducer andactivator oftranscription stat3 in gutinfiltratedlymphocytes and intestinal epithelial cells recentlynicotine was shown to cause a decrease in leukocyte recruitment disease activity index dai and histologicalscore in dss colitis and block tnfmediated expressionof mucosal vascular addresin cell adhesion molecule1 inendothelial cells these authors concluded that nicotinesuppressesinflammation through downregulation ofadhesion molecules in the gut nonetheless clinicalstudies on the efficacy and tolerability of nicotine haveshown thattherapeutic application of nicotine fortreatment of uc is limited because of frequent adverseeffects and nicotine inconsistent efficacy in maintainingremission in uc patients [ ]anatabine is found in plants of the solanacea familywhich includes tobacco peppers tomato and eggplant little is known about the biological properties of anatabinealthough several studies have suggested that this alkaloid is apotential candidate compound for antiinflammatory drugdevelopment [ ] anatabine was marketed in the us asa dietary supplement under the name anatabloc in aninternetbased survey approximately of all users ratedthe effect of anatabine supplementation as good or excellentfor joint pain stiffness and functionality anatabine hasbeen shown to inhibit lipopolysaccharide lpsinducedproinflammatory gene expression as well as nfκb andstat3 phosphorylation in human neuroblastoma shsy5y hek293 human microglia and human bloodmononuclear cells as well as in the brain and spleen ofmouse models of autoimmune encephalomyelitis andalzheimer™s disease in shsy5y cells anatabine alsoreduced the expression of betasecretase ”the rate limitingenzyme for amyloid peptide production which is a majorhallmark of alzheimer™s disease”through inhibition of nfκb activation in this study we aimed to assess the antiinflammatoryeffects of the tobacco alkaloids nicotine and anatabine inthe established dss mouse model of uc our resultsshow that oral administration of anatabine but notnicotine ameliorates the clinical manifestations of dsstreatment in mice the results of gene expression analysisindicated that anatabine had a partial restorative effect onglobal dssinduced gene expression profiles while nicotineonly had minimal effects moreover multianalyte profilingmap showed that anatabine but not nicotine suppressesthe production of il6 il1α tnfα granulocytecolonystimulating factor gcsf and keratinocyte chemoattractant kc while increasing il10 expression in the colon ofdsstreated mice for an overview of the study conceptand analytical procedures see œonline resource  0cruiz castro of inflammation page of resultsanatabine has a protective effect in the dss mousemodel of colitisto study the antiinflammatory properties of nicotineand anatabine c57bl6 mice were provided with nicotine or anatabine in drinking water for a total of dayscolitis was induced by oral administration of dssin drinking water ad libitum during days “ fig 1adsstreated mice developed colitis as evident from thebody weight loss fig 1b increased colon weightlengthratio fig 1c increased dai fig 1d increased stooloccult blood score fig 1e increased intestinal bleedingscore fig 1fincreased diarrhea score fig 1g andincreased colon inflammation score fig 1h in micefig clinical parameters of dsstreated mice administered nicotine or anatabine a mice were orally administered nicotine or anatabine or mgkg for a total of days colitis was induced by oral administration of dss in drinking water ad libitum during days “ b bodyweight changes in mice during the colitis induction and recovery phases body weight changes were calculated as percentage relative to thestarting day of dss treatment day c colon weightlength ratio are represented as mgcm of colon d dai was calculated according to weightloss colon weightlength ratio and intestinal bleeding e stool occult blood score f intestinal bleeding score g diarrhea score h coloninflammatory status data are shown as mean ± sem p p nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg 0cruiz castro of inflammation page of not subjected to dss treatment nicotine and anatabine administration had no significant effect on these parametershowever mice treated concomitantly with anatabine anddss showed a decrease in colitis severity in particular inmice that received concomitant anatabine and dss treatment anatabine improved body weight recovery at mgkg p fig 1b caused a decrease in global dai relative to that in dsstreated mice at daily dose of p and mgkg p fig 1c and reduced the stooloccult blood score at mgkg p fig 1e interestingly nicotine but not anatabine improved the intestinalbleeding score relative to that in dssadministered mice at mgkg p fig 1f the diarrhea score fig 1gand the colon inflammation score fig 1h were notaffected by nicotine or anatabine no variation in waterconsumption was registered across the different experimental groups online resource anatabine reduces dssinduced gene expression changesin the distal colon on a global levelto complement these pathological findings we analyzed the colon transcriptome dsselicited lesions inmost mouse inbred strains including c57bl6 micehave been shown to be more pronounced in the distalcolon [ ] therefore we focused our study on thatportion of the large intestine principal componentanalysis clearly captured the effect of dss treatmenton the first principal component explainedvariance fig 2a while nicotine treatment did notproduce a pronounced effect on the first principalcomponent in the dsstreatment context anatabinetreatmentin combination with dss produced aresponse more similar to that observed in the watercontrols no dssindicating that anatabine had anameliorating effect on dssinduced gene expressionchanges the results of our differential gene expressionanalysis suggested substantial perturbation of the colontranscriptomefdr fig 2b“c application of dss in drinking watercaused significant changes in nearly genes incolon tissues addition of nicotine to dsscontainingdrinking water slightly increased the number of differentially expressed genes in contrast addition of anatabine decreased the number of differentially expressedgenes upon dss treatment by approximately fig 2b and c this alleviating effect of anatabinetreatment on dssinduced gene expression profileswas also apparent in the global gene expression heatmap which showed a global reduction of expressionfold changes upon anatabine treatment fig 2d and eof note in the absence of dss anatabine treatmentdid not result in differential expression of genes andnicotine treatment alone had minor effects fdr pvalue online resource upon dsstreatmentanatabine reduces dssinduced inflammatory geneexpression in the distal colonto gain a more mechanistic understanding of the effectsof nicotine and anatabine treatment we further investigated gene expression changes at the level of functionalgene sets and a ucrelevant causal network model gsaofthe reactome database showed changes acrossmultiple functional categories fig 3b the effects onfunctional categories in dsstreated mice administerednicotine appeared rather similar to those in mice treatedwith dss alone whereas administration of anatabineresulted in a general repression of dssmediated effectswe also evaluated the interaction terms betweennicotineanatabine and dss treatment to more directlyfocus on the modulating effect of anatabine and nicotinetreatment on dss effects fig 3a online resource the following six biological categories showed significant interaction terms upon anatabine and dss treatmentsupporting asignificant suppression of dssinduced effects in thepresence of anatabine œimmune system œextracellularmatrix anization œprotein localization œmetabolism œhemostasis and œsignal transduction fig 3bof these we further explored œimmune system œextracellular matrix anization and œsignal transductionas the most relevant categories in ibdana 20dss fdr allfiginteractiontermsp the hierarchical anization of the reactome database allowed us to investigate the underlying functionalchanges in more detail the œimmune system categoryincludes œadaptive immune system œcytokine signaling in immune system and œinnate immune systemin particular within these immune categories œcytokinesignaling egincluding il6 family signaling andœtolllike receptor cascades were modulated withsignificant3conline resource online resource within thereactome œsignal transduction category œsignaling byreceptor tyrosine kinasesby rhogtpases œsignaling by transforming growth factortgfbeta family members œmapk family signalingcascades œintegrin signaling œsignaling by erythropoietin and œsignaling by gpcr were found to be significantly impacted online resource online resource within the œextracellular matrix anization category almost all subcategories were perturbed includingœdegradation of the extracellular matrix œelastic fiberformation and œextracellular matrix proteoglycansonline resource online resource œsignalingœdegradation ofthe extracellular matrix includesbiological processes such as activation of matrix metalloproteinases mmps and collagen degradationto further follow up on the effects of nicotine andanatabine on inflammatory signaling we evaluatedthe perturbation of the ucrelevant tlril1rtnfr 0cruiz castro of inflammation page of fig transcriptomics results of colon biopsy samples a principal component pc analysis the plot displays all samples in the reduced pc1“pc2plane covering of the total data variance it allows us to examine the relationships between the various groups and treatmentsremarkably pc1 captured the pure dss effect black arrow while pc2 captured the pure exposure effects of anatabine and nicotine blue andred arrows respectively b volcano plots for individual gene differential expressions the horizontal axis represents the log2 differential expressionœfold changes and the vertical axis represents its statistical significance as log10 fdr c number of differentially expressed genes for theselected pairwise comparisons the bar plot displays the number of genes with positive or negative fold changes with corresponding statisticallysignificant fdr ‰¤ note that the lower fdr values observed for the œana dss comparisons do not necessarily prefigure a reduction ofbiological effects because the statistics underlying the fdrp values also depend on the gene expression variance within the experimentalgroups d highlevel heatmap of the statistically significant differentially expressed genes for the selected pairwise comparisons in order toprovide a comprehensible display of the large — foldchange matrix we first normalized its rows to their maximum absolute values andthen reordered them by hierarchical clustering complete linkage method on the basis of their euclidean distances e scatter plot from thecomparisons between the pure and exposuremodified dss effects by using the differential gene expression results the horizontal axis of thescatter plots represents the fold changes of the pure dss effect œwater dss vs water pairwise comparison whereas the vertical axis containsthe corresponding values in case of anatabine or nicotine exposure œanatabinenicotine dss vs water pairwise comparisons in an idealcase included as a reference [first plot] the bestfitted straight line coincides with the diagonal indicated in green and its slope is equal to the transcriptomics effects of anatabine or nicotine exposure were quantified by the slope of the bestfitted straight lines indicated on each plotnic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgnetwork model by using an established networkenrichment approach we inferred the activationstates of the network nodes on the basis of the observedgene expression changes and calculated the overall network perturbation amplitude for each group comparisonfig 3c dss treatment had a strong activating effect onthis signaling network including activation of several coresignaling nodes such as il1rassociated kinase irak1irak4 and myeloid differentiation primary response myd88 online resource œheatmap leadingnodes of note the network perturbation induced bydss treatment was reduced only in the presence ofanatabine with this the results of network analysisfurther supported the ameliorating effect of anatabineon dssmediated inflammation whereas no similareffect was apparent upon nicotine treatment 0cruiz castro of inflammation page of fig biological interpretation of the transcriptomics results a schematic representation of the effects of anatabinenicotine exposure as amodification of the pure dss effect the measured combined œanatabinenicotine dss effect captured by the pairwise comparisons œanatabinenicotine dss vs water can be viewed as the sum of the pure effects of the two factors œdss treatment and œanatabinenicotine exposuretogether with the adjusting twofactor interaction term œanatabinenicotinedss anatabinenicotine exposure is synergistic with dss treatmentif the interaction term has the same sign as the pure dss effect eg both are positive as in the schema in contrast the relationship isantagonistic if the interaction term has an opposite sign to the pure dss effect b gsa results for the top reactome pathway categories theheatmap displays the gsa scores normalized rowwise to their maximum absolute values as well as their competitive q1 statistical significancethe fdrs were calculated only among the top reactome pathway categories which are sufficiently distinct in their gene content to assumeindependence of their enrichment results c hierarchical representation of the gsa results for all pathways contained in the top reactomeœimmune system category and for the twofactor œana 20dss interaction the color map corresponds to the normalized gsa scores containedin the interval [ˆ’ ] whereas their statistical significance competitive q1 p values ‰¤ is indicated by black circles around the nodes theactual labels of the nodes ie the reactome pathway names can be found in online resource the treelike structure of the reactomepathway collection enables topdown investigation within the relevant pathway categories by identifying increasingly specific biologicalprocesses ie involving fewer and fewer genes along the longest paths connecting the statistically significant pathways d npa results for thetlr“il1r“tnfr network model the bar plot displays the npa values for the selected contrasts and their statistical significance is indicated bythe three colored asterisks note that by definition the positive npa values depend on the square of the input genelevel fold changes andtherefore might amplify the differences between the contrasts without preserving the additive relationships among them nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkgexpressionvalues”from theto validate the observations obtained by microarraytranscriptomics we quantified the expression of six œleadingedge genes”genes of the gene set with the highestœimmunedifferentialsystem œextracellular matrix anization and œsignaltransduction reactome categories using realtime quantitative pcr qpcr selected genes included il33 œsignaltransduction and œimmune system categoriesil6œsignal transduction and œimmune system categoriesmmp13 œextracellular matrix anization categoryserpine1 œsignal transduction and œextracellular matrixanization categories thbs1 thrombospondin œsignal transduction and œextracellular matrix anizationcategories and timp1 tissue inhibitor of metalloproteinase œextracellular matrix anization and œimmunesystem categories qpcr results showed a clear decreasein dssinduced expression of il33 il6 mmp13 serpine1thbs1 and timp1 expression in the presence of anatabineat mgkg thereby confirming the findings obtainedfrom the microarray data fig 0cruiz castro of inflammation page of fig validation of microarray transcriptomic results using qpcr a boxandwhisker plots for the distribution of the qpcr expression levels δcqof the selected genes the boxes represent the quartiles while the whiskers extend to the most extreme data point which is no more than times the interquartile range from the box the horizontal brackets indicate the statistical significance of the corresponding comparisons mean pvalue smaller than respectively b scatter plots comparing the mouse colon differential expression values obtained bymicroarray horizontal axis and qpcr vertical axis the following similarity metrics were indicated œbeta is the slope of the best interceptfreelinear fit between microarray and qpcr values œr2 is the coefficient of determination measuring the œgoodnessoffit and œpval is the pvalueassociated to the null hypothesis beta nic nicotine mgkg ana anatabine mgkganatabine decreases dssinduced proinflammatorycytokine production and promotes il10 expressionnext we sought to evaluate the impact of nicotine andanatabine on the expression of colonic inflammatorycytokines by mapin line with the previous dataanatabine but not nicotine significantly reduced theabundance of dssassociated inflammatory cytokines including il6 kc tnfα il1α and gcsf whereas itincreased the levels of the antiinflammatory cytokineil10 at a daily dose of mgkg fig interestinglyanatabine also increased the abundance of il21 andshowed a clear tendency towards increasing colonic il 0cruiz castro of inflammation page of fig map results for colon biopsies statistical assessment of the differences observed in the abundance of selected cytokines between thestudy experimental groups and water control fold changes in the treatment groups nicotine and anatabine at and mgkg dss relative towater control are illustrated with colors ranging from blue decrease to red increase statistically significant differences on the basis of raw pvalues no adjustment has been made for multiple testing grey cells highlight missing estimates on the observed differences due to lackof cytokine quantifications nic nicotine mgkg nic nicotine mgkg ana anatabine mgkg ana anatabine mgkg1 levels fig taken together the map data confirmthe antiinflammatory effects of anatabine in dssinduced colitisdiscussionour study shows that oral administration of anatabinebut not nicotine reduces the clinical manifestations ofdssinduced colitis in a mouse model in line with thesefindings anatabine demonstrated a global downregulatory effect on dssinduced gene expression changes inthe colon whereas the effects of nicotine were morelimited in particular the results of gene expression profiling further supported the reduction of inflammatorysignaling processes upon anatabine treatment includingsuppression of il6 signaling as shown by gsa findingsand tlr signaling as shown by the results of networkperturbation analysis and gsa map also showed asignificant decrease in the abundance of il6 kc tnfαil1α and gcsf and an increase in the expression of il and the antiinflammatory cytokine il10several studies have reported on the antiinflammatoryeffects of nicotine on the dss mouse model of uc subcutaneous administration of nicotine was shown toameliorate tissue injury in dss colitis and il6 expression in cd4t cells via α7nachrs nicotine wasalso shown to decrease the activation of stat3 throughinduction of mir124 in gutinfiltrated lymphocytes andintestinal epithelial cells strikingly alsharari 0cruiz castro of inflammation page of observed that oral but not subcutaneous injection ofnicotine ameliorated intestinalinflammation and colonic tnfα expression in dsstreated mice in spiteof the reported beneficial effects our results do not support a protective effect of orally administered nicotineon dss colitis of note we found that nicotine significantly reduced dssassociated intestinal bleeding whichwas the only clinical parameter affected by this tobaccoalkaloid the vasoconstrictor effects of nicotine are wellstablished in the gut mucosa nicotine decreasesblood flow and cigarette smoking decreases rectalblood flow to normal levels in patients with uc however how changes in blood flow affect the pathophysiology of uc is still unclear the possible therapeuticuse of nicotine to induce remission in uc patients hasbeen evaluated in five clinical studies [“] and twometaanalyses [ ] these studies have demonstrated avariable efficacy of nicotine therapy in induction of remission with several studies showing no effect [ ]moreover a high frequency of adverse events increasedthe withdrawal rate in the nicotine group in some studiesthus limiting the therapeutic benefit of nicotine nucleotidebindingto the best of our knowledge the present study isthe first to assess the impact of anatabine on experimental colitis gene expression analysis of distal colonbiopsy specimens highlighted the antiinflammatoryproperties of anatabine in multiple functional categories including œimmune responses œsignal transduction and œextracellular matrix anization which inturn encompassed several tlr and cytokine signalingpathways genes contributing to the downregulation oftlr cascades included tlr2 tlr4 and tlr6 and anumber of downstream signaling factors shared byseveral tlrs including myd88 ripk2 irak3 irak4and nod1oligomerizationdomaincontaining protein as well as the nuclearfactors elk1 fos cyclic adenosine monophosphatecamp response elementbinding protein creb1activating transcription factor atf1 and atf2 seeonline resource of the respective gene lists for thecytokine signaling pathways the contributing genes included il6 and il6 receptor α ifnγ il4 cxcl10il22 receptor α2 il2 receptor α tnf receptor superfamily member 1a il17α and il17 receptor α jak1jak2 stat3 and stat4 members of the nfκbsignaling pathway also contributed to the overall reducincluding nfκb p65tion in inflammatory cascadesp105 and p100 subunits iκbα and the nfκb activating protein tab3 in agreement with the results oftranscriptional analysis map findings showed a significant decrease in dssassociated il6 kc tnfα il1αand gcsf protein expression and an increase in il10expression in the presence of anatabine strikinglywhile tlr downstream factors modulated by anatabineare shared by most tlrs the majority of cytokineassociated signaling molecules are specific for eachpathway the seemingly pleiotropic regulatory effects ofanatabine suggest that this alkaloid reduces inflammation by inhibiting the expression of several factors involved in different proinflammatory signaling cascadesprevious studies using in vitro and in vivo diseasemodels have demonstrated the antiinflammatory effectsof anatabine [ ] paris showed that this pyridine alkaloid reduced the plasma levels of il1 il6and il17a as well as the expression of il1 infγ andtnfα in the spleen of experimental autoimmune encephalomyelitis mice these authors also showedthat anatabine suppressed stat3 and nfκb phosphorylation in the spleen and brain of these mice anatabine also prevented lps and tnfαinduced nfκb andstat3 phosphorylation in shsy5y and hek cellshuman microglia and human blood mononuclear cells additionally anatabine prevented lpsinduced il1 expression in human whole blood as well as il1il6 and tnfα production in the plasma kidney andspleen in the lps mouse model phosphorylatedstat3 tnfα and il6 were also downregulated in thepresence of anatabine in a transgenic mouse model ofalzheimer™s disease our results on the effects of anatabine in the dssmouse model of uc are also in line with the findings ofa substantial number of studies demonstrating the protective effects of natural alkaloids in several animalmodels of colitis [ ] intraperitoneal administrationof the minor tobacco alkaloid and nicotinic receptoragonist anabaseine was shown to reduce tissue damagemyeloperoxidase activity and colonic tnfα expressionin a tnbs mouse model of colitis these mice alsoshowed reduced nfκb activation in lamina propriamononuclear cells while mice administered a nicotinicreceptor antagonist presented worse colitis symptomsthan those treated with tnbs alone the algaealkaloid caulerpin reduces dss colitis by suppressingnfκb activation and subsequently inhibiting the colonicproduction of tnfα ifnγ il6 ifnγ and il17 oral administration of berberine also ameliorates dssinduced colitis and downregulates the expression oftnfα ifnγ kc and il17 in colonic macrophages the plantderived alkaloid nmethylcytisine andthe tea alkaloid theophylline mitigate colitis by downregulating tnfα il1 and il6 expression in dss andacetic acid models of colitis respectively [ ] induction ofthe antiinflammatory cytokine il10 in thepresence of natural alkaloids has also been reportedthus indirubin ameliorates dssinduced colitis by suppressing the expression of colonic tnfα ifnγ and il2and upregulating il10 additionally indole alkaloids caulerpin and isatin have been shown to increase 0cruiz castro of inflammation page of the expression of il10 in dss and tnbs models of ibdrespectively [ ] of note our results show anincrease in the abundance of il21 and a tendencytowards increase in colonic il1 levels in the presenceof anatabine although il21 expression is increased inmany chronic inflammatory disorders genetic deficiencyof il21 is associated with ibd and inhibition of il21in the early phases of some inflammatory disorders exacerbates disease development suggesting the dual role ofil21 in the control of immune responses il21also promotes il22 expression in mucosal tcellsthrough a mechanism involving stat3 retinoidrelatedorphan receptor γt and aryl hydrocarbon receptorthereby helping protect immunodeficient mice from dsscolitis interestingly il21 has been recently shownto induce il1 production in dendritic cells through astat3dependent but nfκbindependent mechanismthereby suggesting a link between il21 and il1 mounting evidence suggests that alkaloids”in particular isoquinoline alkaloids present in traditional medicineherbs”exertthroughregulation of nfκb and stat3 signaling pathways forexample sanguinarine and cavidine suppress the expression of nfκb p65 subunit thereby reducing colonictnfα and i

curative therapeutic options for a number of immunological disorders remain to be established and approaches for identifying drug candidates are relatively limited furthermorephenotypic screening methods using induced pluripotent stem cell ipscderived immunecells or hematopoietic cells need improvement in the present study using immortalizedmonocytic cell lines derived from ipscs we developed a highthroughput screening htssystem to detect compounds that inhibit il1 secretion and nlrp3 inflammasome activation from activated macrophages the ipscs were generated from a patient with neonatalonset multisystem inflammatory disease nomid as a model of a constitutively activatednlrp3 inflammasome hts of compounds including fdaapproved drugs and compounds with known bioactivity identified compounds as predominantly il1 inhibitorssince these compounds are known inflammasome inhibitors or derivatives of these resultsprove the validity of our hts system which can be a versatile platform for identifying drugcandidates for immunological disorders associated with monocytic lineage cellsintroductionone of the main cell types affected by immunological disorders are white blood cells such aslymphocytes monocytes and neutrophils although our understanding of the cellular pathophysiology of immunological disorders has greatly benefited from in vitro studies usingpatientderived primary hematopoietic cells or in vivo animal models these approaches haveseveral limitations patientderived hematopoietic cells cannot be obtained in sufficient quantities and their in vitro phenotypes can be affected by the in vivo conditions of the patient suchas the cytokine milieu or the administration of therapeutic agents while animal models haveprovided important insights into these disorders species differences in the immunologicala1111111111a1111111111a1111111111a1111111111a1111111111open accesscitation seki r ohta a niwa a sugimine y naitoh nakahata t induced pluripotentstem cellderived monocytic cell lines from anomid patient serve as a screening platform formodulating nlrp3 inflammasome activity one e0237030 101371 pone0237030editor xiaoping bao purdue university unitedstatesreceived march accepted july published august peer review history recognizes thebenefits of transparency in the peer reviewprocess therefore we enable the publication ofall of the content of peer review and authorresponses alongside final published s theeditorial history of this is available here101371 pone0237030copyright seki this is an openaccess distributed under the terms of thecreative commons attribution license whichpermits unrestricted use distribution andreproduction in any medium provided the originalauthor and source are crediteddata availability statement all relevant data arewithin the manuscript and its supportinginformation files one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfunding this work was supported by the grant forthe core center for ips cell research of researchcenter network for realization of regenerativemedicine from the japan agency for medicalresearch and development amed [tn and mks] the program for intractable diseases researchutilizing diseasespecific ips cells of amed and [tn and mks]practical research project for allergic diseasesand immunology research on allergic diseasesand immunology of amed [mks]practical research project for rareintractablediseases of amed [mks] and thedevelopment causes discrepancies in the function and phenotype of the immune cells [“]overall highthroughput screening hts of therapeutic compounds using patientderivedcells or animal models is usually not feasiblethe establishment of disease or patientspecific induced pluripotent stem cells ipscs has led to the development of a new field of disease modeling owing to their pluripotencyand capacity for selfrenewal ipscs can function as an unlimited source of patientderivedsomatic cells and progenitor cells ipscs have also been used as a source of phenotypebasedhts [“] however several roadblocks remain for ipscbased hts as follows obtaining alarge number of differentiated progenies from pscs is cost and laborintensive and theyield and function of the differentiated cells often vary among clones and experimentalbatchesjapan society for the promotion of science jspswe have established ipscs from patients with autoinflammatory syndromes including neokakenhi grant number [mks]nippon shinyaku co ltd provided support inthe form of salaries to the authors [rs and hn]the specific roles of rs and hn are articulated inthe ˜author contributions™ section the funders hadno role in study design data collection andanalysis decision to publish or preparation of themanuscriptcompeting interests rs and hn are employeesof nippon shinyaku co ltd this employmentdoes not alter our adherence to one policieson sharing data and materialsnatalonset multisystem inflammatory disease nomid also known as chronic infantile neurological cutaneous and articular [cinca] syndrome nakajonishimura syndrome and blau syndrome for disease modeling in these studies ipscderived myeloid cells wereimmortalized by transducing lentiviral vectors that encoded myc bmi1 and mdm2 anddisease phenotypes were recapitulated in vitro thus ipscderived immortalized myeloid celllines ipsmls can be expanded from one experimental batch with reduced financial and laborcosts they also can be stored and differentiated into terminally differentiated progenies therefore ipsmls can overcome the roadblocks associated with ipscbased hts nomid is the most severe form of cryopyrinassociated periodic syndrome caps anautoinflammatory disease caused by heterozygous mutations in the nlrp3 gene [ ]nacht lrr and pyd domainscontaining protein nlrp3 is expressed mainly in myelomonocytic lineage cells and acts as a sensor of cellular stress induced by various pathogens andsterile stimuli in normal macrophages a œpriming stimulus such as lipopolysaccharidelps induces the expression of nlrp3 and prointerleukin il1 an inactive form of theproinflammatory cytokine il1 then an œactivating stimulus such as adenosine triphosphate atp enhances the assembly of a protein complex known as nlrp3 inflammasomethis inflammasome contains the protease caspase1 which processes proil1 to the matureform on the other hand lps stimulation of monocytic cells obtained from untreated capspatients induces robust il1 secretion without secondary activating signals due to autoactivation of nlrp3 inflammasome indeed antiil1 therapy for caps patients has beenproven effective [ ] however antiil1 therapy has several weak points the efficacy ofantiil1 therapy is often inadequate for patients with severe phenotypes il1 maturation is mediated not only by nlrp3 inflammasome but also other inflammasomes and proteases [ ] thus a complete blockade of il1 may result in excessive immunosuppressionmoreover the cost and lifelong injection of biologics worsen the patients™ quality of life therefore other therapeutic approaches such as the direct inhibition of nlrp3 inflammasomeactivity are under considerationnlrp3 inflammasome is an attractive drug target because nlrp3 inflammasome activation is associated with the pathogenesis of various chronic inflammatory conditions recently several selective nlrp3 inhibitors entered the clinical phase their chemicalstructures are undisclosed but presumed to be sulfonylureas or their derivatives mcc950 asulfonylureabased potent selective inhibitor of nlrp3 inflammasome activation wasalso recently identified as a direct nlrp3 inhibitor by binding to the walker b atphydrolysismotif of the nacht domain [ ] given that capsrelated mutations frequently occursin the nacht domain it is not surprising that most capsrelated nlrp3 mutantsescape efficient mcc950 inhibition therefore novel nlrp3 inflammasome inhibitorseffective for diseases including caps are sought one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningin the present study we developed an hts system to identify compounds that regulate theactivity of nlrp3 inflammasome using ipscs generated from a nomid patient we established ipsmls which we used as a prototype of nlrp3activated immune cells by immortalizing ipscderived monocytic progenitor cells we conducted hts of approximately compounds and validated their inhibitory effect on the secretion of il1resultsfunctional monocytic cells on feeder and serumfree monolayer culturewe previously reported that macrophages derived from ipscs carrying diseaseassociatedmutations in the nlrp3 gene showed excessive secretion of il1 without secondary signals these macrophages were considered functional based on compatible appearances withprimary macrophages under an electron microscope the secretion of proinflammatory cytokines and the phagocytosis of listeria monocytogenes macrophages in that study wereobtained via a differentiation protocol using op9 feeder layers recently we have updated ourmonocytemacrophage differentiation system to a defined condition without feeder cells andserums which can also produce functional macrophages we therefore examined whethermonocytes obtained with the feeder and serumfree monolayer differentiation system exhibited a similar in vitro phenotypefor this a nlrp3mutated ipsc clone derived from a nomid patient with a somaticnlrp3y570c mutation was differentiated into monocyticlineage cells since thenlrp3 mutation was a somatic mosaicism the nlrp3nonmutated wildtype clonederived from the same donor was used as an isogenic control ipsc line the differentiatedcells showed a mononuclear and slightly foamy appearance consistent with the appearance ofin vitro differentiated monocytes fig 1a and expressed the hematopoietic cell marker cd45the myeloid cell marker cd11b and the monocytic cell marker cd14 fig 1bwe collected floating monocytic cells from the culture supernatant and evaluated theircytokine production we used lps as a priming signal and silica as a secondary inflammasomeactivating signal as expected the wildtype ipscderived monocytes required twosequential signals to secrete il1 fig 1c while the monocytes carrying the nlrp3 mutationshowed excessive il1 secretion without a secondary signal fig 1d both clones showedrobust secretion of il6 confirming the appropriate downstream signal transduction of lpsstimulation fig 1c and 1d overall the monocytic cells differentiated under feeder andserumfree condition showed the abnormal il1 secretion associated with the in vitro phenotype of nomidestablishment of ipsmls from monocytic progenitor cellswe next established an ipsml line from the ipscderived monocytic cells for this we recovered monocytic lineage cells and introduced lentiviral vectors encoding myc bmi1 andmdm2 after “ days culture in the presence of macrophage colonystimulating factormcsf the cells started to continuously proliferate in an exponential manner fig 2a theipsmls showed a small mononuclear appearance consistent with the appearance of monocyticlineage cells fig 2b the integration of transgenes into the genome of ipsmls was confirmed fig 2c the ipsmls expressed cd45 cd11b and cd14 similar to the originalipscderived monocytic cells fig 2d karyotype analysis of the ipsml demonstrated thatmost of the cells maintain normal karyotype except for some normal variations s1 tablebecause monocytes are heterogeneous progenitor cells that can differentiate into macrophagesand dendritic cells [ ] they might be composed of a heterogeneous population potentiallymaking their cytokine production insufficient we therefore compared the cytokine profiles of one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig functional monocytic cells on feeder and serumfree monolayer culture a a representative maygiemsa staining image of monocytic cells derived fromipscs b a flow cytometric analysis of monocytic cells the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are showncd il1 and il6 secretion from monocytic cells with wildtype c and mutant d nlrp3 cells were stimulated with lps ngml for hours and silica μgml for an additional hour bars show the mean ± sem of four experiments � p paired ttest101371 pone0237030g001ipsmls and terminallydifferentiated ipsmlderived macrophages the ipsmls carrying anlrp3 mutation nomidmls were differentiated into macrophages mlmps fig 2esince mlmps produced an increased amount of il1 and il6 compared with nomidmlsfig 2f and 2g and showed less variability than ipscderived monocytes fig 2h wedecided to use mlmps to construct our htsestablishment of an hts platform using mlmpsto establish our hts system we differentiated nomidmls into mlmps and disseminatedthem onto 384well plates we added the compounds at the same time as when we disseminated the cells into the culture wells then the mlmps were cultured with appropriate concentrations of compounds for hours in order for the cells to firmly adhere to the plate surfaceand they were stimulated with lps for another hours fig 3a after incubation the supernatant was collected and the relative concentrations of cytokines were measured using ahomogeneous time resolved fluorescence htrf assay in this protocol a caspase1 inhibitor one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of ipsmls from monocytic cells a cumulative growth curve of ipsmls the number of cells was counted and cumulated in every passageculture the day of lentiviral transduction was regarded as day b a representative maygiemsa staining image of ipsmls c a reverse transcriptionpcr analysisof ipsmls transgenespecific primer pairs were used my myc b bmi1 md mdm2 as a positive control lentiviral expression vectors were used d a flowcytometric analysis of ipsmls the staining profiles of specific antibodies thick lines and isotypematched controls gray areas are shown e a phase contrastimage of mlmps f g il1 f and il6 g secretion from nomidmls and mlmps cells were stimulated with lps ngml for hours the bars show themean ± sem of four experiments �� p paired ttest h the coefficient of variation cv of lpsstimulated ipscderived monocytes fig 1c and 1dnomidmls and mlmps f g101371 pone0237030g002acyvadcho specifically inhibited the secretion of il1 in a dosedependent mannerfig 3b in contrast the proteasome inhibitor mg132 which inhibits the nfκb pathway byblocking the degradation of iκb decreased the production of both il1 and il6 in a one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig establishment of an hts platform using mlmps a schematic illustration of the compound screening b dosedependent inhibition of il1 closedcircles and il6 open circles secretion by acyvadcho mg132 and mcc950 bars show the mean ± sem of five experiments c the quality of the assaysystem was evaluated signaltobackground ratios and z™factors of plates for il1 closed and il6 open are shown d criteria and number of hitcompounds [il1] and [il6] indicate the percent inhibition of il1 and il6 secretion respectively e doseresponse curves of the hit compounds bars showthe mean ± sd of four wells101371 pone0237030g003dosedependent manner fig 3b these data proved the specificity of the hts system wealso evaluated the inhibitory effect of mcc950 mcc950 inhibited the secretion of il1 withan approximate ic50 at μm without inhibiting the secretion of il6 fig 3b overall theipsmlbased cytokine assay in combination with the htrf system successfully detected the one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeninginhibitory effect of previously reported compounds proving the application of our system tohtshts using annotated compoundsto identify compounds that inhibit the activation of mutant nlrp3 we next performed htsusing compounds including fdaapproved drugs and compounds with known bioactivity to evaluate the quality of the assay system the z™factor and signalbackground sb ratio of each plate were calculated using the data from dmso controls with or without stimuli s1 fig regarding il1 the z™factor and sb ratio were consistently high for each384well plate z™factor ± and sb ratio ± mean±se fig 3c since a z™factor over indicates an excellent assay these data proved the appropriateness of our hts system the z™factor and sb ratio for il6 were slightly lower than those for il1 z™factor± and sb ratio ±in the first screening we assessed the inhibitory effects of all compounds influencing cytokine secretion at μm s2 table and s2 fig we first selected the compounds thatinduced a more than reduction in il1 secretion fig 3d of these the compoundsthat also nonspecifically inhibited or enhanced il6 secretion were excluded we adopted amore relaxed criterion for il6 modulators because the z™factors of the il6 assay were lowerthan those of the il1 assay we also excluded duplicate compounds and known steroidswith broad antiinflammatory effects for the remaining compounds we evaluated thereproducibility of the inhibitory effects at different dose twelve compounds showed ic50values at less than μm and predominant il1 inhibition at least at two different dosesfinally seven compounds consistently showed predominant il1 inhibition fig 3e allseven compounds have previously been reported to inhibit nlrp3 inflammasome [“]indicating their inhibitory effects on both wildtype and mutant cellsvalidation of hit compounds with primary human cellswe also wondered if the effect of these compounds could be recapitulated in primary humancells we used primary peripheral blood mononuculear cells pbmcs obtained from healthyvolunteers for the validation to obtain prompt il1 secretion from nonmutant cells westimulated the pbmcs with lps and atp compounds modulating the activity of hsp9017aag and herbimycin a showed a selective inhibitory effect on il1 secretion comparable to the effects seen in mlmps fig on the other hand the remaining compoundsaside from the pancaspase inhibitor zvadfmk inhibited both il1 and il6 fig indicating that they had offtarget effects or their il1 inhibitory effects were nonspecificdiscussionwe established a diseaseassociated phenotypic hts system for nomid using ipsmls ourhts platform showed a stable z™factor and sb ratio for the amount of il1 to avoid falsepositives such as nonspecific inhibitors and cytotoxic compounds we also measured theamount of il6 an inflammasomeindependent cytokine we identified compounds as predominant inhibitors of il1 secretion from among candidates the compounds identified here have already been reported as inhibitors of nlrp3 inflammasome proving thevalidity of our strategy screening a larger number of compounds may help identify novel andmore specific compoundswe could efficiently immortalize monocytic progenitor cells derived from human ipscsmoreover these ipsmls could differentiate into terminally differentiated macrophagesmlmps as previously reported [ ] mlmps showed enhanced cytokine secretion and one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningfig hit validation with pbmcs dosedependent inhibition of il1 closed circles and il6 open circles secretion from pbmcs of a healthy donor cellswere stimulated with lps ngml for hours and atp mm for an additional hour bars show the mean ± sd of four wells101371 pone0237030g004therefore were deemed suitable for hts in this manner the financial and labor costs of htscan be dramatically decreased our strategy can be applied to other immunological disordersin which monocytemacrophagelineage cells play critical pathological roles in addition theimmortalization of ipscderived hematopoietic cells for hts can be expanded to otherhematopoietic disorders one concern associated with the immortalization of monocytic cellsis that the immortalization can affect the characteristics of the cells especially regarding thecell cycle and death which may affect the immunological functions to avoid any potentialchanges in the cellular phenotype we first expanded the ipsmls generated a larger numberof frozen stocks and then used the same passage number of the stocks for a series of htssessionsmodulating the function of the inflammasome by small compounds is a promising frontierfor controlling diseases associated with inflammasomemediated chronic inflammation somecompounds such as mcc950 have already been shown to be markedly effective without anysignificant side effects in animal models nevertheless identifying more candidates to modulate nlrp3 inflammasome is desirable as mcc950 inefficiently inhibits capsrelated nlrp3mutants interestingly we identified several compounds effective in mutant cells that were previously reported to inhibit the activation of nlrp3 inflammasome indicating that the activation of mutant nlrp3 shares activating signals to some degree with wildtype nlrp3however several compounds that exerted predominantly il1 inhibitory effects on mutantcells were nonselective on pbmcs which could be explained by offtarget effects or false positives offtarget effects including cytotoxicity could be the result of a higher sensitivity bypbmcs to the true positives the clear reason is difficult to elucidate however because of theheterogeneity of the pbmc population on the other hand ipsmls consist of relativelyhomogenous cell populations and may therefore be more useful than primary cells in in vitro one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningexperiments as for nonspecific inhibitors and false positives in hts the upregulation of certain signaling pathways irrelevant to the inflammasome in ipsmls might be one cause cytotoxic compounds may also suppress cytokine secretion in a nonspecific manneradditionally our hts system relies on the amount of secreted cytokines which can varybetween cells and only indirectly describes the inflammasome activity thus a combination ofmeasuring the cytotoxicity and direct detection of the inflammasome activity should improvethe accuracy of our hts systemto conclude although phenotypic screenings have been performed with various cell typesdifferentiated from human pscs the number of screenings with immune cells is still limitedour hts system provides a large number of functional monocytic lineage cells and a versatileplatform for compound screening to modify diseaseassociated phenotypes therefore it canbe used to screen candidate compounds for the treatment of congenital immunological disorders associated with monocytic lineage cellsmaterials and methodsstudy approvalthis study was approved by the ethics committee of kyoto university r0091g0259 andwritten informed consent was obtained from the patient™s guardians in accordance with thedeclaration of helsinkimaintenance and differentiation of human ipscswe previously established ipscs from a nomid patient cira188ai with nlrp3 somaticmosaicism y570c undifferentiated ipscs were maintained on mitotically inactivatedsnl feeder cells with primate es cell medium reprocell japan supplemented with ngml bfgf wako pure chemicals industries japan feeder and serumfree monocytic celldifferentiation was performed in accordance with previously described protocols with somemodifications [ ]lentivirus productionlentiviral constructs encoding myc bmi1 and mdm2 in the csiiefrfa vector and twoplasmids for lentiviral vector packaging pcmvvsvgrsvrev and pcaghivgp werekindly provided by dr satoru senju kumamoto university kumamoto japan and hiroyukimiyoshi riken bioresource center tsukuba japan plasmids were transfected into 293tcells crl3216 atcc usa by lipofection lipofectamine ltx thermo fisher scientificusa and days later viral ps in the culture supernatants were concentrated by centrifugation at rpm for hours at ˚cgeneration of ipsmlsipsmls were generated as previously described with some modifications [ ] briefly onday of the monocytic cell differentiation from ipscs floating cells were collected andinfected with lentiviruses the cells were cultured in stempro34 serumfree medium thermofisher scientific containing mm lglutamine in the presence of gmcsf ngml andmcsf ngml rd systems usa after approximately days proliferating ipsmlsappeared for macrophage differentiation ipsmls within passages after thawing were cultured in rpmi1640 medium sigmaaldrich usa containing fetal bovine serum equitechbio usa and mcsf ngml for days with a medium change on day adherent one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningcells were collected by treatment with accumax innovative cell technologies usa and usedfor the subsequent experimentsmaygiemsa stainingcells were seeded onto glass slides by cytospin thermo fisher scientific and stainedwith maygrunwald and giemsa staining solution merck kgaa germany in accordancewith the manufacturer™s instructions the slides were examined using a biorevo bz9000keyence japan a planapo × objective nikon japan and the bzii viewer software program keyence were used for the image acquisitionflow cytometrycells were treated with fcr blocking reagent miltenyi biotec germany and stained with primary antibodies cd45fitc becton dickinson and company usa cd11bpe andcd14apc beckman coulter usa at dilutions of for negative controls primary antibodies were replaced with mouse igg1 bd dapi sigmaaldrich was used to exclude deadcells the flow cytometric analysis data were collected using a macsquant analyzer miltenyibiotec and analyzed using the flowjo software program treestar usareverse transcriptionpolymerase chain reaction pcrrna samples were prepared using an rneasy mini kit qiagen germany total rna ng was reverse transcribed into cdna using a primescript rt master mix kit takarajapan pcr was performed on a veriti thermal cycler thermo fisher scientific withtakara ex taq hs polymerase using approximately ng cdna the forward primers targeting the coding region of the genes were ™gatcagcaacaaccgaaaat3™ myc™ccattgaattctttgaccagaa3™ bmi1 and ™gctgaagagggctttgatg3™mdm2 the reverse primer targeting wpre was ™gttgcgtcagcaaacacagt3™measurement of cytokinescells were seeded at × cellswell onto a 96well cell culture plate in rpmi1640 mediumcontaining fbs the cells were stimulated with ngml lps from e coli k12 invivogen usa for hours and μgml silica us silica usa for an additional hour aftercentrifugation the supernatants were collected the concentrations of cytokines in the culturesupernatants were analyzed using a flowcytomix kit ebioscience usa and a macsquantanalyzer in accordance with the manufacturer™s instructioncompound screeningcells were seeded at × cellswell onto 384well cell culture plates in rpmi1640 mediumcontaining fbs and compounds four hours later the cells were stimulated with ngmllps for hours after centrifugation the supernatants were transferred to 384well smallvolume white plates using a biomek nxp beckman coulter the relative cytokine levels weremeasured using an htrf kit cisbio bioassays france and a powerscan4 microplatereader ds pharma biomedical japan with excitation at nm and emission at and nm the htrf signals were calculated as the htrf ratio ×em nmem nmand then the percent inhibition was calculated using dmso controls cell seeding and reagentdispensation were performed with a multidrop combi thermo fisher scientific thereagents purchased are as follows acyvadcho mg132 merck kgaa mcc950sigmaaldrich fdaapproved drug library iccb known bioactives library enzo life one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningsciences usa usdrug collection international drug collection microsource discoverysystems usa lopac1280 sigmaaldrich tocriscreen mini tocris bioscience uk andkinase inhibitor libraries enzo life sciences merck kgaa selleck chemicals usahit validation with pbmcscryopreserved human pbmcs were purchased from cellular technology limited usa thecells were thawed in accordance with the manufacturer™s instructions and seeded at ×cellswell onto 384well cell culture plates in rpmi1640 medium containing fbs andcompounds four hours later the cells were stimulated with ngml lps for hours and mm atp sigmaaldrich for an additional hour the relative cytokine levels were measuredusing the htrf kit as described abovestatistical analysesstatistical analyses were performed using the excel and graphpad prism software programsgraphpad software usa statistical significance was evaluated using the paired ttestp was considered statistically significant the z™factor and sb ratio were calculated asfollowsz0 ¼ 00 ð3shc þ 3slcþðmhc 00 mlcþsb ¼ mhcmlcwhere σ is the standard deviation sd μ is the mean hc is the high control and lc is the lowcontrolsupporting informations1 fig htrf ratios of the assay controls each of the plates contained high stimulated and low not stimulated controls treated with dmso values falling outside of themean ± 3sd were excluded as outliers red crosses except when z™factors and sb ratios werecalculatedtifs2 fig scatter plot of the compound screening percent inhibitions for il1 xaxis and il yaxis within a range of to are shown seven hit compounds are markedtifs1 table karyotype analysis of ipsmlspdfs2 table a total list of compounds with the assay dataxlsxs1 raw imagepdfacknowledgmentswe thank drs takayuki tanaka mitsujiro osawa and yohei nishi cira kyoto universitykyoto japan for fruitful discussions and technical assistance dr peter karagiannis cirakyoto university kyoto japan for proofreading the paper ms harumi watanabe cira one 101371 pone0237030 august one 0cipscderived monocytic cell lines serves a platform for drug screeningkyoto university kyoto japan for providing administrative assistance and dr akitsu hottacira kyoto university kyoto japan for providing 293t cellsauthor contributionsconceptualization ryosuke seki megumu k saitodata curation akira ohta megumu k saitofunding acquisition megumu k saitoinvestigation ryosuke seki akira niwa yoshinori sugiminemethodology akira ohtaresources akira ohtasupervision akira niwa haruna naito tatsutoshi nakahata megumu k saitowriting “ original draft ryosuke sekiwriting “ review editing megumu k saitoreferences mestas j hughes cc of mice and not men differences between mouse and human immunology jimmunol “ epub 104049jimmunol17252731 pmid holsapple mp west lj landreth ks species comparison of anatomical and functional immune system development birth defects res b dev reprod toxicol “ epub 101002bdrb10035 pmid geissmann f manz mg jung s sieweke mh merad m ley k development of monocytes macrophages and dendritic cells science “ 101126science pmid takahashi k tanabe k ohnuki m narita m ichisaka t tomoda k induction of pluripotent stemcells from adult human fibroblasts by defined factors cell “ 101016jcell200711019 pmid takahashi k yamanaka s induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors cell “ 101016jcell200607024pmid lo cicero a jaskowiak al egesipe al tournois j brinon b pitrez pr a high throughput phenotypic screening reveals compounds that counteract premature osteogenic differentiation of hgpsipsderived mesenchymal stem cells sci rep epub 101038srep34798 pmid pubmed central pmcid pmc5064407thorne n malik n shah s zhao j class b aguisanda f highthroughput phenotypic screening of human astrocytes to identify compounds that protect against oxidative stress stem cellstransl med “ epub 105966sctm20150170 pmid pubmed central pmcid pmc4835244 darville h poulet a rodetamsellem f chatrousse l pernelle j boissart c human pluripotentstem cellderived cortical neurons for high throughput medication screening in autism a proof ofconcept study in shank3 haploinsufficiency syndrome ebiomedicine “ epub 101016jebiom201605032 p

patients who have diabetes suffer from Type DMT2DM Many studies suggest using the significant role of lncRNAs to improve thediagnosis of T2DM Machine learning and Data Mining techniques are tools that canimprove the analysis and interpretation or extraction of knowledge from the dataThese techniques may enhance the prognosis and diagnosis associated withreducing diseases such as T2DM We applied four classification models including Knearest neighbor KNN support vector machine SVM logistic regression andartificial neural networks ANN for diagnosing T2DM and we compared thediagnostic power of these algorithms with each other We performed the algorithmson six LncRNA variables LINC00523 LINC00995 HCG27_201 TPT1AS1 LY86AS1DKFZP and demographic dataResults To select the best performance we considered the AUC sensitivityspecificity plotted the ROC curve and showed the average curve and range Themean AUC for the KNN algorithm was with standard deviation SD themean sensitivity and specificity were and respectively After applying theSVM algorithm the mean AUC obtained after stratified 10fold crossvalidationand the SD obtained The mean sensitivity and specificity were and respectively The mean AUC for ANN and the SD were and also the meansensitivity and specificity were and At last for the logistic regressionalgorithm our results showed of mean AUC and the SD of the meansensitivity and specificity were and respectively According to the ROCs theLogistic Regression and SVM had a better area under the curve compared to theothersContinued on next page The Authors Access This is licensed under a Creative Commons Attribution International License whichpermits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit tothe original authors and the source provide a link to the Creative Commons licence and indicate if changes were made Theimages or other third party material in this are included in the 's Creative Commons licence unless indicated otherwisein a credit line to the material If material is not included in the 's Creative Commons licence and your intended use is notpermitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyrightholder To view a copy of this licence visit httpcreativecommonslicensesby40 The Creative Commons Public DomainDedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unlessotherwise stated in a credit line to the data 0cKazerouni BMC Bioinformatics Page of Continued from previous pageConclusion We aimed to find the best data mining approach for the prediction ofT2DM using six lncRNA expression According to the finding the maximum AUCdedicated to SVM and logistic regression among others KNN and ANN also had thehigh mean AUC and small standard deviations of AUC scores among the approachesKNN had the highest mean sensitivity and the highest specificity belonged to SVMThis study™s result could improve our knowledge about the early detection anddiagnosis of T2DM using the lncRNAs as biomarkersKeywords Data mining Gene expression Machine learning algorithms Type diabetes mellitusBackgroundDiabetes mellitus DM is one of the most prevalent chronic noncommunicable diseases NCD around the world about of the patients who have diabetes sufferfrom Type DM T2DM [] The risk of developing T2DM is strongly associated withmany predispositions behavioral and environmental risk factors and also genetic factors [“] Besides the genetic factors strong evidence indicates that factors such asobesity and physical inactivity are the main nongenetic determinants of the disease [] T2DM can range from predominant insulin resistance with relative insulin deficiency to dominant defective secretion with insulin resistance [] It is often related tometabolic syndrome problems Individuals who have impaired glucose tolerance arehighrisk subjects of type diabetes []Studies demonstrate a drastic increase of the disease in recent decades The trendsestimate that by more than million people will be affected by the disease []People who suffer from T2DM are susceptible to many forms of complications leadingto morbidity and mortality in these patients Many studies emphasize the genetic factors in the pathogenesis of T2DM [ ] Long noncoding RNAs long ncRNAslncRNA are subsets of RNA specified as being transcripts with lengths exceeding nucleotides that could not be translated into protein [] Long noncoding RNAslncRNAs belong to a heterogeneous class of regulatory lncRNAs with transcriptlengths nucleotides which have a positive role in the development and growth ofseveral various diseases including T2DM supporting the hypothesis that abnormal expression of LncRNAs is related to various diseases [] Besides considering the significant role of lncRNAs in disease pathogenesis increasing researches suggest using themto improve diagnosis prognosis and clinical management of T2DM Genomewide association studies GWAS have recently introduced several particular diabetesrelatedloci in the human genome [] Also many studies discovered the relationship betweenmore than susceptible loci and T2DM at a genomewide significant level [ ]Deregulation of genes located in GWAS defined loci may be risk factors for human diseases concerning which we applied the GWAS catalog to select six lncRNAsLINC00523 LINC00995 CG27_201 TPT1AS1LY86AS1 DKFZP as our gene targets for the present study [] Knowledge Discovery in Databases KDD or data miningare techniques for the computational process of discovering patterns in large datasetscontaining various approaches such as artificial intelligence machine learning statisticsand database systems [] These methods are applied to recognize patterns in dataprediction association and classification problems [ ] Considering the 0cKazerouni BMC Bioinformatics Page of importance of early detection of T2DM machine learning and Data Mining techniquesare tools that can improve the analysis and interpretation or extraction of knowledgefrom the data [ ] These techniques may enhance the prognosis and diagnosis associated with life quality reducing diseases such as T2DM [ ]To date several other studies tried to predict diabetes mellitus using outstandingdata mining techniques [“] Vijayan et al[] applied the expectationmaximization algorithm KNN algorithm Kmeans algorithm amalgam KNN algorithm and ANFIS algorithm to predict and diagnose Diabetes Mellitus They usedthe UCI dataset containing blood test and demographic variables and their resultsshowed that EM possessed the least classification accuracy and amalgam KNN andANFIS provided better classification accuracy of more than and respectively Another study conducted by Saravananathan [] used popular classifiincluding J48 Support Vector Machines SVM Classificationcation algorithmsand Regression Tree CART and kNearest Neighbor kNNfor diabetic dataTheir performance indicators were accuracy specificity sensitivity precision errorrate They found that the J48 technique™s performance was remarkably superior tothe other three techniques for the classification of diabetes data Meng []compared three data mining models of logistic regression ANN and decision treefor predicting diabetes mellitus or prediabetes by risk factors They gathered information about demographic characteristics family diabetes history anthropometricmeasurements and lifestyle risk The decision tree model C50 had the best classification performance with an accuracy of with a sensitivity of andspecificity of Another study performed by Saeidi [] used logistic regression to assess the diagnostic value of LY86AS1 and HCG27_201 as biomarkersfor T2DM They obtained a sensitivity of and specificity of Anotherstudy [] used two other lncRNAs including LINC00523 and LINC00994 expressions for the evaluation of their potential diagnostic value for T2DM They appliedlogistic regression and achieved a sensitivity of and specificity of Inour study we combined six lncRNAs as variables for the first time and appliedfour classification models including classification algorithms like Knearest neighbor KNN support vector machine SVM logistic regression and artificial neuralnetworks ANN for diagnosing T2DM and we compared the diagnostic power ofthese algorithms with each other In the present study we aimed to find the bestdata mining approach for the prediction of T2DM using six lncRNA expressionThe result of this study could improve our knowledge about the early detectionand diagnosis of T2DM using the lncRNAs as biomarkers []MethodsThe primary aim of the present study was to implement four models to predict DT2Mapplying data mining techniques based on the lncRNA variables The research objectives of our study wereImplementing data mining techniques for prediction of the DT2M Comparing the applied methodsselecting the best model for the T2DM prediction 0cKazerouni BMC Bioinformatics Page of We used the variables for predicting T2DM and comparing the performance of thevarious data mining techniques For the implementation of the algorithms we usedANACONDA3“ bit a free and source platform distribution of pythonprogramming language with a vast number of modules packages and rich libraries thatprovide various methods for classification problems For obtaining the best amount ofperformance in the models 10fold crossvalidation performed on the dataset In dealing with the small data sets crossvalidation is a prominent strategy for estimating theperformance CrossValidation is a performance evaluation technique commonly usedin practice Here the data set is repeatedly partitioned into two nonoverlapping partsa training set and a holdout set For each partitioning the holdout set is used fortesting while the remainder is used for training The two most popular variants aretenfold crossvalidation 10fold CV where the data is split into ten mutually disjointfolds []Since our samples were more than and to be sure that each fold contains thesame proportion of healthy and diabetic individuals we used the stratified 10foldcrossvalidation approach [] Therefore the results are reliable and more credibleWe applied four popular data mining approaches on the lncRNA variables regression knearest neighbors SVM and neural network classification algorithmsKNN algorithmThe knearest neighbor™s algorithm kNN is an algorithm for classifying variables regarding the closest training data in the feature space KNN uses an instancebasedlearning method which is one of the simplest algorithms among data mining techniques This method considers the nearest neighbors to each object and decides todedicate the object to classes [ ]SVM algorithmSupport Vector Machine SVM is a supervised algorithm which divides the featurespace called hyperplanes considering the target classes SVM computes classification bymaximizing the margin of the hyperplane that intercepts classes This algorithm plots amultidimensional hyperplane that divides classes and increases the margin betweenclasses to enhance the accuracy of classification We used different kernel functionsembedded in the SVM class of SVC library in python framework as a quadratic polynomial radial basis etc to classify the instance and to detect the best accuracy amongthem [“]Artificial neural networkArtificial Neural Network is a data processing algorithm that simulates the biologicalneural network in its computations A common problem in using ANN is that they actfundamentally as a black box and the parameters are set by the model so we cannotdemonstrate them [] we can just apply the model in our problems and obtain thehigh performance We used Multilayer Perceptron Neural Networks MLPNN Thestructure of a multilayer perceptron neural network has been demonstrated in Fig It maps a set of input data into a set of appropriate output classes It includes threelayers input layer hidden layer output layer The principal function of neurons of the 0cKazerouni BMC Bioinformatics Page of Fig Artificial Neural Network structureinput layer is to divide input Xi into neurons in the hidden layer The neuron of thehidden layer adds the appropriate weights of Wij to the input variables The output formula isYj ¼ fWji XiX 10 11Where f is a simple threshold function that we considered sigmoid and hyperbolictangent function []In the present study a Multilayer Perceptron Neural Networks MLPNN was performed The structure of MLPNN is as shown in Fig It makes a map of input dataonto a set of suitable output dataThe RBF networks are another type of neural network In MLP each neuron considers the weighted sum of its input values in which each input value is multiplied by acoefficient and the results are the sum of values RBF is a more intuitive approach toMLP An RBFN classifies the inputs by calculating the input™s similarity to examplesfrom the training set Each RBFN neuron stores one of the examples from the trainingset as a œprototype for classification of new input in each neuron the Euclidean distance between the input and its prototype is calculated The input is dedicated to aclass when it has more similar to that class than the other classesLogistic regressionLogistic regression is a common approach for predictive modeling practices The functionpX provides probability output between and for all values of X where X1“Xp are thepredictors The coefficients β0“βp are estimated using maximum likelihood estimationþβp Xð Þ ¼ eβ þ eβ1X1 þ‹¯Ã¾Î²Ã¾Î²pXp1X1 þ‹¯Ã¾Î²pXpDatasetThis study was based on the data obtained from three previous research conducted bySaeidi and Mansoori [ ] and the research of Parvizi and colleagues which 0cKazerouni BMC Bioinformatics Page of is not published yet We integrated these three studies and our data mining analysiswas implemented in their studies The data were collected from unrelated Iraniansubjects T2DM patients and healthy individuals matched for age and sexT2DM patients were recruited from individuals who referred to the Diabetic Clinic atShohada Hospital Tehran Iran In the current study we applied six lncRNAs expression and also six demographic variables including sex age weight height BMI andFBS for analysis and inputs of algorithms For the preprocessing phase we normalizedthe data inputs for KNN SVM and ANN models We also had low missing variablesand we replaced them with zero Table lncRNA extraction and selectionIncreasing evidence has suggested several lncRNAs are implicated in T2DM pathogenesis Recently human βcell transcriptome analysis showed lncRNAs dynamic regulation and abnormal expression of lncRNAs in T2DM [] However the extent oflncRNA deregulation in T2DM has yet to be determined To date more than100 susceptibility loci have been identified as being associated with T2DM at a genomewidesignificant level [ ] Considering this into account and by querying the GWAS catalog we candidated lncRNAs LY86AS1 HCG27_201 LINC00523 LINC00994TPT1AS1and DKFZP as target genes for this studyThe large scale GWAS have recognized approximately SNPs that were susceptibleto T2DM [] From there we used the GWAS catalog access in June to create alist of SNPs associated with T2DM In the current study we selected six lncRNA forexpression analysis according to the scan carried out in the study of Mansoori []and Saeedi [] We selected variants that had associations with increased risk ofT2DM We applied a quantitative PCR analysis of lncRNA expression levels in the samples We calculated the respective amount of each lncRNAs applying the 2ΔΔct asmeans of duplicate measurementsTable The lncRNAs as inputs of algorithmsnumberLncRNA VariablesDemographic VariablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPSexAgeWeightHeightBMIFBS 0cKazerouni BMC Bioinformatics Page of Analysis and evaluation criteriaTo select the best performance data mining algorithms in predicting diabetic patientswe considered AUC sensitivity specificity and plotted ROC curve for the folds we ranand showed the average curve and its range [ ]ResultsTable shows the significant downregulation of PBMC expressions of the variables inthe T2DM group compared with the control group The AUC of each classificationtechnique has been demonstrated in Table AUC stands for œArea under the ROC Curve AUC computes the entire twodimensional area under the whole ROC curve According to the finding the maximumAUC dedicated to SVM and logistic regression among others knn also had the highestmean AUC and minimum standard deviation of AUC scores among the approachesThe mean and standard deviation for AUC sensitivity and specificity of each algorithmis given in Table Apart from classification AUC sensitivity and specificity the Receiver Operating Characteristic ROC with stratified crossvalidation is shown for eachapproach in Figs and ROC curves generally plot true positive rate on the Yaxis and false positive rate onthe Xaxis In other words a false positive rate of zero and a true positive rate of onein the top left corner of the plot is called the ideal point It means that a larger areaunder the curve AUC is usually better According to the demonstrated ROCs theKNN and SVM have a better area under the curve in comparison with the othersDiscussionFor a medical diagnosis optimized approaches to gain useful and accurate outcomesare essential Applying machine learning and data mining methods to automate theprocess of diagnosis may assist practitioners to enhance the quality of their clinical decisions [ ]Since T2DM is one of the prevalent diseases with severe consequences [] developing efficient methods for early detection of the disease was the primary purpose of ourresearchRegardless of high number of lncRNAs in the RNA profile of human a few numbersof them has been proved to be biologically active The role of the few lncRNAs hasbeen identified but several studies discussed the significant impact of lncRNAs in diabetic people which may represent the role of abnormal expression of lncRNAs in thelncRNAs in theincidence of T2DM [] According to the possible function ofTable Relative expression of the variablesVariablesLINC00523LINC00995HCG27_201TPT1AS1LY86AS1DKFZPDiabetesΔCT ± SEM “ “ “ “ “ “ControlΔCT ± SEM “ “ “ “ “ “pvalue 0cKazerouni BMC Bioinformatics Page of Table The AUC of algorithms for each iterationNumberof foldsAUCKNNSVMANNLogistic Regressiondevelopment of T2DM we considered the expression levels of six lncRNAs in additionto the demographic data in diabetic and healthy individuals for our study Tomeasure the expression of the lncRNAs we applied PBMCs which demonstrate an extensive proportion of the genes encoded in the human genome [] Several studies haveinvestigated different machine learning and data mining methods to predict differentdiseases [ ] such as heart diseases thyroid tumors and also diabetestype diabetes prediction In the present study we combined four commonly used datamining algorithms KNN SVM neural networks and regression to predict type diabetes using Long noncoding RNAs expression and the demographic variables for thefirst time because most of the previous studies used blood test variables or the demographic data for their analysis Receiver operating characteristic ROC analysis AUCsensitivity and specificity measure was used to assess the diagnostic value of the sixbiomarkers for T2DM The mean AUC for the KNN algorithm was obtained andwith standard deviation and we obtained the highest sensitivity with thestandard deviation of among other approaches After applying the SVM algorithm the mean AUC obtained after 10folds with the standard deviation of and the highest specificity among other approaches obtained with the standarddeviation of For the ANN we applied a multilayer perceptron with five hiddenlayers and the mean AUC of folds was and the standard deviation was Atlast for the logistic regression algorithm our results showed of mean AUC andthe standard deviation of The lower standard deviations in the AUC scores ofcomputed folds means the algorithm has worked with more performance [ ]Other studies investigated data mining algorithms for several diseases Saravananathanand Velmurugan [] applied several classification algorithms in their study to analyzeincluding KNN Sadri Sa™di [] compared three data miningdiabetes dataTable The mean and standard deviation of AUC sensitivity and specificity of algorithmsAlgorithmKNNMean AUC std ˆ’ ˆ’ ˆ’ ˆ’ Mean sensitivity ˆ’ std ˆ’ ˆ’ ˆ’ ˆ’ Mean specificity ˆ’ std ˆ’ ˆ’ ˆ’ ˆ’ SVMANNLogistic Regression 0cKazerouni BMC Bioinformatics Page of Fig The ROC for KNNalgorithms to predict T2DM and gained precision for ANN Sidiq [] reported about accuracy for KNN and accuracy for SVM algorithms applyingfor the Diagnosis of Various Thyroid Ailments In another study for the heart diseasesThe data mining algorithms indicated more than accuracy The investigated studies are in line with the findings of our study that these algorithms have a strong powerfor prediction and early detection of many diseases including T2DM and we obtainedremarkably better accuracy for prediction for example the SVM and logistic regressionaccuracy were In our study we also obtained a better accuracy for logistic regression that was and in comparison with other studies is a strong point for exampleSaeidi [] conducted a study to review two Long noncoding RNA expressions intype diabetes mellitus and with applying regressions reported about accuracyAnother research [] used two different Long noncoding RNA expressions in type Fig The ROC for SVM 0cKazerouni BMC Bioinformatics Page of Fig The ROC for MLPdiabetes mellitus and found of accuracy with the regression algorithm In thepresent study for the first time we performed four data mining algorithms on six Longnoncoding RNAs and compared their power with each other We demonstrated thatLong noncoding RNAs are effective biomarkers for data mining algorithms and have afeasible power to be applied for prediction of T2DM Also in this research we optimized the parameters of every algorithm and used stratified 10fold crossvalidation togain the best performance To be mentioned in the nearest neighbor™s algorithm theparameter k was varied between one and nine to find the bestoptimized method andwe selected k to have the best performance and the lowest standard deviation in theaccuracy of the folds In addition in choosing the parameters of the artificial neuralnetwork the number of hidden layer neurons significantly affects the accuracy of thenetwork so we set the parameters with two hidden layers with five and three neuronsFig The ROC for logistic regression 0cKazerouni BMC Bioinformatics Page of respectively to yield the best accuracy Considering the standard deviation of scores foreach algorithm the KNN had the lowest std Moreover the highest accuracy amongthe algorithms was the SVM algorithm and Logistic regression which had the maximum accuracy in folds among others We should mention that the strong points ofour study are using demographic data and six Long noncoding RNAs and combiningthem to get the best detection power of T2DM and performing four outstanding datamining algorithms and comparing their performances As the limitations of this studywe should account for the limited number of samples which is due to the high costs ofmeasuring the Long noncoding RNAs No doubt the higher number of samples wouldlead to higher performance and more reliable resultsConclusionIn this paper the performance of conventional data mining classification techniqueshas been calculated and compared for a dataset of patients referred for the screeningof type diabetes to the Shohada Hospital Iran The biomarker applied in this studydemonstrated high diagnostic value and the diagnostic process is suitable which couldhelp in the diagnosis of prediabetes and T2DMThe classification techniques compared were support vector machine artificial neuralnetwork decision tree nearest neighbors and logistic regression In data mining it isnot possible to say one classification technique will always work best and it often depends on the number of samples their distribution and the choosing of the right algorithm In this research work SVM and Logistic Regression had the best Area UnderCurve among methods of classification with the mean AUC of KNN and ANNalso had the high mean AUC and small standard deviations of AUC scores among theapproaches KNN had the highest mean sensitivity and the highest mean specificitybelonged to SVMFor future works performing other data mining and machine learning methods andusing higher numbers of samples are recommended to enhance the performanceAcknowledgmentsNot applicableAuthors™ contributionsAB and FA designed the study FA and AB collected the data and performed the statistical analysis AB and NPinterpreted the data FK ZM and LS wrote and revised the paper All authors read and approved the final manuscriptFundingNot applicableAvailability of data and materialsThe datasets used andor analyzed during the current study are available from the corresponding author onreasonable requestEthics approval and consent to participateEthical approval was obtained from the Shahid Beheshti University of Medical Sciences Ethics CommitteeIRSBMURETECHREC13951036 We informed all participants that their participation was voluntary and the study didnot state any potential risk and their identities will be private Informed written consent forms were taken from allparticipants before participationConsent for publicationNot applicableCompeting interestsThe authors declare that they have no competing interests 0ccoding RNAs LINC00523 and LINC00994 in type diabetes in an Iranian cohort Mol Biol Rep “Saeidi L Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Mansoori Z Kazerouni F Long noncodingRNA LY86AS1 and HCG27_201 expression in type diabetes mellitus Mol Biol Rep “Petersmann A Nauck M MüllerWieland D Kerner W Müller UA Landgraf R Freckmann G Heinemann L Definitionclassification and diagnosis of diabetes mellitus Exp Clin Endocrinol Diabetes “Armoon B Karimy M Epidemiology of childhood overweight obesity and their related factors in a sample of preschoolchildren from Central Iran BMC Pediatr Tuomilehto J Lindström J Eriksson JG Valle TT Hämäläinen H IlanneParikka P KeinänenKiukaanniemi S Laakso MLouheranta A Rastas M Prevention of type diabetes mellitus by changes in lifestyle among subjects with impairedglucose tolerance N Engl J Med “Guariguata L Whiting DR Hambleton I Beagley J Linnenkamp U Shaw JE Global estimates of diabetes prevalence for and projections for Diabetes Res Clin Pract “Leti F DiStefano J Long noncoding RNAs as diagnostic and therapeutic targets in type diabetes and relatedcomplications Genes Heydari M Teimouri M Heshmati Z Alavinia SM Comparison of various classification algorithms in the diagnosis of type diabetes in Iran International Journal of Diabetes in Developing Countries “Kazerouni BMC Bioinformatics Page of Author details1Department of Laboratory Medicine School of Allied Medical Sciences Shahid Beheshti University of MedicalSciences Tehran Iran 2Department of Health Information Technology and Management School of Allied MedicalSciences Shahid Beheshti University of Medical Sciences Tehran Iran 3Department of Clinical Biochemistry School ofMedicine Tehran University of Medical Sciences Tehran Iran 4Department of Genetics Faculty of Medicine BabolUniversity of Medical Sciences Babol IranReceived December Accepted August ReferencesLi X Zhao Z Gao C Rao L Hao P Jian D Li W Tang H Li M The diagnostic value of whole blood lncRNAENST00000550337 for prediabetes and type diabetes mellitus Exp Clin Endocrinol Diabetes “ Mansoori Z Ghaedi H Sadatamini M Vahabpour R Rahimipour A Shanaki M Kazerouni F Downregulation of long non Perkel JM Visiting œnoncodarnia In Future Science Kapranov P Cheng J Dike S Nix DA Duttagupta R Willingham AT Stadler PF Hertel J Hackermüller J 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classifier models using stratified tenfold cross validation techniques InInternational Conference on Computing and Communication Systems Springer “ Abdar M Kalhori SRN Sutikno T Subroto IMI Arji G Comparing Performance of Data Mining Algorithms in PredictionHeart Diseases International Journal of Electrical Computer Engineering “ Sambyal RS Javid T Bansal A Performance analysis of data mining classification algorithms to predict diabetesInternational Journal of Scientific Research in Computer Science Engineering and Information Technology “ Pradhan M Kohale K Naikade P Pachore A Palwe E Design of classifier for detection of diabetes using neural networkand fuzzy knearest neighbor algorithm International Journal of Computational Engineering Research “Tzeng FY Ma KL ing the black boxdata driven visualization of neural networks IEEE Morán I Akerman Ä° Van De Bunt M Xie R Benazra M Nammo T Arnes L Nakić N GarcíaHurtado J RodríguezSeguí SHuman β cell transcriptome analysis uncovers lncRNAs 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increasing relevancy of geospatial technologies such as geographic information system GIS inthe public health domain particularly for the infectious disease surveillance and modelling strategies Traditionally thedisease mapping tasks have faced many challenges” authors rarely documented the evidence that were used to createmap before evolution of GIS many errors aroused in mapping tasks which were expanded extremely at global scalesand there were no fidelity assessment of maps which resulted in inaccurate precision This study on infectious diseasesgeosurveillance is divided into four broad sections with emphasis on handling geographical and temporal issues to help inpublic health decisionmaking and planning policies geospatial mapping of diseases using its spatial and temporalinformation to understand their behaviour across geography the citizen™s involvement as volunteers in giving healthand disease data to assess the critical situation for disease™s spread and prevention in neighbourhood effect scientificanalysis of healthrelated behaviour using mathematical epidemiological and geostatistical approaches with capacitybuilding program To illustrate each theme recent case studies are cited and case studies are performed on COVID19 todemonstrate selected modelsKeywords Geospatial technology 01 Citizen Science 01 Public health 01 COVID19 01 Mathematical epidemiologyIntroductionThe public health sector™s increasing demand for mappinganalytics and visualization had started a date back in thelast years which has resulted in a growing informationage technology for communicable disease surveillance andepidemiology Baker Bos and Blobel Friede Friede Khan Reeder Yu and Edberg This continuous publichealth burden with advances in information technology Sameer SaransameeriirsgovinPriyanka SinghPriyankaiirsgovinVishal KumarVishalkumariirsgovinPrakash ChauhanprakashiirsgovinIndian Institute of Remote Sensing Indian Space Researchanisation Kalidas Road Dehradun Indiacombined with spatial data led to the development ofvarious tools and systems that provides visualization ofdisease data in space and time Dredger Kothari Robertson and Nelson Schriml et alThe first integral definition of public health was given byWinslow as ˜˜science and art of preventing diseaseprolonging life and promoting health through the anized efforts and informed choices of society anizations public and private communities and individuals™™The American Public Health Association APHA mentioned public health as a practice of preventing the spreadof disease and an aim of promoting good health from smallcommunities to across the world Turnock Advances in information technology and spatial features resultedin geospatialtechnology which is acute for mappingsurveillance predicting outbreaks detecting clustering andanalysing spread patterns of infectious diseases with epidemic or pandemic potential in communities and acrossterritories AvRuskin Carpenter Castronovo Dominkovics Gao 0c Heymann and Brilliant Hills Klompas Reis Geospatial technology has provided visualization and analytical tools topublic health professionals and decision makers to executediseases control programs in affected andor suspectedregions and make analysis and predictions possible thatwas once technologically out of reachGeospatial technology includes geographical information systems GIS global positioning systems GPS andsatellitebased technologies such as remote sensing RSGIS is known for geographic data capture input updatemanipulation transformation analysis query modellingand visualization of all forms of geographically referencedinformation through the set of computer programs BonhamCarter GPS provides positioning navigationand timing PNT services by capturing data from satellitesand providing it to users Eldredge and RS isan earth observation instrumentthat delivers regionalinformation on climatic factors and landscape featuresTherefore GPS and RS provide regional and spatialinformation while GIS provides geospatial data integrationas well as accurate geospatial analysis in realtime mannerZhen Geospatial Technology and InfectiousDisease SurveillanceInfectious diseases mostly adapts antimicrobial andmobility features later formed in a shape of pandemic andor epidemic Chen Cheng Lee andNishiura which forced public health authorities tounderstand not only the diseases virulence but also itsdemographic and environmentalfactors that helps inmaking spread patterns though space and time domainCroner For example the global spread of highlypathogenic avian ‚uenza HPAI H5N1 in “ withno effective vaccines led to concern among public healthdecision makers in spite of many international programsRappole and Huba´lek The reason behind theirconcern was they were lacking of disease surveillance toolin its initial stage which caused inaccessibility to populations atrisk and faced difficulties in implementingimmunization strategies at a global scale Kitler Stoto However the impact of environmentaland demographic factors also plays a major role as this caninform about the interaction between hosts and pathogensand patterns of spread in space and timeThe GIS provides dynamic maps to understand geographical distribution of diseases for analysis on frequencyof cases disease mapping spatial cluster of diseases disease association with environmentalfactors networkanalysis etc With such a visualization and analyticalJournal of the Indian Society of Remote Sensingforservice frameworkcapabilities GIS technology is holding a widespreadgrowth in public health Ahmad 2011a b Booman HanafiBojd Kolivras Martin Nykiforuk and Flaman Abdul Rasam Zhang Zhen Theseamless integration of GIS with realtime infectious diseaserelated diverse datasets through webbased mappingto the development of geospatial dashboardleadsgeospatialinfectious diseasesurveillance Dent Gao Yun Theinfectious diseaserelated data mightinclude diseasesurveillance data activeconfirmed cases and health system data hospital visits emergency services availabilitynursedoctor availabilityICUbed availability Many source geospatialstandards of GeospatialConsortium OGC are used as a Web Map ServiceWMS Web Coverage Service WCS Web ProcessingService WPS Web Feature Service WFS etc Bulatovic´ Gao to visualize accesspublish and manipulate geospatial resources Also manyother popular industrial geospatial standards are developedby ESRI Google Yahoo and MapInfo Granell to fetch locationbased data and provide infectiousdisease surveillance dashboard to monitor and control thegeographically spread of disease Zhang TheGeocoded Really Simple Syndication GeoRSS taggedXML files from GeoRSS services can also be used toprovide geocoded infectious disease news from socialmedia platform Tolentino KassHout andAlhinnawi 2013a b Kodong Historical ContextThe mapping of infectious diseases using geospatial andinformation technology to benefit public health is not a newway of tracking the diseases Ahmad Cui Hirsch Hornsby Matthew May Mujica Nicholson and Mather Noble Perl and Moalem Williams The historical disease mapping has faced manychallenges” authors rarely documented the evidencethat were used to create map after mapping had beenimplemented before the beginning of geographical information systems many errors arouse which were expandedextremely at global scales and there were no fidelityassessment of maps which resulted in inaccurate precisionBut nowadays wide range of geospatial applications areavailable in public health community with a possibilities ofvisualization analysis detection of clusters formed andcalculate diseaserelated metrics such as incidence andprevalence rate Beck Clarke Hay Jacquez Kleinschmidt Lawson and 0cJournal of the Indian Society of Remote SensingLeimich Moore and Carpenter Robinson Wilkinson The earliest mapping for visualisation ofthe linkbetween disease and place was done in on plagueepidemic in Italy Dent During cholera outbreak in the study of physician John Snow had made a novelcontribution in history of public health and epidemiologyby using cartography applications and geographic visualization in fighting cholera After years the maps wereidentified as a communication tool in understanding andtracking of infectious diseases such as the ‚uenzapandemic yellow fever and cholera Since then revolutionof webbased tools started in applied health geographyBoulos The trend of infectious disease mappingcould be seen from review of the Health GIS literature which demonstrated that research papers out of were focused on infectious disease mapping Lyseen Covid19The ongoing pandemic outbreak targeting humans™ respiratory system was recently discovered in December by the name of Coronavirus Disease Covid19 WorldHealth anization from a cluster of patients with acuterespiratory distress syndrome in Wuhan Hubei ProvinceChina Huang Lu 2020a b and spreadglobally by March This pathogenic disease is structurally related to the Coronavirus CoV which belongs tofamily Coronaviridae and the order Nidovirales Thisfamily is classified into four genera”AlphacoronavirusBetacoronavirus Gammacoronavirus and Deltacoronavirus on the basis of their phylogenetic and genomicanalysis The species of Alphacoronavirus and Betacoronavirus infect mammals causes respiratory illness inhumans and gastroenteritis in animals while species ofGammacoronaviruses and Deltacoronaviruses infect birdsbut some of them can also infect mammals Woo et alfrom Betacoronavirus The two virusgenus”Severe Acute Respiratory Syndrome SARSCoVor Middle East Respiratory Syndrome MERSCoV”hadearlier demonstrated that coronaviruses can cause significant public threat Ge The COVID19 is categorizedby World Healthanization WHO on the basis of genomic sequencinganalysis ofrespiratory tract samples which isobtained from total of nine patients Huang Lu 2020a b COVID19 has started behaving like theonceinacentury pandemic by affecting healthy adults aswell as elderly people with some health issues and byinfecting others at an exponential rate of increase thanSARS or MERSinto BetacoronavirusspecieslowerGeospatial TechnologyDuring occurrence of diseases geospatial technologies andservices could help in representing the spatiotemporalinformation and in analysing the dynamic spread of diseases As mentioned by Boulos geospatial technologies and services which performs in real time mannerare tremendously relevantto create a ˜˜spatial healthinformation infrastructure™™ In this section a review onmany geospatial technologies with enabled IT services iscarried out to understand and analyse the spread and outbreak of disease with a case study on COVID19 pandemicCitizen Scienceissues and concernsThe expansion of Citizen Science from biodiversity andecological domain Haklay MillerRushing to public health community across spatial extentsmade an urgent need to study its different forms Crowl The indepth report of EU describes taxonomyof Citizen Science in three levels European Commission described in Roy Wiggins andCrowston and Haklay Roy categorized Citizen Science by participant™s number and oftheir spread ˜˜local™™ and ˜˜mass™™ and ˜˜thoroughness™™time and resource investment or King described ˜˜for the people with the people or by the people™™ about Citizen Science activities Wiggins andCrownston classified Citizen Science projects inconservation managing natural resources action addressing localinvestigation answering scientific questions and education providingknowledge to citizens Haklay classified CitizenScience into four levels based on participant™s engagement” level is crowdsourcing in which citizens withless or no knowledge on activity perform as sensors tocomplete computing tasks level is distributed intelligence where citizens are being trained with skills forinterpretation of collected data level is participatoryscience in which citizens decide about research questionsand types of data to be collected and level is extremewhere citizens are fully involved in defining researchstrategies data collection data interpretation and performing scientific analysis Apparentlythe concept ofCitizen Science is rare in public health domain but some ofits contribution seen in some studies which not only helpsin predicting disease risks but also in combating theinfectious diseases CurtisRobles Palmer Smolinski Wilson Another approach similar to Citizen Science is ˜popularepidemiology™ in which experts and laypersons jointly 0ccollect environmental data responsible for particular healthconsequences Brown or ˜street science™ as a process in which general public communities actively engagedin defining problems framing of research questions anddecisionmaking activities about research design CorburnCrowdsourceVGI Mobile AppsDespite technological and computational developments inGeoWeb many web technologies such as jQuery andAJAX mapping APIs like Google and GPS devicesresulted in a new revolution of neogeography Turner where mapping is done by crowd and can bereached by anyone from general public members groupSuch revolution brought a trend of Volunteered GeographicInformation VGI which is first coined and explained byGoodchild 2007a According to Goodchild 2007b VGIhighlighted the human capabilities in collecting geospatialinformation by using five senses and then integrating withexternal sensors of mobile devices like GPS accelerometer camera digital compass and microphone gives valuable datasets which can neither be retrieved from satelliteimagery nor collected with any GPS receivers Anothersuccessful term in geospatial mapping using mobile technology is crowdsourcing Heipke HudsonSmith which was coined by Howe thatinvolves the collection of geospatial information or mapping of any particular activity by an undefined crowd ornetwork of people Both terms VGI and crowdsourcingslightly differ but they are usually recognized as a synonyms or even as a combined term ˜˜crowdsourcing geographic information™™ Sui Over the lastdecade VGIoriented source mobileareEpiCollect Aanensen for ecology and epidemiology NoiseTube Maisonneuve httpnoisetubenet and Noise Battle GarciaMartı´ for noise monitoring Skywatch Windoo httpwindoochfor weather monitoring Mappiness httpwwwmappinessukfor behavioural analysis MacKerron andMourato appsThe source mechanism for data collection usingAndroid devices can be performed by Data KitODK suite107 https datakit which is composed of ODK Collect and ODK Aggregate ODK Collecthttps datakitusecollect provides a customizable framework for geospatial data collection and ODKAggregate is a web application that runs on ApacheTomcat server httptomcatapache to store collecteddata through a synchronization with a databaseforexample PostgreSQL Brunette As suchsuite™s performance can be seen in various activities likeagricultural monitoring Krosing and Roybal Journal of the Indian Society of Remote Sensingmonitoring of deforestation and school attendance documentation of war crimes and health programs Anokwa Digital Contact TracingNowadays COVID19 has become the greatest threat forpublic health in last years and due to such pandemicvarious levels of lockdown are issued across the world tobreak its chain of infection transmission However this isthe first approach to invade the contagion but once itwould be lifted this pandemic would start in a new wayand might reach its highest peak by infecting more andmore population Ferguson Thereforetocombat with such a global pandemic threat anotherapproach is discovered by a group of researchers known asdigital contact tracingSmartphonebased contact tracing is known as a digitalcontact tracing which presents a sustainable solution tolimit the transmission of infectious disease by tracing theirpotential transmission routes in a population howeversuch an app presents significant concerns regarding privacy The digital contact tracing works on the principle of˜crowdsource data™ by measuring the proximity to aninfectious person In previous diseases risk surveillancethe contact tracing apps were used to pool location timestamped data to determine the exposures to risk of infectionsSacks Such data are highly personal and leadmany privacy concerns Smith but they werenot always accurate to infer the exposure risks due to noisydata Farrahi Therefore various smartphoneapps are developed in COVID19 pandemic in which someapps use location for proximity and some of them are notusing location services of mobile device subject to theprivacypreserving natureCOVID19 Contact TracingIn order to illuminate the epidemiology of COVID19 andto characterize its severity Lipsitch there is anurgent need of digital platform that captures realtimeaccurate information on COVID19 patients diseasesdiagnosis treatment and clinical reports and whom theyget interacted at which place to detect clusters and generatealerts Such information may help in understanding riskfactors of infection and in predicting the next generation ofinfectious persons FitzGerald Addressing thisunprecedented challenge many mobile apps have beendeveloped and are being used at large scale and some ofthem are as follows 0cJournal of the Indian Society of Remote Sensing¢ COVID Symptom Study COVID Symptom Tracker”This mobile app is developed in collaboration of ZoeGlobal Ltd a digital health care company and a groupof academic scientists from Massachusetts GeneralHospital and King™s College London which waslaunched in UK on March and becameavailable after days in USA This app enquires aboutage location and other diseases risks and also a selfreporting function is enabled which is associated withCOVID19 infection and exposure Drew This app retrieve updates on healthcare worker™sexperiences who are on COVID19 duty their stressand anxiety and use of personal protective equipmentPPE kits are being surveyed through this app toobserve intensity of health care workers Drew et alappimplemented¢ Aarogya Setu”This mobile app is launched on April by Government of India to aware general publicon COVID19 symptoms government advisory measures online consultation facility and dynamics ofdisease Thiscrowdsourcingapproach by which general public members enter theirdetails for selfassessment and this assessment is thenused to trace the infectious contacts or agents as adigital contact tracing concept This app uses locationservices to geolocate the users and Bluetooth tomaintain the log of contacts when one userdevicecomes in contact with another userdevice and as suchdigital contact tracing activity helps in identifying thecluster of diseases and communities which are at risk ofinfection The Aarogya Setu app was downloadedby million users within days of its launchUpadhyay and by using app™s crowdsourcedata the Indian government detected approx positive casesinformed probable users ofbeing at risk and identified potential clusters TheTimes of IndiaNumerous digital contact tracing apps are in use indifferent parts of world”TrackCOVID Yasaka TraceTogether Bay WeTrace De Carli and Google and Apple™s recently announcedjoint initiative Li and Guo COVID19 Data Visualization and ExploratoryData AnalysisWith early experiences of epidemics such as “SARSCoV Boulos and the “ MERSCoVGikonyo and other seasonal flu™s online realtime or nearrealtime mapping of diseases™ occurrencesusing geospatial technologies and web applications havealways been used as a pivotal webbased tools in trackinghealth threats and combating infectious diseases Thissection described a range of mapping dashboards based ongeospatialtechnologies for tracking and unfolding thecoronavirus disease around the world Some of the globaland national geospatial initiatives with an aim to supplyinformation faster than diseases are as summarized inTable Infectious Diseases ModellingThe intention of infectious diseases surveillance is to detectepidemics in their early stages so that the countermeasurescould be taken for preventing its wide spread Suchsurveillance tasks require many epidemiological and statistical methods with geospatial features in investigatingepidemics preferably from localized areas The reason forpreferring the local areas for investigation is because epidemics generally emerged in small areas and then spreadwidely if they are not controlled However some methodsrequire rigorous conventions in their underlying modelsand are too problematical to be applied on small areasThereforefordetecting diseases prevalence with case studies on smalldatasets which would be more useful for public healthactivitiessection discussessimple methodsthisClusteringClustering deals with the study of spatialtemporal patternsof the spread of communicable diseases and identificationof other diseaserelated aspects allied with heterogeneousgeographical distribution which might be helpful in elucidating the diseases™ spread mechanism Such study andanalysis on spacetime patterns is a kind of diseasesurveillance which involves detecting the outbreak clustersof active cases monitoring of localisation and isolation ofinfectious agents and relative risks assessment of affectedsites at early stage Clements Cromley Kulldorff This study on geographical clustering ofinfectious diseases with temporal features helps in makingstrategies that dynamically update on emergence source ofdisease outbreak to help epidemiologists and decisionmakers for identification of spread and risk zones Thusclustering helps to enable timely prevention and containment measures and timely resource allocation to mitigatethe diffusion of diseasesBased on spacetime surveillance of diseases spacetime scan statistic Kulldorff is one of the clusterdetection tools which is widely used in geographicalsurveillance of diseases during epidemic andor pandemicThe spacetime scan statistic comes with two versions”prospective and retrospective Desjardins 0cTable Summary table for geospatial dashboards for COVID19Project nameDatasetsScopePurposeJournal of the Indian Society of Remote SensingWHO Coronavirus Disease COVID19WHO™s official dataDashboard Dong httpscovid19whointGlobal Visualization of official daily counts ofconfirmed cases and deaths related toCOVID19 with time stamps using EsriArcGIS Online serviceExploratory data analysis using 3D graphto perform countrywise analysis usingpopulation confirmed cases cumulativecases deaths and cumulative deathsProvides daily aggregate case and deathcount in CSVJohns Hopkins University COVID19Aggregated data from WHO EuropeanGlobal Dashboard for visualizing realtimeDashboard Dong httpscoronavirusjhuedumaphtmlCentre for Disease Prevention andControl ECDC WorldoMeters BNONews US CDC 1Point3Arces COVIDTracking Project and DXYmapping of COVID19 with graphs onconfirmed and daily casesCritical trend analysis on new cases perday mortality and fatality analysis inpopulation timeline of outbreak etcISRO™s BHUVAN COVID19 GeospatialSolution httpsbhuvanapp3nrscgovincoronacorona_dashboarddashboarddashboardphptypecitizenCOVID19 data Source MoHFWIndiaTime series visualization of activerecovered and deceased cases fromMarch to till dateGraphical analysis on spread trend ofCOVID19 daywise and statewiseCOVID19INDIA httpswwwCM Health M handles Press Trust ofIndiaVisualization of cumulative and dailycovid19indiaIndia state press bulletins PBI and ANIreportsnumbers of confirmed active recovereddeceased and tested statewise casesthrough maps and graphsProvides daily COVID19 cases in stateand district and cases reassigned to statesthrough APIMapmyIndia COVID19 httpsmapsCOVID19 data Source MoHFWIndiaProvides API on corona dashboards tomapmyindiacomcoronadistricts_containment_zonecontainment_zone_gradientHunger Relief Centres Source MyGovHunger Night Shelter Source MyGovNDMAvisualize cases at district and state levelhotspots treatment centres testing labsquarantine centres containment zoneslockdown issues hunger relief hunterand night sheltersMonthly climate explorer for COVID19httpscdsclimatecopernicuseuappsc3sappc3smonthlyclimatecovid19explorerMonthly COVID19 cases Source JHUGlobal Visualization of COVID19 fatalities withCSSEAtmospheric composition”PM10 and NO2Source CAMS EAC4Meteorological data”humidity hPaand surface air temperature on hourly andmonthly average rate Source ERA5reanalysisclimatic and atmospheric variations onmonthly basisExploratory analysis on correlation ofpollutants and specific humidity withCOVID19 deathsExperimental COVID19 and GlobalCOVID19 cases Source JHU CSSEGlobal Visualize earthquake as a cause of increaseSeismic Risk Map httpsmaps quakemapcovid1920200520v3grm234900Global earthquake risk map Source GEMGlobal Earthquake Modelin COVID19 cases due to people™sdisplacement from damaged buildingsOwusu and difference between both is thatprospective neglects historical clusters which may havepreviously occurred before the most current time period ofanalysis with no health threat Kulldorff Thereforethe prospective version of spacetime scan statistic iscommonly used to detect statistically significant active orevolving clusters of diseases for the present time periodand when more data become available the tool can be rerun to detect new evolving clusters with update on relativerisks for each affected sites Previously the prospectivespacetime scan statistic was used in thyroid cancerKulldorff shigellosis Jones measlesYin syndromic surveillance Yih and many other diseases However cluster analysis of 0cJournal of the Indian Society of Remote Sensingdiseases can be performed through several packages andlibraries in R Go´mezRubio and Pythonsoftware Yeng The contribution of cluster detections and analysis inCOVID19 pandemic is becoming useful nowadays as itdetects active and emerging clusters of COVID19 andnotify epidemiologist decision makers and public healthcare officials which can help in eradicating infections fromaffected sites and improving interventions quarantine andisolation measures The significant applications of clustering with respect to infectious diseases modelling aredemonstrated across the world Zarikas forexample India Bhosale and Shinde USA Desjardins Hohl Brazil Martines Italy Cereda China Ji Liu 2020a b Qiu Zhang Singapore Bhosale and Shinde Pung SouthKorea Shim French Alps Danis Germany Pfefferle Sergipe Andrade etcOutlier AnalysisThe outlier is defined by Hawkins as ˜˜an observationwhich deviates so much from the other observations as toarouse suspicions thatit was generated by a differentmechanism™™ In other words when data generation processstarts behaving abnormal and reflects the abnormalities orerrors in data such abnormalities are known as outliersBansal However the outliers generally holdadvantageous information about the systems unusual characteristics and entities which impact the data generationprocess Some of the useful applications of outliers in diseases are Cleynen Dai and Bikdash Krishnan Lo Prensner Washington Wu and Krishnan Clusteringalgorithms are optimized to find clusters rather than outliersand the accuracy of outlier detection depends on how goodthe clustering algorithm captures the structure of clustersMaximum Entropy Modelling Maxent ApproachIn context of disease systems disease transmission risksdepend on distribution of pathogens species in space andtime in some complex environmental conditions Townsend and as such treatments are focused mainly on spatialdimensions therefore diseases transmission risks are purelyhandled through geographical phenomena Such geographical link with diseases leads to the challenge of spatialmapping of disease transmission which overcame throughthe branches of biodiversity science”ecology and biogeography Such approach of ecological and biogeographical modelling can be seen from various studies on diseasetransmission risks mapping for example Arboleda Deka and Morshed Ferreira Holt Mweya Nakazawa Reeves Samy Qian Zhao Zhu Following recent studies on geographical mapping ofpathogens causing disease transmission machine learningbased maximum entropy method Maxent Elith Phillips is applied on spatial records ofCOVID19 with a set of bioclimatic environmentalvariables from WorldClim Poggio Ramı´rezVillegas and Bueno Cabrera to analyse theirfavourable environmental conditions as shown in Fig and Table required in maintaining its population TheMaxent principle is to estimate the target probability distribution by applying the maximum entropy to distributionwhich is most spread or closest This study is carried out inR software Ihaka and Gentleman and a geographical dataset consists of latitude and longitude of thoseregions which were affected till March Figure depicts the habitat suitability map of virus withprobability range in colour scale to visualize the highsuitability light and dark green colour medium suitabilityyellow and dark brownlow suitability light browncolour and unsuitable grey colour Table lists thefavourable bioclimatic variables and their contribution inpercent in maintaining the suitability of virusSusceptibleInfectiousRecovered SIR ModelEpidemiology deals with the study of pattern and occurrence of diseases in space and time associated with otherfactors such as environment demography and the translation of epidemiology into mathematical equations todescribe the spread of infectious diseases is known asmathematical epidemiology Allen Rayner andBender The mathematical epidemiology model isimplemented to understand the transmission dynamics ofcommunicable diseases by categorizing population intosusceptible infectious and recovered compartments Thefirst basic model known as SusceptibleInfectiousRecovered SIR model was proposed by Kermack andMcKendrick to describe the transmission of epidemic diseases from individual to individual The SIRmodel is a set of nonlinear ordinary differential equationswhich is mathematically defined as follows¼ l N þ SðÞ 00 bSI¼ bSI 00 cI 00 lI¼ cI 00 lRdSdtdIdtdRdtð1Þð2Þð3Þ 0cJournal of the Indian Society of Remote SensingFig Predicted suitability of Betacoronavirus using data till March Table Responsible bioclimatic variables in suitability modellingHereS is the class of susceptible individuals who are not yetcontracted to diseaseI is the class of infectious people who are now infectedwith disease and become infectious to infect others¢ R is class of recovered individuals who have recoverednow and are removed from class S¢ N is a total population size N S I R and t istime in days or weeks¢ b is the contact rate of infected person with suspected¢¢¢Bioclimatic variablesPercent contributionMean temperature of coldest quarterPrecipitation of wettest monthMean diurnal rangeIsothermalityAnnual mean temperatureMax temperature of warmest monthPrecipitation of coldest quarterPrecipitation of wettest quarterAnnual precipitationPrecipitation of driest quarterMean temperature of driest quarterMean temperature of wettest quarterPrecipitation seasonalityTemperature seasonalityPrecipitation of warmest quarterMean temperature of warmest quarterTemperature annual rangePrecipitation of driest monthMin temperature of coldest monthperson per dayc is the infectious period and average infectious periodis 1c¢ l is the per capita death rate which is adjusted by birthrate lNThere are many other compartment models derived fromthe basic epidemic model SIR with more compartmentsand transitions” SusceptibleExposedInfectiousRecovered SEIR Li and Muldowney SusceptibleInfectiousExposedRecoveredDeadSEIRDPiccolomiini and Zama SusceptibleInfectiousExposedRecoveredSusceptible SEIRS Liu and Zhang SusceptibleInfectiousQuarantineRecoveredSIQR Erdem

Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedObjective To investigate whether preventive administration of a proton pump inhibitor PPI can reduce the occurrence anddevelopment of traumatic granuloma TG following type IVVI cordectomy Methods We retrospectively analyzed the statusof postoperative granulomas in patients who underwent type IVVI cordectomy due to glottic cancer and determinedwhether postoperative administration of a PPI had any impact on granuloma formation and development Results Thepercentage and number of patients with granuloma in the PPI treatment group experimental group at the 1st 2nd 3rd and6th month following surgery were and respectively The percentageand number of patients with granuloma in the noPPI group control group were and respectively The granuloma percentage of the PPI treatment group was lower than that of thecontrol group at all postoperative time points assessed The diï¬erences were not statistically significant at the 1st monthp but were statistically significant at the 2nd and 3rd months after surgery p p ConclusionPreventive use of a PPI in patients after type IVVI cordectomy can shorten the TG recovery duration and may reduce theseverity of TG but it cannot prevent TG from occurring Our results should be confirmed by prospective randomized controlledtrials with large sample sizes IntroductionLaryngeal squamous cell carcinoma LSCC is a commonhead and neck cancer It had an incidence of approximately in China between and [] and new cases were reported worldwide in [] Itis estimated that there will be new cases worldwidein [] Approximately twothirds of LSCCs originatein the glottic area The presence of hoarseness in patientswith earlystage glottic cancer GC prompts the patients toseek medical treatment Anatomically the larynx is surrounded by cartilage and has sparse lymphatic tissue As aresult patients with GC are mostly diagnosed at an earlystage which is clinically defined as T12N0M0 []In recent years surgery has been gradually abandoned in the treatment of earlystage GC and has beenreplaced with transoral microsurgery TM or radiotherapyTM has the advantages of being minimally invasive and having a high laryngeal preservation rate and high costeï¬ectiveness Transoral laser microsurgery TLM is the mostcommon surgical method used for GC although a few studies in the literature have adopted transoral coblation microsurgery TCM Although TM methods diï¬erthermalinstruments are often needed for the surgery [“]Traumatic granuloma TG is a common complication ofTM especially after type IVV cordectomy Mild TG mayonly manifest as hoarseness foreign body sensation and frequent throat clearing The severe granuloma may cause or becomplicated by perichondritis which leads to severe symptoms such as dyspnea Additionally this granulation isbelieved to be an important factor in the formation of glotticweb and larynx stenosis [“] Reflux is an important 0cBioMed Research Internationalfactor that aï¬ects granuloma formation Proton pump inhibitors PPI are often used empirically in the treatment ofpatients with postoperative granulomas [ ]We found in our previous clinical practice that patientswith a wide range of resections such as type IVVI cordectomies have a higher risk of postoperative granuloma Thiscan sometimes be very severe even requiring temporary tracheotomy to alleviate dyspnea which greatly and adverselyimpacts patients™ quality of life PPIs have been empiricallyused for the treatment of patients with postoperative granuloma and evidence of its eï¬ectiveness has been published[] However there has not been any research on the prevention of postoperative granuloma formation with PPIsTherefore we initiated PPI treatment for patients who hadundergone type IVVI cordectomies and compared theresults with those of patients who had also undergone thistype of surgery but were not treated with PPIs to evaluatethe eï¬ect of PPI treatment for preventing postoperative granuloma formation in this patient population Materials and Methods Patients This was a retrospective study Patient inclusioncriteria i patients with vocal cord cancer who agreed toundergo type IV V and VI cordectomies ii patients whodid not undergo preoperative and postoperative radiotherapy Exclusion criteria i patients who were complicatedwith diabetes and were not treated routinely ii patientswho used PPIs regularly iii patients who continued tosmoke and drink after surgeryA total of patients between January and December were recruited as the control group who did not use PPIsimmediately after surgery and did not take PPIs persistentlyA total of patients between January and June were recruited as the experimental group PPI treatment group who took PPIs immediately after the surgeryand routinely Since this was a retrospective study only thepatients in the experimental group underwent preoperativereflux symptom index RSI and reflux finding score RFSassessments Patients with an RSI score points andoran RFS ‰¥ points were considered to have laryngopharyngeal reflux disease LPRD [ ]All patients signed an informed consent form prior to thesurgery All clinical experiments conformed to the guidelinesissued by the committee on clinical research of Peking UnionMedical College Hospital PUMCH Ethics Committeeapproval was obtained at PUMCH and all patients providedspecific written informed consent Surgical Procedure According to the European Laryngological Society classification endoscopic cordectomies includethe following types type IV total cordectomy type Vaextended cordectomy encompassing the contralateral cordtype Vb extended cordectomy encompassing the arytenoidtype Vc extended cordectomy encompassing the ventricularband type Vd extended cordectomies encompassing thesubglottis and type VI extended cordectomies encompassing the anterior commissure [ ] During the surgerythe larynx was fully exposed with a selfretaining laryngo°scope Karl Storz Tuttlingen Germany and the tumor°was resected en bloc under direct visualization of a orlaryngoscope Karl Storz Tuttlingen Germany using amodel coblator ArthroCare Corp Sunnyvale CA witha coblation level of and a coagulation level of Postoperative Treatment Procedure and FollowUp Thepatients in the experimental group were treated with intravenous omeprazole mg per day before the recovery of oralfeeding cases recovered oral feeding on the first day aftersurgery and cases recovered oral feeding on the third dayThen they were given mg oral omeprazole twice daily minutes before breakfast and dinner for consecutiveweeks The patients in the control group were not treatedwith PPIs but if severe granulomas formed during followup and required intervention they were also given PPIs routinely patients in the control group developedgranulomas But only patients developed severe granulomas on the 2nd 3rd and 3rd month after surgery respectively and began to be given PPIs for weeks the same asthe experimental group while the other cases in the control group were not given PPIs throughoutthe wholefollowup period All patients were treated with cefuroximeat mg twice daily for week to prevent infection Afterthe surgery the patients were required to quit smoking anddrinking and engage in reasonable vocal useThe followup procedure included regular checkups at and months after surgery If a granuloma was detectedduring the 3month checkup the patients were followedmonthly until the granuloma disappeared Postoperativegranuloma was defined as relatively smooth tissue in the surgery region that protruded from the surrounding area thatmay be attached to a pseudomembrane The granulomaand the surrounding area did not have obvious vascularhyperplasia The proportion of patients with postoperativegranuloma was used as an observation indicator Additionally the number of unscheduled visits and the rate of reoperationincluding tracheotomy and granulectomy wererecorded as indicators of granuloma severityDuring followup if the granuloma was found to severelyimpact vocalization andor breathing or if patients were suspected of having a recurrent tumor the granuloma was surgically resected and the specimen was sent for examination Statistical Analysis Data were statistically analyzed withSPSS software SPSS Chicago IL Nonnormally distributed quantitative data are represented as median and interquartile range and were subjected to the Wilcoxon ranksumtest Normally distributed measurement data are representedas mean ± standard deviation and were subjected to theindependent sample t test Count data were subjected to thechisquared test Diï¬erences with p were consideredstatistically significant Results Baseline Characteristics The baseline data of the patientsin the PPI treatment group and the control group are shownin Table Among the patients in the PPI treatment 0cBioMed Research InternationalTable General information DiscussionVariableSex MFAge yearsTumor staging T1aT1bT2Surgery type IVVVIPPIn ± Controln ± p valuegroup patients were male and patient was female withan average age of ± years patients were at theT1 stage and patients were at the T2 stage patients underwent type IV surgery patients underwent type V surgeryand patients underwent type VI surgery Among the patients in the control group patients were male and patient was female with an average age of ± years patients were at the T1 stage and patients were at theT2 stage patients underwent type IV surgery patientsunderwent type V surgery and patients underwent typeVI surgery The two groups of patients did not diï¬er significantly in the baseline conditions Table Postoperative Granulation The numbers and percentageof patients with granuloma in the PPI treatment group atthe 1st 2nd 3rd and 6thmonth followup were and respectively The numbers and percentage of patients with granuloma in the control group at those time points were and respectively Although the percentage of granuloma in the PPItreatment group was lower than that of the control group ateach stage only the diï¬erences at the 2nd and 3rd monthsafter surgery were statistically significant p and p respectively Only one patient in the PPItreatment group required a second surgery due to persistentgranulation patients in the control group underwent a second surgery among whom patients had granuloma complicated with chondronecrosis and required totracheotomy due to dyspnea Figure However the diï¬erence between the two groups was not statistically significantp In the PPI treatment group only patients hadtwo unscheduled visits In the control group patients had unscheduled visits among them one patient had visitsdue to dyspnea The diï¬erence in the number of unscheduledvisits was not statistically significant p Table Eï¬ects of PPI Treatment in Patients with LPRD in theExperimental Group Among the patients in the experimental group were diagnosed with LPRD according to preoperative RSI and RFS scores and patients did not have LPRD The numbers of LPRDpatients with granuloma at the 1st 2nd 3rd and 6th monthsafter surgery were and respectively and the numbers of LPRD patients with granuloma at these time pointswere and While the data showed that the percentageof granuloma in the LPRD patients was higher than that inthe LPRD patients only the diï¬erence at the 3rd month aftersurgery was statistically significant p Table Transoral microsurgery TM for earlystage glottic cancerGC can achieve oncological therapeutic eï¬ects similar tothose of radiotherapy TM has the advantages of being minimally invasive and having a high laryngeal preservation rateand a low tracheotomy rate its disadvantage includes postoperative complications such as bleeding infection airwayburns and granuloma formation Therefore currently thetreatment selected for patients with earlystage GC is determined by the disease conditions as well as patient needs[ ] Postoperative traumatic granuloma TG is a common complication during the healing process after TLM surgery Severe TG may cause or be complicated by chondritis orchondronecrosis leading to severe complications includingdyspnea With the increase in the range of cordectomy theincidence of TG is also significantly increased Therefore itis necessary to investigate ways to reduce the incidence andseverity of postoperative TG [ ] Like TLMthecoblationassisted endoscopic cordectomy or TCM used inour study is also based on thermal damage whose working°C much lower than laser™s workingtemperature is °°temperature which is C1000C and as a result its healing process and the mechanism of TG formation are alsosimilar to those of TLM Current literature indicates thatreflux may be an important factor in the occurrence of postoperative TG [ “] Therefore we aimed to investigatewhether antiacid therapy could reduce TG through an analysis of the eï¬ects of PPI treatment in patients who underwenttype IVVI cordectomy which has rarely been reported°C70Our study showed that the incidences of TG after transoral surgery were as high as and in the twogroups which were much higher than the incidences of reported by Nerurkar and Shah and reported by Wang The reason for this discrepancy may be that the patients in our study all underwent typeIV or higher surgeries Enlarged wounds and damage to theperichondrium or cartilage may lead to increased TG andchondronecrosis Nerurkar and Shah reported that the incidence of TG in patients who have undergone type IV TLMwas and the study by Wang showed that the incidences of TG in patients who have undergone type IV andtype V TLM were and respectively [ ]Meanwhile the incidence of chondronecrosis was not rare especially on whom the surgeon had to expose thethyroid cartilage during tumor resection in TLM [] Thesestudies suggest that type IV or higher surgeries lead to a highlikelihood of TG occurrenceOur study showed that PPI treatment did not suppressthe formation of TG at the 1st month after surgery but thepercentage of TG in the PPI treatment group graduallybecame lower than that of the control group and the diï¬erences were statistically significant at the 2nd and 3rd monthsafter surgery At the 6th month granulomas disappeared inboth groups Our findings suggest that although PPI treatment cannot reduce the incidence of TG it can significantlyshorten the duration of granulomas a finding that is similarto the study by Wang [] Additionally we did notobserve any severe cases of TG that were complicated with 0cBioMed Research InternationalabcdefghiFigure a“f Shows a typical case in the control group who was a male patient for years old with glottic cancer T2N0M0 at the left sidefollowed by type V cordectomy a One month after surgery a granuloma was found in the left vocal cord under a fibrolaryngoscope b months after surgery the granuloma enlarged and the right vocal cord become edema obviously c months after surgery d monthsafter surgery e Computerized tomography CT scan taken before surgery f months after surgery chondronecrosis was found in the CTscan where the white arrow points out g“i Shows a typical case in the experimental group who was a male patient for years old withglottic cancer T2N0M0 at the right side followed by type V cordectomy g h and i were taken under a fibrolaryngoscope in month months and months after surgery respectively A granuloma could be found in g but disappeared in h and idyspnea in the PPI treatment group however TG in patients in the control group caused or was complicated bychondritis or chondronecrosis which led to severe dyspneaThere were only unscheduled visits among the PPI treatment group compared to unscheduled visits in the controlgroup Unfortunately the diï¬erences in the two indicators ofTG severity between the two groups were not statistically significant although we believe PPI treatment did alleviate the 0cBioMed Research InternationalTable Percentage and severity of granuloma in the two groups1st month2nd month3rd month6th monthResurgeryNumber of unscheduled visits asmedian and interquartile rangePPI treatment groupControl groupp valueNote aindicates that the diï¬erence is statistically significant0005a0037a Table Percentage of granuloma in patients with or without LPRDin the experimental group1st month 2nd month 3rd month 6th month”LPRD n LPRD n p valueNote aindicates that the diï¬erence is statistically significant0029aseverity of TG In this study postoperative PPI was used for weeks referring to the antiacid duration weeks in amedical routine of vocal process granulation and laryngopharyngeal reflux disease J R Lechien™s report in andChinse experts consensus on diagnosis and treatment of laryngopharyngeal and reflux disease in [] The resultsshowed that PPI could shorten the recovery time and potentially prevent severe complicationsOur study showed that the percentage of TG did not differ significantly between the LPRD and LPRD patientsp However it took longer for granulomas to disappear in LPRD patients than in LPRD patients and the difference in the number of patients with granulomas wasstatistically significant between the two groups at monthsafter surgery p The reason for this phenomenonmay be that during the early stage of recovery after vocalcord injury changes in the extracellular matrix are the mainmanifestation and injury impacts a lot while acid reflux hasa little eï¬ect Acid reflux may impact the repair process in themiddle and late stages [ ]This was a retrospective nonrandomized controlledstudy with a small sample size We only analyzed granulomaformation in the patients and did not assess their oncologicaloutcome We based the diagnosis of LPRD on RSI and RFSscores and did not perform dualprobe 24hour pH monitoring Therefore we were unable to determine whethernonacid reflux had an impact on the formation and development of postoperative granuloma ConclusionThe preventive use of PPI in patients who have undergonetype IVVI cordectomy cannot reduce the incidence of TGwhile it can shorten the TG recovery duration and may alsoreduce the severity of TG Our findings should be confirmedby prospective randomized controlled studies with largersample sizesData AvailabilityAccess to these anonymized data will be made available bythe corresponding author Dr Jian Wang upon reasonablerequestConflicts of InterestThe authors declare that they have no conflicts of interestfinancial or nonfinancial to discloseAuthors™ ContributionsXiaofeng Jin and Yanyan Niu contributed equally to thisstudyAcknowledgmentsThis study was funded by the Nature Science Foundation ofBeijing China Grant No References[] L B Du W M Mao W Q Chen œIncidence and mortality of larynx cancer in China during  ZhonghuaLiu Xing Bing Xue Za Zhi vol no pp “ [] F Bray J Ferlay I Soerjomataram R L Siegel L A Torreand A Jemal œGlobal cancer statistics GLOBOCAN estimates of incidence and mortality worldwide for cancers in countries CA A Cancer Journal for Clinicians vol no pp “ [] B Gupta N W Johnson and N Kumar œGlobal epidemiology of head and neck cancers a continuing challenge Oncology vol no pp “ [] M F Vaculik C A MacKay S M Taylor J R B Trites R DHart and M H Rigby œSystematic review and metaanalysisof T1 glottic cancer outcomes comparing CO2 transoral lasermicrosurgery and radiotherapy Journal of Otolaryngology Head and Neck Surgery vol no p [] W Steiner œResults of curative laser microsurgery of laryngealcarcinomas American Journal of Otolaryngology vol no pp “ [] M S Strong œLaser excision of carcinoma of the larynxLaryngoscope vol no pp “ [] A S Carney M S Timms C N Marnane S KrishnanG Rees and S Mirza œRadiofrequency coblation for the resection of head and neck malignancies Otolaryngology and Headand Neck Surgery vol no pp “ [] M Lee M A Buchanan F Riï¬at and C E Palme œComplications after CO2 laser surgery for early glottic cancer an 0cBioMed Research Internationalinstitutional experience Head Neck vol no S1 pp E987“E990 [] B Liu L Cheng H Ming and C Zhong œTreatment of theearlystage glottic cancer using lowtemperature radiofrequency coblation Journal of Cancer Research and Therapeutics vol no pp “ [] Y Zhang B Wang G Sun G Zhang L Lu and G LiangœCarbon dioxide laser microsurgery versus lowtemperatureplasma radiofrequency ablation for T1a glottic cancer asingleblind randomized clinical trial BioMed Research International vol Article ID pages [] M Remacle H E Eckel A Antonelli œEndoscopic cordectomy A proposal for a classification by the Working Committee European Laryngological Society European Archivesof OtoRhinoLaryngology vol no pp “ [] L Wang S Sun S Wang D Liang and W Ji œClinical observation of traumatic granuloma after CO‚‚ laser cordectomy andlaryngopharyngeal reflux Zhonghua Er Bi Yan Hou Tou JingWai Ke Za Zhi vol no pp “ [] M Canis F Ihler A Martin C Matthias and W SteinerœTransoral laser microsurgery for T1a glottic cancer reviewof cases Head Neck vol no pp “ [] A Galli L Giordano D Sarandria D di Santo and M BussiœOncological and complication assessment of CO2 laserassisted endoscopic surgery for T1T2 glottic tumours clinicalexperience Acta Otorhinolaryngologica Italica vol no pp “ [] N K Nerurkar and R Shah œFactors responsible for the development of carbon granuloma post transoral laser cordectomy Lasers in Medical Science vol no pp “ [] P C Belafsky G N Postma and J A Koufman œThe validityand reliability of the reflux finding score RFS Laryngoscopevol no pp “ [] P C Belafsky G N Postma and J A Koufman œValidity andreliability of the reflux symptom index RSI Journal of Voicevol no pp “ [] M Remacle C van Haverbeke H Eckel œProposal forrevision of the European Laryngological Society classificationof endoscopic cordectomies European Archives of OtoRhinoLaryngology vol no pp “ [] J Yoo C Lacchetti J A Hammond R W Gilbert and Headand Neck Cancer Disease Site Group œRole of endolaryngealsurgery with or without laser versus radiotherapy in themanagement of early T1 glottic cancer a systematic reviewHead Neck vol no pp “ [] C M Chiesa Estomba F A Reinoso A O Velasquez J LFernandez J L Conde and C S Hidalgo œComplications inCO2 laser transoral microsurgery for larynx carcinomas IntArch Otorhinolaryngol vol no pp “ [] J R Lechien F Mouawad M R Barillari œTreatment oflaryngopharyngeal reflux disease a systematic review WorldJournal of Clinical Cases vol no pp “ [] M K Wani and G E Woodson œLaryngeal contact granuloma Laryngoscope vol no pp “ [] C Ling M Yamashita J Zhang D M Bless and N V Welham œReactive response of fibrocytes to vocal fold mucosalinjury in rat Wound Repair and Regeneration vol no pp “ 0c'

Molecular Medicine The role of selenium metabolism andselenoproteins in cartilage homeostasisand arthropathiesDonghyun Kang Jeeyeon Lee Cuiyan Wu3 Xiong Guo3 Byeong Jae Lee24 JangSoo Chun5 andJinHong Kim AbstractAs an essential nutrient and trace element selenium is required for living anisms and its beneficial roles in humanhealth have been well recognized The role of selenium is mainly played through selenoproteins synthesized by theselenium metabolic system Selenoproteins have a wide range of cellular functions including regulation of seleniumtransport thyroid hormones immunity and redox homeostasis Selenium deficiency contributes to various diseasessuch as cardiovascular disease cancer liver disease and arthropathy”Kashin“Beck disease KBD and osteoarthritisOA A skeletal developmental disorder KBD has been reported in lowselenium areas of China North Korea and theSiberian region of Russia and can be alleviated by selenium supplementation OA the most common form of arthritisis a degenerative disease caused by an imbalance in matrix metabolism and is characterized by cartilage destructionOxidative stress serves as a major cause of the initiation of OA pathogenesis Selenium deficiency and dysregulation ofselenoproteins are associated with impairments to redox homeostasis in cartilage We review the recently exploredroles of selenium metabolism and selenoproteins in cartilage with an emphasis on two arthropathies KBD and OAMoreover we discuss the potential of therapeutic strategies targeting the biological functions of selenium andselenoproteins for OA treatmentIntroductionSelenium Se is an essential trace element in humans12Selenium is generally taken up from the diet through foodor other forms of external supplementation Dietaryselenium is obtained in the form of selenomethionineSeMet selenocysteine Sec selenite and selenate Significant health benefits have been attributed to seleniummetabolic systems that play major physiological roles inthyroid hormone metabolism immunity and antioxidantdefense23 Selenium is required for the production ofthyroid hormonemetabolizing enzymes and seleniumCorrespondence JinHong Kim jinhkimsnuackr1Center for RNA Research Institute for Basic Science Seoul South Korea2Department of Biological Sciences College of Natural Sciences Seoul NationalUniversity Seoul South KoreaFull list of author information is available at the end of the These authors contributed equally Donghyun Kang Jeeyeon Leesupplementation is thought to improve the function ofthyrocytes and immune cells4 Selenium supplementationdemonstrated immunostimulant effects such as enhancedproliferation of activated T cells activation of naturalkiller cells and tumor cytotoxicity mediated by cytotoxiclymphocytes56 In contrast selenium deficiency is associated with the occurrence virulence and disease progression of viral infections7Selenium inadequacy can lead to various types ofdiseases most notably cardiovascular disease8“ cancer13“ hepatopathy1617 and arthropathy Cardiovascular diseases are associated with systemic seleniumlevel with a higher risk at or μgL seleniumconcentration in the blood10 A type of endemic cardiomyopathy Keshan disease is linked to selenium deficiency811 Keshan disease occurs in lowselenium areasin Chinasodium seleniteand is prevented by The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproductionin any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate ifchanges were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material Ifmaterial is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this license visit httpcreativecommonslicensesby40Official journal of the Korean Society for Biochemistry and Molecular Biology 0cKang Experimental Molecular MedicinePage of studiesEpidemiologicalsupplementation12 Lowselenium status is correlatedwith a significantly increased risk of cancer incidenceand mortality13“haveprovided evidence on the cancerpreventing effects ofselenium18“ Selenium deficiency is also characterizedby elevated levels of oxidative stress markers in the liver21which significantly contribute to liver injury17 The oxidative stress caused by selenium deficiency further plays adetrimental role in joint development Selenium deficiency is the main cause of endemic Kashin“Beck diseaseKBD which is mainly reported in lowselenium areas ofChina North Korea and the Siberian region of RussiaMoreover there is a growing body of evidence suggestingthat the pathogenesis of osteoarthritis OA the mostcommon form of arthritis may be associated with selenium deficiency by resulting in oxidative stress22“However it is noteworthy that excessive selenium intakecan also cause selenosis2930 which accompanies adversesymptoms including fatigue diarrhea nausea increasedheart rate necrosis in liver and kidney and neurologicaldamage Chroniccompromisesimmune and reproductive systems in humanseventuallyselenosisOA is characterized by progressive loss of cartilageextracellular matrix ECM and pathological changes inother joint tissues such as subchondral bone sclerosisosteophyte formation and synovial ‚ammation31 Cartilage destruction is considered a hallmark of OA and is aresult of increased production of catabolic effectors32“and reduced matrix biosynthesis by chondrocytes36 OA isassociated with multiple etiologies involving systemicfactors such as age37 as well as local factors such asmechanical stress38 driven by weightbearing and jointinstability Both OAcausing factors have been found tocause oxidative stress in chondrocytes Oxidative stressresults from the abnormal production of reactive oxygenspecies ROS and the loss of cellular antioxidant capacityMany preclinical and clinical studies have indicated theaccumulation of oxidative burden in chondrocytesundergoing osteoarthritic changes3940 Emerging evidence suggests that oxidative stress is mechanisticallylinked to the initiation of osteoarthritic changes inchondrocytes through the acquisition of senescent phenotypes36 Therefore restoring redox homeostasis canserve as a rational therapeutic strategy to alleviate OAprogression Here we review the role of selenium metabolism in cartilage and bone and the significance ofmaintaining its homeostasis in the context of joint diseases such as KBD and OAOverview of the selenium metabolic systemThe selenium metabolic system and the biosynthesis ofselenoproteinsSelenium metabolism is a systemic process that includesandtransformationtransportationabsorptiontheOfficial journal of the Korean Society for Biochemistry and Molecular Biologyexcretion of selenium Fig Selenium is obtained inanic forms”SeMet and Sec”and inanic forms”selenite and selenate”from diet Selenium is taken up bythe liver that synthesizes and exports SELENOP whicheventually circulates through the bloodstream SELENOPwith multiple Sec residues41 transports selenium to othertissues and ans42 and the transported selenium isconverted to selenophosphate by intracellular seleniummetabolic pathways Selenium is excreted through exhalation and urine in the form of smallmolecule metabolites formed by sequential methylation4344Selenium plays biological roles predominantly in theform of selenoproteins synthesized by the seleniummetabolic system Ingested inanic selenium is firstreduced to hydrogen selenide H2Se via glutathioneGSH and thioredoxin TXN systems Selenide is furtherconverted to Sec amino acids for incorporation intospecific sites of selenoproteins such as the catalytic sites ofa selenoenzyme Mechanistically selenophosphate synthetase SEPHS2 catalyzes the production of selenophosphate through the reduction of hydrogen selenideThe subsequent reaction with phosphoseryltRNA PSertRNA[Ser]Sec yields SectRNA[Ser]Sec Sec amino acids areincorporated into polypeptidethrough themachinery utilizing the UGA codon Selenocysteineinsertion sequence binding protein SBP2 binds toselenocysteine insertion sequence SECIS element whichis located in the ²untranslated region ²UTR of selenoprotein mRNA and mediates the transfer of SectRNA[Ser]Sec to the Asite of ribosome which recognizesthe UGA codon as the Sec integration codon Collectivelythe selenoprotein translation machinery consists of SECISelement SBP2 Secspecific eukaryotic elongation factorEEFSEC and aminoacylated SectRNA[Ser]Sec therebyenabling UGA to be recognized as a Sec codon and utilized for translation into the growing polypeptidechainsSelenoproteinssome ofSelenoprotein is defined as a protein containing Secamino acid residue The biological functions of seleniumare mostly exerted through selenoprotein domains thatcontain Sec residues Twentyfive selenoprotein geneshave been identified in the human genome45 In mice atotal of selenoproteins have been characterized46 andtargeted deletion ofthese selenoproteinsdemonstrated their essential roles in developmental processes and in disease pathogenesis Selenoproteins can beclassified into subfamilies based on their cellular functionssuch as those implicated in antioxidation GPX1 GPX2GPX3 GPX4 redox regulation TXNRD1 TXNRD2TXNRD3 MSRB1 SELENOH SELENOM SELENOWthyroid hormone metabolism DIO1 DIO2 DIO3 selenium transport and storage SELENOP selenophosphatesynthesis SEPHS2 calcium metabolism SELENOK 0cKang Experimental Molecular MedicinePage of Fig Selenium metabolic system in mammals Selenium is absorbed from the diet undergoes several conversion steps and is incorporated intopolypeptide chains completing selenoprotein synthesis Dietary sources of selenium uptake exist in inanic form such as selenate and selenite andanic form such as Sec and SeMet Inanic forms are reduced by TXNRDTRX or GRXGSH systems and anic forms are cleaved by SCLYforming selenide Selenophosphate is synthesized from selenide by SEPHS2 and the subsequent reaction with PSertRNA[Ser]Sec mediated by SEPSECSyields SectRNA[Ser]Sec SectRNA[Ser]Sec is transferred to the Asite of ribosome mediated by SBP2 which binds to SECIS located in the ²UTR of aselenoprotein mRNA Finally the UGA codon is recognized as the Sec integration codon Abbreviations SeMet selenomethionine Secselenocysteine GRX glutathione reductase TRX thioredoxin TXNRD thioredoxin reductase GSH glutathione MGL methionine gammalyase SCLYselenocysteine lyase SEPHS2 selenophosphate synthetase SARS seryltRNA synthetase PSTK phosphoserylSeptRNA kinase SEPSECS SeptRNASectRNA synthase EEFSEC Secspecific eukaryotic elongation factor SBP2 SECIS binding protein SELENOT myogenesis SELENON protein foldingSELENOF SELENOI SELENOS and protein AMPylation SELENOO4748 The functions of other selenoproteins such as GPX6 and SELENOV still remain unclearGlutathione peroxidases GPXs such as GPX1 cytosolicGPX GPX2 gastrointestinal GPX and GPX4 phospholipid hydroperoxide GPX catalyze the decompositionof a great variety of peroxides thus protecting cellsagainst oxidative damage4950 Thioredoxin reductasesTXNRDs employ NADPH as an electron donor to revertoxidized TXN to a reduced dithiol the oxidation status ofwhich is critically implicated in regulating various cellbehaviors including proliferation and apoptosis51 Thephysiological significance of TXNRDs is further supported by the embryonic lethality of Txnrd1 or Txnrd2knockout mice5253 Deiodinases DIOs regulate thyroidhormone metabolism by catalyzing the conversion ofthyroid hormones from precursor thyroxine T4 to biologically active triiodothyronine T3 or inactive reverseT3 rT354 The expression levels of several selenoproteinsOfficial journal of the Korean Society for Biochemistry and Molecular Biologyare ‚uenced by the extent of selenium uptake Forexample seleniumdeficient animals and human cell linesexhibit reduced transcription of selenoproteins such asGPX1 DIOs SELENOI and SELENOW55“ A subset ofselenoproteins such as GPX1 and SELENOW is moresensitive to selenium supplementation or deficiency Thehierarchy of selenoprotein expression is more apparentwhen the intracellular level of selenium is limited1Seleniumresponsive genesgenesareseleniumcontainingSeleniumresponsivethe genes whoseexpression patterns are ‚uenced by supplementationwith selenium orcompoundsTreatment of a cancer cell line with methylseleninic acidin genes58 Theseinduced expression changesresponsive genes were closely associated with annotationsrelated to cell cycle regulation androgenresponsive genesand phase II detoxification pathway Selenium supplementation of macrophages diminished the expression oflipopolysaccharide LPSinduced pro‚ammatory genes 0cKang Experimental Molecular MedicinePage of such as cyclooxygenase2 COX2 and tumor necrosisfactorα TNFα59 suggesting that selenium has anti‚ammatory effects on the immune system The CTDdatabase httpctdbase reports the effect of environmental chemicals including selenium on gene expression profiles in various human tissuesThe role of selenium and selenoproteins incartilage development and KBDSelenium levels and its role in joint tissuesJoints are composed of various types of connective tissues including cartilage bone synovium meniscus andligament Among these tissues cartilage is the maincomponent that absorbs mechanical stress cushioningbones from impacting each other during various weightbearing activities In the human knee joint the seleniumconcentration in cartilage is approximately μgkg dryweight whereas the selenium concentrations in ligamentand meniscus are and μgkg dry weight respectively6061 The requirement of adequate physiologicalselenium levels for maintaining cartilage homeostasis hasbeen recognized Selenium deficiency retards the growthand development of cartilage and bone62“ Growthretardation was observed in rats after two generations ofselenium deficiency62 Mice fed a diet deficient in selenium resulted in fibrocartilage formation at the articularsurface ultimately showing degeneration of articularcartilage63 Selenium deficiency induced the expression ofthe chondrocyte hypertrophy marker gene type X collagenCOLX in articular cartilage64 The expression of parathyroid hormonerelated protein PTHrP which controlschondrocyte maturation during endochondral ossification was enhanced in both articular cartilage andhypertrophic growth plate following selenium deficiencyThese changes were in line with the phenotypic changesobserved in the cartilage of KBD patients64 However itshould be noted that growth retardation caused by selenium deficiency may also be associated with the deregulation of bone metabolism65 In a study by Cao et alselenium deficiency severely compromised bone microarchitecture as a result of increased bone resorption66Abnormalities in selenium metabolism and skeletaldevelopment diseasesSelenium deficiency is regarded as one of the initiatingfactors of KBD which is an endemic osteoarthropathycaused by the premature closure of epiphyseal plate andthe impaired skeletal development Skeletal deformities inhands fingers knees and elbows and in severe casesdwarfism and movement disorders are the symptoms ofKBD22 The KBD area roughly coincides with lowselenium areas including a geological belt extendingfrom northeast to southwest China North Korea andeastern Siberia22 A metaanalysis showed that seleniumOfficial journal of the Korean Society for Biochemistry and Molecular Biologylevels in the water soil cereal and corn in KBD endemicregions were lower than they were in nonendemicregions supporting the fact that the level of selenium intissue is predominantly affected by dietary intake23 In linewith this finding selenium levels in the whole bloodserum hair and urine of KBD patients were markedlylower than those of healthy controls24Selenoprotein gene polymorphisms are associated withincreased susceptibility to KBD There were significantdifferences in the allelic frequency of GPX1 Pro198Leurs1050450 between the KBD and control group67 Inaddition the mRNA level of GPX1 and enzyme activity oftotal GPX in blood were lower in the KBD group thanthey were in the control group67 Haplotypes of TCCTTC and TTT of rs1050450 rs3811699 and rs1800668in GPX1 gene also had a significant link to KBD68 Asinglenucleotide polymorphism SNP in the promoterregion of SELENOS rs28665122 ˆ’105G A was relatedto the increased risk of KBD and upregulation of PI3KAktsignaling in patients with KBD69 In this study tertbutylhydroperoxide tBHPtreatmentinduced chondrocyteapoptosis was mitigated by selenium supplementation viasodium selenitetreatment which suppressed thePI3KAkt pathway The minor Aallele of SELENOFrs5859 was associated with a significantly higher incidenceof KBD70The animals fed a seleniumdeficient diet recapitulatedsome of the pathological manifestations of KBD stronglysupporting the notion that selenium deficiency is criticallyassociated with the development of this endemic arthropathy Selenium deficiency impaired bone and cartilagegrowth with the exhibition of premature chondrocytehypertrophy as evidenced by an increased expression ofCOLX compatible with the phenotypes in KBD cartilage64The lowselenium condition in combination with threemycotoxins deoxynivalenol DON nivalenol NIV and T yielded procatabolic changes and hypertrophic phenotype of chondrocytes as evidenced by the loss of aggrecanand type II collagen COLII and the increase in COLX andmatrix metalloproteinases MMPs expressionrespectively71 In contrast selenium supplementation partiallyalleviated these mycotoxininduced damages in chondrocytes71 In rats dietary selenium deficiency over twogenerations caused the onset of physiological seleniuminsufficiency72 In this condition pathological changes inthe epiphyseal plate were observed with the decreasedexpression of COLII and GPX1 in the chondrocytes suggesting a possible association of reduced chondrocyte anabolism and antioxidant capacity with the epiphyseal platelesions observed in KBD72 The relevance ofimpairedselenium metabolism to the onset of KBD was furthervalidated using a mouse genetic deletion model Targeteddeletion of SectRNA[Ser]Sec Trsp gene in osteochondroprogenitor cells from embryonic stage caused the 0cKang Experimental Molecular MedicinePage of depletion of selenoproteins in skeletal systems causinggrowth retardation abnormalities in the epiphyseal growthplate delayed endochondral ossificationand chondronecrosis which recapitulated the major pathologicalfeatures of KBD73As a prophylactic treatment selenium supplementationswere given to children living in a KBD area The supplemented group showed elevated physiological seleniumlevels in their hair samples and exhibited a substantiallylower prevalence of KBD74 A metaanalysis including fiverandomized controlled trials RCTs and ten prospectivenonRCTs statistically demonstrated the benefits of selenium supplementation in preventing KBD in children75Selenium metabolism and OAPhysiological significance of oxidative stress inchondrocytesOA is the most common form of arthritis and is primarilycharacterized by the loss of cartilagespecific ECM and otherpathological changes in joints including subchondral bonesclerosis osteophyte formation and synovial ‚ammation31Articular cartilage is composed of abundant proteoglycans inwhich sulfated glycosaminoglycan chains such as chondroitinsulfates are bound to a core protein such as aggrecan Loss ofcartilage matrix during OA progression is a combined resultof increased catabolic process in cartilage and reduced anabolic activity of chondrocytes The molecularlevel understanding of OA pathogenesis has led to the identification ofmajor catabolic enzymes ADAMTS576 MMP377 andMMP1378 which mediate the degradation of cartilagematrix Pro‚ammatory cytokines drive the expression ofthese catabolic factors in chondrocytes through the activationof transcription factors such as HIF2α32 and NFκB79Abnormalities in various metabolic pathways such as glucose80 or amino acid metabolic system81 in chondrocyteshave been implicated in activating catabolic cascades inosteoarthritic cartilage82 Moreover increased cellular uptakeof Zn2 through the upregulation of zinc transporter ZIP8activates metalregulatory transcription factor1 MTF1which in turn induces the expression of matrixdegradingenzymes in chondrocytes3383 Regulation of catabolism bythefurthershowed the association of metabolic abnormalities with thecatabolic process of OA34cholesterol“CH25H“CYP7B1“RORαaxisMeanwhile the upstream regulatory mechanism eliciting an imbalance in OA matrix homeostasis needs furtherinvestigation OAcausing factorssuch as age andmechanical stress lead to excessive oxidative stress inchondrocytes3738 Consistently clinical and preclinicalOA studies indicated a cumulative oxidative burden inosteoarthritic chondrocytes3940 Emerging evidence suggests that oxidative stress plays a significant role in OAdevelopment and the disease progression can be mitigatedby counteracting oxidative stress3684“In generalOfficial journal of the Korean Society for Biochemistry and Molecular Biologyoxidative stress results from the abnormal production ofROS and the loss of cellular antioxidant capacity Synovialfluid from patients with latestage OA who were undergoing knee joint replacement had a lower level of oxidoreductases than that from healthy controls87 In partthe increase in oxidative stress is attributable to mitochondrial dysfunction in OA chondrocytes8889 OAchondrocytes displayed reduced mitochondrial DNAcontent mitochondrial dysfunction and diminishedexpression of NRF2 which regulates the transcription ofoxidoreductase genes89 Similarly chondrocytes fromaged individuals exhibited increased ROS burden andmitochondrial and genomic DNA damage90“ Therefore the proper maintenance of redox homeostasis canpotentially serve as a rational therapeutic strategy toprotect against OA progressionPotential roles of selenium metabolism in OAThe protective effect of selenium in OA has beenexplored in a large number of epidemiological and geneticstudies Table The concentration of selenium in serumwas significantly lower in OA patients than that of normalcontrols25 Similarly the results from a populationbasedcohort study demonstrated the linkage between lowselenium levels in toenails with OAassociated pain anddisease severity2627 Several studies have indicated thatcartilage matrix homeostasis is impaired in seleniumdeficiency Lowselenium status diminished COLIIexpression level regulated by SOX9 which is known as amaster regulator required for maintaining cartilage matrixIn fact SOX9 was destabilized by thehomeostasisdownregulation ofseleniumresponsive PRMT5 thatsustains SOX9 stability via methylation93 In anotherstudy rats fed a seleniumdeficient diet exhibited lowsulfotransferase activity which resulted in diminishedforcontents ofmechanicalcartilagematrix28 In contrast selenium supplementation ameliorated the spontaneous degeneration of articular cartilagein STR1 N mice by increasing the expression of GPXs94In cultured chondrocytes pretreatment with SeMetmarkedly inhibited nitric oxide NO and prostaglandinE2 PGE2 production in response to pro‚ammatorycytokine IL1β95 Expression of SBP2 a factor recognizingSECIS element had a positive correlation with GPX1 andGPX4 expression and antioxidant capacity in chondrocytes96 Oxidation resistance mediated by SBP2 wasdiminished in response to IL1β treatment in vitro and indamaged regions of cartilage in OA patients96 Downregulation of selenoprotein mRNAs including GPX397GPX1 and GPX49698 and Selenop99 was observed inhuman and mouse OA chondrocytessulfated glycosaminoglycan essentialstressabsorbingpropertyofGenetic factors such as SNPs in selenoproteins wereidentified to be risk factors for OA development A GAG 0cKang Experimental Molecular MedicinePage of Table List of selenoproteins associated with the pathogenesis of arthropathies KBD and OAGeneGPX1GPX3GPX4DIO2DIO3SELENOFSELENOPSELENOSFunctionExpression in OASNPAntioxidantReduction of hydrogen peroxide and anic peroxidesDownregulatedPlasma antioxidantDetoxification of lipid hydroperoxidesMetabolism of lipidsActivation of hormonesDeiodination of T4 to T3Inactivation of hormonesConversion of T4 to rT3Protein foldingStorage and transport of SeAntioxidant propertiesProtein foldingERassociated protein degradationDownregulatedDownregulatedUpregulatedDownregulatedrs1050450 KBDrs3811699 KBDrs1800668 KBDrs225014 OArs12885300 OArs945006 OArs5859 KBDrs28665122 KBDRef“haplotype in SELENOS gene was significantly associatedwith increased levels of‚ammatory factors in OApatient plasma100 SNPs in DIO2 which converts precursor thyroid hormone T4 to its active form T3 were alsorelated to genetic susceptibility to OA developmentLevels of DIO2 mRNA and protein were markedly upregulated in OA cartilage101 A common DIO2 haplotypecomposed of the minor Callele of SNP rs225014 and thecommon Callele of SNP rs12885300 was significantlyassociated with advanced hip OA as indicated by a higherodds ratio101“ Locus rs225014 which confers risk toOA was associated with the differential methylation ofCpG located in the upstream region of DIO2 gene andwas correlated with upregulated DIO2 expression inOA104 Meanwhile DIO3 depletes the resources that canbe utilized for the production of active thyroid moleculesby catalyzing the conversion of T4 and T3 into inactivemetabolites The minor Gallele of the DIO3 variantrs945006 was associated with a protective effect againstOA development105However a few aspects regarding the relationshipbetween selenium and OA remain controversial Firstseveral studies indicate that there are no differences inselenium levels between OA and normal tissues Theselenium concentrations in synovial fluid and plasma of OA patients were not significantly different from thoseof healthy controls106 Similarly no significant difference in selenium concentration was noted between sixdogs with posttraumatic OA and six control dogs107Second the beneficial effect of selenium supplementationin alleviating OA symptoms has been debated The resultsfrom a controlled doubleblind trial of patientsOfficial journal of the Korean Society for Biochemistry and Molecular Biologyrevealed that the supplementation of a formulation containing selenium with vitamins A C and E SeACE didnot have any remarkable curative effect compared to aplacebo108 In a study with an independent cohort theprevalence of radiographic knee OA was not significantlyassociated with dietary selenium intake109Nonethelessit is apparent that selenium deficiencydysregulation of selenoproteins and genetic variations inselenoprotein genes serve as potential risk factors for OAThe vital role of selenium metabolism in maintainingcartilage homeostasis is expected considering its criticalinvolvementin regulating cellular processes such aschondrogenic differentiation of progenitor cells maintenance of redox homeostasis and DNA damage repair inchondrocytes which are covered in the next sectionIntracellular roles of selenium metabolism andselenoproteins in cartilageChondrogenic differentiation programs of progenitor cellsSelenium exerts various beneficial effects to promoteproliferation and differentiation of chondrogenic progenitorcells110111 Selenium supplementation stimulated the proliferation of ATDC5 chondrogenic cells even under serumdeprivation by inducing cyclin D1 expression110 Deficiencyof SELENOO interfered with the chondrogenic differentiation of ATDC5 cells by suppressing the expression ofchondrogenic genes SOX9 COLII and aggrecan anddecreasing the activity of alkaline phosphatase112 Knockdown of Gpx1 reduced the chondrogenic differentiation ofATDC5 cells by modulating intracellular GSHoxidizedGSH GSSG ratio113 Selenop was differentially upregulatedduring the chondrogenic differentiation of micromass 0cKang Experimental Molecular MedicinePage of culture of mesenchymal cells isolated from mouse limbbuds114 In line with the effects of selenium metabolism andselenoproteins in chondrogenic progenitor cells observedin vitro deficient uptake of selenium severely affectedchondrogenic differentiation of mesenchymal lineage cellsin mice64andOsteochondroprogenitorspecific deletion of Trsp genesignificantly impaired chondrogenic programs causingabnormalities in bone and cartilage development in mice73endochondralossificationthusAntioxidant defense and redox homeostasisfunction ofattributed to theThe protective effects of selenium on cartilage are primarilyantioxidantdefense115“ The metabolism and survival of chondrogenic progenitors and chondrocytes are greatly compromised by ROS including free radicals peroxides andsuperoxide anions118“ Recent studies strongly supportthe notion that mitochondrial dysfunction and oxidativestress are the main drivers of OA pathogenesis37Although ROS play essential roles in the maintenance ofbasal cellular activities such as chondrocyte proliferationand matrix remodeling in cartilage excessive oxidativestress causes detrimental events such as cellular senescence36121 dedifferentiation122 and apoptosis123 ROScause oxidative damage to various cellular componentsand disrupt the balance between ECM catabolism andanabolism119 ROS suppress mitochondrial oxidativephosphorylation and ATP production which are essentially required to sustain cartilage matrix synthesis124 Inaddition ROS induce matrix degeneration through theupregulation of matrixdegrading enzyme expressionwhile this effecttreatment123125 The detrimental effects of ROS on cartilagehomeostasis can be effectively alleviated by augmentingcellular antioxidant activity under stress conditions andseveral attempts have been made to treat OA by targetingthe regulators involved in oxidative stress production incartilage84“is abolished by antioxidantThe protective role of selenium metabolism is thoughtto be exerted through the neutralization of ROS viaantioxidant activities of selenoproteins including GPXsand TXNRDs Bone marrow stromal cells cultured inmedium supplemented with low selenite concentrationexhibited ROS accumulation along with the reducedexpression of GPXs TXNRDs and other seleniumindependentinmicronuclei generation which is an indication of chromosome damage126 Both GPX1 expression and activitywere substantially lower in mice fed a seleniumdeficientdiet than those in mice fed a normal dietleading todecreased trabecular number reduced femoral trabecularvolumetotal bone volume ratio and trabecular separation66 The rats exposed to a seleniumdeficient diet withT2 toxin showed increased lipid peroxidation level andoxidoreductaseenzymesresultingOfficial journal of the Korean Society for Biochemistry and Molecular Biologydecreased antioxidant GPX activity in their serum andcartilage127 A seleniumdeficient dietinduced theexpression of miR1385p which in turn suppressed theexpression of SELENOM that has antioxidant functionand caused mitochondrial dysfunction and apoptosis ofchondrocytes128 Lead Pbinduced oxidative stress andtoxicity reduced the expression of selenoprotein mRNAsand the effect was mitigated by selenium supplementation129 In summary the antioxidant properties of selenoproteins showed therapeutic potential by counteractingthe accumulation of damage induced by oxidative stress incartilageDNA damage repairIt is well known that DNA damage pathways play substantial roles in the progression of arthropathies119 Theexpression of genes related to DNA damage was changedin the cartilage of KBD patients130131 Chronic DNAdamage induces the initiation of apoptosis or cellularsenescence in chondrocytes36132133 Selenium has apotential to reduce DNA damage and increase DNArepair capacity134 In part the beneficial effect of seleniumon genomic stability is associated with the antioxidationeffect of selenoproteins such as GPXs and TXNRDswhich remove ROS before they cause DNA damage134Cancer cells supplemented with selenium nM sodiumselenite or μM SeMet showed elevated levels of GPX1and TXNRD1 enzyme activity effectively protectingagainst DNA strand breaks induced by ultraviolet A orH2O2induced oxidative stress135 SeMet reduced theextent of DNA damage and enhanced DNA repair capacity by inducing repair complex formation in DNAdamaged cells through U

"annual meeting of the european associationfor the study of diabetes“ september sindex of oral presentationsop diabetes complications new insights from cutting edge epidemiologyop news on the insulin secretion frontop insulin sensitivity and biomarkersop central actions in diabetesop glucoselowering therapies and the liverop uncomplicating the pathogenesis of diabetes complications inhumansop smoke on the water is bat still hotop charting human beta cell failure in type diabetesop novel agents in type diabetesop developing better insulinsop from diagnostics to the endstage of diabetic kidney diseaseop nafld is it all about the liverop diabetic retinopathy see what's newop taking the long view of diabetesop pregnancy in diabetes prediction and outcomesop signals and networks in beta cell failureop broken heart in diabetesop unlocking the potential of digital healthop decoding the heritable basis of type diabetesop feeding the pipeline from drugs to surgeryop sglt2 inhibitors at the heart of the matterop new treatments for nafld hope or hypeop addressing potential new treatments of diabetic kidney diseaseop glucagon and hormones beyondop incretin based therapiesop unusual forms of diabetesop macrovascular complications and beyondop linking inflammation to metabolismop what's new in automated insulin deliveryop understanding the mechanisms of diabetic kidney diseaseop novel aspects of diabetic neuropathyop reducing the burden of hypoglycaemiaop what exercise doesop back to the future risk markers in diabetesop diet not only quantity mattersop on the road to human islet failure in type diabetesop a deep dive into the mechanisms of diabetesop triggers and drivers of beta cell failure in type diabetesop gastroentero pancreatic factors anoids mice and menop new aspects of novel therapiesop fatty mattersop diabetes care is expensiveop developing beta cellsop modelling metabolism lessons from animalsop diabetic foot new developments in wound healingop challenges in delivering diabetes care new solutionsop thinking about diabetes complications in the brainop insulin secretion in various subgroupsindex of poster sessionsps diabetes and early deathps living with chronic diabetes complicationsps micro and macrovascular complications of diabetesps global view on diabetes complicationsps type diabetes treatment irlps unusual forms of diabetesps molecular insights into glucose abnormalitiesps pathophysiology of glucose homeostasisps the inner workings of the pancreasps islets and antibodies in type diabetesps markers and phenotypes of glucose traitsps global aspects on the epidemiology of type diabetesps risk factors for type diabetesps prevalence of type diabetes around the worldps risk factors in type diabetesps islet transplants revisitedps islets in type diabetes new playersps beta cells under stressps to live and let die a beta cell perspectiveps job description insulin secretionps further down the road to human islet failure in type diabetesps sitting and exercising does it allps the ins and outs of carbohydrate metabolismps pregnancy in vitro and in vivo studiesps pregnancy epidemiologyps pregnancy who is at riskps incremental studies on gut hormonesps the fundamentals of insulin resistanceps studies on insulin resistanceps treatment of hyperglycaemia in pregnancyps pancreatic hormonesps insulin secretion in mice and menps something more about obesityps more about metabolismps inflammation in type diabetesps models of prediabetes and diabetesps models of obesity and insulin resistanceps lipid metabolismps adipokine signallingps drugs and environment in obesityps weight loss interventionsps brain mattersps sglt2 inhibitors clinical aspectsps different aspects of sglt2 inhibitorsps basic aspects of incretinbased therapiesps clinical outcome of incretinbased therapies 0cdiabetologiaps glycaemic control and incretinbased therapiesps various clinical aspects of incretinbased therapiesps various aspects of nutrition and dietps oral therapies metformin sensitizers and other nonsecretagoguesps novel agents to treat diabetes and its consequencesps novel glucoselowering agents in type diabetesps key issues in improving outcomes in people with diabeteseducation and costsps how to improve diabetes careps the impact of new basal insulinsps insulin therapy real world studiesps insulin therapy fast acting insulin analoguesps the challenges of insulin therapy in type diabetesps different aspects of insulin therapyps the continued advance of continuous glucose monitoringps insulin pump therapyps automated insulin deliveryps the varied use of technologies in type diabetesps novel applications of technology in diabetesps novel therapies to reduce hypoglycaemiaps mechanisms and clinical consequences of hypoglycaemiain diabetesps emerging topics in hypoglycaemiaps investigating diabetes distress and depressionps aspects of quality of life and well beingps digital health in type diabetesps is telehealth the answer to improving care in diabetesps predicting prognosis of diabetic kidney diseaseps clinical aspects of diabetic kidney diseaseps the rock and role of experimental kidney diseaseps new tools to view diabetic retinopathyps diabetic retinopathy screening and interventionps focus on diabetic foot ulcersps hypertension and vascular diseaseps cure the pain of diabetic neuropathyps understanding clinical neuropathyps from artificial intelligence to treatment of diabetic footps from biomarkers to genetics of diabetic kidney diseaseps treatment of nafld and diabetes from food to pharmacologyps mechanisms and prevalence of nafldps lipids everywhere lipid metabolism in the liver and the heartps all about coronary arteries and diabetesps lipids and glucose not so good for the heartps cardiac complications of mice rats and cellsps atherosclerotic complications stemming from cells to the kidneyps stiff arteries and how to avoid themps cardiac function and dysfunctionps cardiovascular complications in humans through and throughps diabetes and neoplasiaps contemplating cognitive dysfunction in diabetesps endothelial cell circulation and the heartps tradition no nontraditional complications of diabetes 0cdiabetologiaop diabetes complications new insightsfrom cutting edge epidemiologycirculating metabolites significantly improve the prediction of renaldysfunction in type diabetesm scarale1 s de cosmo1 c prehn2 f schena3 j adamski2 vtrischitta4 c menzaghi11fondazione irccs œcasa sollievo della sofferenza san giovannirotondo italy 2helmholtz zentrum m¼nchen germany 3universityof bari bari italy 4sapienza university roma italy and aims chronic kidney disease ckd mainly indicated by a reduced glomerular filtration rate gfr remains one of theleading causes of reduced lifespan in patients with type diabetest2d discovering novel biomarkers able to predict low gfr will helpidentify highrisk patients to be targeted to more aggressive and burdensome preventive and treatment strategiesmaterials and methods we measured serum metabolites byabsoluteidqtm p180 kit biocrates life sciences ag innsbruckaustria and investigated their association with egfr calculated with theckdepi formula in a discovery sample of patients with t2d cases and controls with egfr60 and ‰¥70mlmin173m2 respectively a threshold p value of 28x104 ie followingbonferroni's rule was used as statistical significance in a model comprising age sex smoking bmi hba1c diabetes duration albumintocreatinine ratio acr and ongoing treatments metabolites associatedin the discovery sample were validated threshold p value of 005numberof surviving validation metabolites in a second cohort comprising diabetic patients cases and controls for egfr60 or ‰¥70mlmin173m2 respectively standardized values of each validated metabolitesweighted for the effect size ie observed in the discovery samplewere then summed up in a metabolic score metscore to be used as agfr prediction tool to this purpose metscore was used on top of anestablished clinical model comprising sex age bmi hba1c and acrand then discrimination [δ area under the receiver operating characteristic roc curve auc and the relative integrated discriminationimprovement ridi] and reclassification [the categoryfree net reclassification index cnri] measures were evaluatedresults thirteen metabolites six acylcarnitines six biogenic amines andone amino acid were independently associated to low egfr [ors range for 1sd increase p range 13x107 20x104] in the discoverysample all of them but one a biogenic amine were validated in thereplication sample [ors range for 1sd increase p range32x1018 43x106 below the threshold of 0051242x103] theauc of the abovementioned clinical model was and in the discovery the replication and the pooled sample respectively the addition of metscore on top of the clinical model improvedboth discrimination and reclassification measures in the discovery δauc4 p14x103 ridi29 p20x1011 ½cnri54p15x1014 the replication δ auc39 p16x103 ridi28p38x108 ½cnri30 p22x1010 and the pooled samples δauc39 p40x106 ridi29 p22x1017 ½cnri35p19x108conclusion we have discovered and validated metabolites that arestrongly associated with low egfr in patients with t2d a metscorecomprising these metabolites improves an established clinical prediction model of low egfr in terms of both discrimination and reclassification encouraged by these findings we are now investigating the ability ofmetscore to improve prediction of gfr decline in prospective cohorts oft2d with the aim of improving risk stratification and therefore refiningprevention efforts of kidney dysfunction in diabetic patientssupported by italian ministry of health rf201302356459disclosure m scarale noneassociation between insulinlike growth factor binding protein2 andinsulin sensitivity metformin and mortality in patients with newlydiagnosed type diabetesmr kristiansen12 js nielsen12 i brandslund3 da olsen3 jvstidsen2 sk nicolaisen4 r hjortebjerg25 j frystyk561danish centre for strategic research in type diabetes dd2odense 2steno diabetes center odense odense 3irs lillebaelthospital biochemistry and immunology vejle 4department ofclinical epidemiology aarhus 5department of clinical researchuniversity of southern denmark odense 6department ofendocrinology odense university hospital odense denmark and aims insulinlike growth factor binding protein2igfbp2 is engaged in metabolism circulating concentrations ofigfbp2 are positively correlated to insulin sensitivity overexpressionof igfbp2 protects against obesity and diabetes in mice and metforminincreases igfbp2 gene expression indicating that igfbp2 is a target ofmetformin action interestingly igfbp2 appears to predict mortalityindependently of insulin sensitivity this study aimed to investigate theassociation between indices of insulin sensitivity metformin treatmentand mortality in patients with newly diagnosed type diabetes t2dmaterials and methods in this crosssectional study we included newlydiagnosed patients with t2d enrolled in the danish centre for strategicresearch in type diabetes dd2 cohort patients were continuouslyenrolled from to throughout denmark and followed usingdanish healthcare registries unbound fractions of igfbp2 were determinedin serum from fasting drug na¯ve n864 and metformin treated ‰¥ twoprescriptions months prior enrollment patients n558 using an inhouseassay developed on the simoa platform values are given as medians iqrassociation was analyzed using a pearson™s regressioncox regression amultivariable model was used to adjust for age bmi and homasresults a total of patients with median age of medianbmi of and median diabetes duration of yearswere included igfbp2 level was positively correlated with homasr2026 and p0005 and inversely correlated with cpeptide r2018and p0005 both associations persisted following adjustments for ageand bmi the igfbp2 level in metformin treated patients was slightlylower ngml than in drug na¯ve patients ngml p0026 a total of patients suffered from one or morecomorbidities from charlson comorbidity index their igfbp2 levelswere higher than patients with no comorbidity vs ngml p0001 during a median of years offollowup a total of patients died igfbp2 level was significantly higher at baseline in patients that died vs not died vs ngml p0001 igfbp2 was associated withmortality with a hazard ratiohr ci per doubling in proteinconcentration of p0001 this association was notobserved when analyzing patients without comorbidities but was significant in patients with other comorbidities hr p0001conclusion this is the first larger study to confirm that igfbp2 isassociated with indices of insulin sensitivity but is not largely affectedby metformin treatment interestingly increased igfbp2 level is associated with high mortality rates but the association was mainly driven bythe presence of comorbidities at baselinesupported by university of southern denmark and region of southerndenmarkdisclosure mr kristiansen nonebuilding clinical risk score systems for predicting allcause andcardiovascularspecific mortality among type diabetes patientscs liu1 tc li2 cc lin1 ci li11china medical university hospital taichung 2china medicaluniversity taichung taiwan 0c and aims no prior prediction model for mortality considered the effect of glycemic variability and blood pressure variabilitywhich have been broadly reported as the important clinical predictors ofmortality especially in diabetes patients the aim of this study was todevelop and validate risk score systems with considering the effects ofglycemic and blood pressure variability on allcause and cardiovascularspecific mortality in persons with type dmmaterials and methods this is a retrospective cohort study consistingof type diabetic patients aged years during allparticipants were randomly allocated into two groups derivation andvalidation sets in ratio and were followed up until death or august cox proportional hazards regression were used to develop allcauseand cardiovascularspecific mortality prediction model prediction modelperformance was assessed by the area under the receiver operating characteristics curve aurocresults overall deaths were identified after a mean of years offollowup the prediction accuracy measured by auroc of and 15year allcause mortality based on a model containing the identifiedtraditional risk factor biomarkers and variability in fasting plasmaglucose and hba1c and diastolic blood pressure variability were and respectively in derivation set and the corresponding values forcardiovascularspecific mortality were and respectively the predictionaccuracy in the validation set for allcause mortality were and respectively and for cardiovascularspecific mortality were and respectivelyconclusion our prediction model considering glycemic and blood pressure variability had good accuracy of prediction of cardiovascularspecific and allcause mortality in patients with type diabetessupported by ministry of science and technology of taiwandisclosure c liu noneincident cardiovascular disease by clustering of favourable riskfactors in type diabetes the eurodiab prospectivecomplications studys soulimane1 yd vogtschmidt12 m toeller3 b balkau4 nchaturvedi5 jh fuller6 ss soedamahmuthu121department of medical and clinical psychology center of research onpsychological and somatic disorders corps tilburg universitynetherlands 2institute for food nutrition and health university ofreading reading uk 3heinrichheineuniversity d¼sseldorfd¼sseldorf germany 4clinical epidemiology universit© parissaclayuvsq inserm cesp villejuif france 5institute of cardiovascularscience university college of london london uk 6department ofepidemiology and public health eurodiab london uk and aims the incidence of cardiovascular diseases cvdis up to eight times higher in people with type diabetes t1d greaterclustering of adverse risk factors is thought to contribute to excess cvdrisks in type diabetes though not explored in t1d the aim of this studywas to examine a cvd risk reduction for those in the most favourablethird of individual risk factors compared to the least favourable two thirdsand b cvd risk reduction by clustering of favourable cvd risk factorsmaterials and methods we analysed data of participants from theeurodiab prospective complications study a european t1d cohortrecruited in countries between were men with a meanage of ± years we studied seven cvd risk factors namely hba1csmoking bmi combined systolic and diastolic bp ldl cholesterolphysical activity pa and diet table cox proportional hazards analyses were used to calculate hazard ratios hr [95ci] of incident cvdfor each cvd risk factor adjusted for age sex retinopathy comparingthose in the most favourable tertiles with the least favourable two tertilesdiabetologiawe then scored each individual by the number of risk factors for whichthey occupied the most favourable tertilesresults there were incident cvd cases after a mean followup of± years multivariable cox models showed that participants withthe most favourable hba1c57 [39mmolmol] had a significantlylower cvd risk hr [95ci] [] than the least favourabletwo tertiles nonsignificant inverse associations were found withfavourable bmi [] pa [] diet score[] and bp [] no associations were foundwith smoking or ldlcholesterol greater clustering of favourablecvd risk factors was associated with a lower risk of cvd in univariatemodels with a significant linear trend in multivariate models the resultswere partly attenuated with the lowest hr of [ ] in peoplewith clustering of favourable cvd risk factors tableconclusion greater clustering of favourable cvd risk factors was associated with a lower risk of incident cvd in people with t1d with a doseresponse relationship hba1c remained the most protective factor againstcvd in t1d targeting combined risk factors could be more effective inpreventing cvd risk than targeting single risk factorssupported by welcome trust the european community and diabetesukdisclosure s soulimane nonebidirectional association between type diabetes and obstructivesleep apnoea a metaepidemiological studyt karagiannis1 e athanasiadou1 a tsapas12 e bekiari11clinical research and evidencebased medicine unit aristotleuniversity of thessaloniki thessaloniki greece 2harris manchestercollege university of oxford oxford uk and aims individual epidemiological studies suggest acomplex relationship between type diabetes and obstructive sleepapnea we aimed to assess whether there is a bidirectional associationbetween the two conditions by conducting a metaanalysis of longitudinalcohort studiesmaterials and methods we included cohort studies that evaluated theassociation between type diabetes and obstructive sleep apnea in eitherdirection published until january we pooled cohortspecific estimates by means of random and fixed effects metaanalyses and calculatedodds ratios ors with confidence intervals cis to measure theassociation of prevalent obstructive sleep apnea with incident type diabetes and of prevalent type diabetes with incident obstructive sleepapnearesults out of records identified through the search cohortstudies were included in the metaepidemiological analysis ten studiesevaluated the association between prevalent obstructive sleep apnea andincident type diabetes one study assessed the association betweenprevalent type diabetes and incident obstructive sleep apnea while fourstudies evaluated a bidirectional association duration of study followupranged between and years median years the random effectsmetaanalysis for prevalent obstructive sleep apnea and incident type diabetes patients yielded an or of ci to 0cdiabetologiaresults were consistent in the fixed effects metaanalysis figureprevalent type diabetes increased the odds of incident obstructive sleepapnea patients with an or of ci to and ci to for the randomeffects and fixedeffects metaanalysis respectively metaanalyses of effect estimates adjusted forconfounding factors were similar to those of the main analysisconclusion pooled evidence from large cohort studies suggests thatpresence of obstructive sleep apnea at baseline is associated withincreased risk for developing type diabetes while presence of type diabetes is associated with increased risk for developing obstructive sleepapnea thus effective management of either condition could preventdevelopment of the otherfigure odds ratio for developing type diabetes in patients with obstructive sleep apnea versus those without obstructive sleep apneaalzheimer hr [ ic ] vascular dementia hr [ ic ] and nonvascular dementia hr [ ic ] when a 3years landmark analysis was conducted the associations remained similar for vascular and nonvascular dementia but disappeared for alzheimer™s diseasesconclusion the association of t2d with neurodegenerative diseasesdiffer by type of dementia the strongest detrimental association wasobserved for vascular dementia moreover t2d patients with polycaemic control have an increased risk of developing vascular andnonvascular dementiadisclosure c celismorales nonesupported by greece and the european social fund esfdisclosure t karagiannis noneglycated haemoglobin type diabetes and the links to dementia andits major sub types findings from the swedish national diabetesregisterc celismorales1 s franz©n2 am svensson3 n sattar1 sgudbjornsdottir21institute of cardiovascular and medical sciences university ofglasgow glasgow uk 2department of molecular and clinicalmedicine university of gothenburg gothenburg sweden 3swedishnational diabetes register gothenburg sweden and aims type diabetes t2d has been associated withhigh dementia risk however the links to different dementia subtypes isunclear we examined to what extent t2d associated with alzheimervascular and non vascular dementia incidence and whether such associations differed by glycaemic controlmaterials and methods in this swedish national diabetes registerstudy we included patients with t2d and matchedcontrols the outcomes were incidence of alzheimer vascular and nonvascular dementia the association of t2d with dementia was stratifiedby baseline glycated haemoglobin hba1c concentrations cox regression was used to study the excess risk of outcomesresults the followup median years t2d patientsand controls developed dementia the strongest association was observed for vascular dementia here patients with t2d had ahr of [ ci ] compared to controls the association oft2d with nonvascular dementia was more modest hr [ ci ] however risk of alzheimer was lower in t2d patientscompared to controls hr [ ci ] when the analyseswere stratified by circulating concentrations of hba1c a doseresponseassociation was observed compare to patients with t2d with hba1c mmolmol those with hba1c mmolmol had a higher risk of 0cop news on the insulin secretion frontwhat makes beta cells 1st responders and are they temporallyconsistentv kravets we schleicher jm dwulet am davis rkpbenningerbioengineering university of colorado aurora usa and aims calcium ca2 uptake drives glucosestimulated insulin secretion from the pancreatic cells functionalsubpopulations of cells disproportionally control the oscillatory phaseof ca2 uptake which is disrupted with ageing and in diabetes less isknown about cells which impact the 1st phase of ca2 uptake disruptedin early diabetes here we determine whether œ1stresponder cells thatlead the 1st phase of ca2 uptake are the same as œhub cells that coordinate oscillatory ca2 2nd phase we study what makes cell a1st responder and whether 1st responders are a transient state or a distincttemporally stable subpopulationmaterials and methods we used mipcreer gcamp6s mouse modelwhich expresses ca2sensitive gfp specifically in cells weperformed simultaneous recording of ca2 dynamics and gap junctionpermeability in individual islets we stimulated islets with glucosekatp channel blocker glibenclamide and kcl based on ca2 dynamicswe defined the of cells responding to the glucose stimulation soonerthan the rest of the islet as œ1st responders and the of cellsresponding slower as œlast responders we tested their temporal consistency over and hours we used laser ablation to remove specificcells from the islet we performed computational modelling of the isletelectrophysiologyresults we found that ca2 wave coordination of the 1st responders wasnot greater than the isletaverage and hence they are not overlapping withhighlycoordinated œhub cells in fact according to our gap junctionpermeability data 1st responders had lower than average electricalcoupling p00157 furthermore our computational model showedlower electrical coupling conductance in both 1st and last respondersp00447 p00279 this may be explained by our finding that1st responders are located at the islet™s periphery at ± of the islet™sradius we found 1st responders to be consistent under glibenclamidestimulation cells which respond first to the glucose remained in the15th percentile of the time response distribution when stimulated withglibenclamide sem this is consistent with our computationalresults 1st responders had lower katp conductance hence highermembrane depolarization probability p00086 glucose elevationswith 1h period showed that 1st responders remained consistent withreaction time within the of the reaction time distribution for all cellswith an elevation period of hours their reaction time shifted to thesecond quartile of the distribution and with hours to the medianunlike 1st responders last responder cells were not consistent at any timeinterval ablation of the 1st responders discoordinated but did notdisrupt the ca2 response of the islet a different cell took over the roleof the 1st responder postablation this new 1st responder was a cell whichoriginally preablation was within a leading 7th percentile of the timeresponse distribution sem conclusion in conclusion 1st responders are distinct from œhub cellsubpopulation have higher membrane depolarization probability and areless strongly coupled to other cells after the laser ablation of a1st responder new 1st responder taking on it™s role comes from a poolof original leading cells while initially consistent over a short 1h periodof time 1st responders may be losing temporal consistency over longertime periodssupported by nr01 dk102950 dk106412 jdrf 3pdf2019741andisclosure v kravets nonediabetologiabetaarrestin is absolutely required for the potentiation of insulinsecretion by gipma ravier1 j obeid1 m leduc1 s costes1 p gilon2 s dalle1 gbertrand11igf univ montpellier cnrs inserm montpellierfrance 2universit© catholique de louvain brussels belgium and aims the scaffold protein betaarrestin2 arrb2 isknown to uncouple g protein coupled receptors gpcrs from the gprotein and to recruit new signaling pathways such as the erk12pi3k fak‹¯ in non beta cells arrb2 interacts with a wide rangeof gpcrs but its interaction with the gip receptor gipr is still unclearour aim is to determine if arrb2 is involved in the signaling of thegipr in pancreatic beta cellsmaterials and methods the experiments were carried out in beta cellsfrom fivemonthold arrb2 and arrb2 male mice camp productioncampsepac endogenous pka akar3 and erk12 ekaractivations [ca2] in the cytosol [ca2]c fura2lr and in the endoplasmic reticulum [ca2]er d4er were assessed by live cell imagingin mouse pancreatic beta cells epac2 epac2gfp recruitmentbeneath the plasma membrane was monitored by total internal reflectionfluorescence microscopy factin depolymerisation was evaluated byphalloidin staining alexa fluor 488conjugated phalloidin and thep h o s p h o r y l a t i o n o f f o c a l a d h e s i o n k i n a s e f a k b yimmunofluorescenceresults insulin secretion from arrb2 islets was reduced by compared to arrb2 islets in response to gip 100pm10nm p001when arrb2 arrb2gfp was reexpressed in arrb2 beta cells insulin secretion in response to gip was restored to a similar level thanin arrb2 islets surprisingly upon gip stimulation 10pm10nm thecamp production pka activation and epac2 recruitment were similarin arrb2and arrb2 beta cells both [ca2]c and [ca2]er remainedcomparable finally the activation of erk12 was also similarin arrb2 and arrb2 beta cells by contrast the factin depolymerisationinduced by 10nm gip was significantly reduced p001 in arrb2 beta cells pi3kγ and fak have been reported to be involved in factindepolymerisation in response to gip and glucose respectively and to berequired for optimal insulin secretion as expected the pi3kγ inhibitoras604850 1μmoll reduced factin depolymerisation p001by gip stimulation in arrb2 beta cells but no additional effect wasobserved in arrb2 beta cells moreover gipinduced fak activationwas also reduced by in arrb2 beta cellsconclusion our study revealed that arrb2 is required for the potentiation of insulin secretion by gip through factin depolymerisation probably via fak activation and pi3kγ recruitment but independently fromthe canonical camp signalling pka and epac2 and the erk12 pathway therefore any variation in the expression of arrb2 as observed indiabetic states should functionally affect the incretin effect produced bygipsupported by soci©t© francophone du diabete sfddisclosure ma ravier nonepancreatic beta cellselective deletion of the mitofusins and mfn1and mfn2 impairs glucosestimulated insulin secretion in vitro andin vivoga rutter1 e geiadou1 t rodriguez2 c muralidharan3 mmartinez3 p chabosseau1 a tomas1 g carrat1 a di gregorio2 ileclerc1 ak linnemann31cell biology functional genomics faculty of medicine imperialcollege london london uk 2national heart and lung instituteimperial college london london uk 3center for diabetes andmetabolic diseases indiana university school of medicineindianapolis usa 0cdiabetologia and aims mitochondrial metabolism of glucose is essential for the initiation of insulin release from pancreatic beta cellsalthough altered in subjects with type diabetes whether mitochondrialultrastructure and the proteins controlling the fission and fusion of theseanelles are important for glucose recognition is unclear here wegenerated mice with beta cellselective adultrestricted deletionof mfn1 and mfn2 essential for mitochondrial fusion and studied theimpact on insulin secretion and glucose homeostasis in vivo and in vitromaterials and methods c57bl6 mice bearing mfn1 and mfn2 alleleswith floxp sites were crossed to transgenic animals carrying aninducible cre recombinase under pdx1 promoter control pdxcreertrecombination was achieved by daily tamoxifen injections for one weekislets were isolated and used for live beta cell fluorescence imaging ofcytosolic cal520 or mitochondrial rgeco free ca2 concentrationand membrane potential tetramethyl rhodamine methyl ester tmrmusing spinning disc confocal microscopy nikon ti2 mitochondrialnetwork characteristics were quantified using super resolution fluorescence zeiss lsm and transmission electron microscopy intravitalimaging was performed in mice injected with an adenoassociated virusto express the cytosolic ca2 sensor gcamp6s selectively in beta cellsunder the control of the rat insulin promoter using multiphoton microscopy leica tcs sp8 dive blood flow through the islet was visualisedsimultaneously after injection of fluorescent albumin647results mitochondrial length was sharply to ± of controlsp00001 reduced in the mfn12 ko mice and these animals displayedhigher fasting glycaemia than control littermates at weeks vs mmoll p005 in vivo an increase in circulating glucose levelswas also observed p005 at min and p001 at min and wasassociated with a substantial fivefold decrease in plasma insulin min p00001 postintraperitoneal glucose injection mitochondrialca2 accumulation and membrane potential were significantly reducedp001 in response to high glucose in the ko animals examined byintravital imaging of the exteriorised pancreas antiparallel changes incytosolic ca2 and mitochondrial membrane potential observed incontrol animals were largely suppressed after mfn12 deletionconclusion mitochondrial fusion and fission cycles are essential in thebeta cell to maintain normal mitochondrial bioenergetics and glucosesensing both in vitro and in the living mouse such cycles may bedisrupted in some forms of diabetes to impair mitochondrial functionand consequently insulin secretio

This study was performed assess the clinical outcomes of elderly patients withosteoporotic femoral neck fractures FNFs AOOTA 31BC treated by initial uncementedtotal hip arthroplasty UTA or cemented total hip arthroplasty CTAMethods This study involved consecutive elderly patients with osteoporotic FNFs AOOTA31BC treated by initial UTA or CTA in our medical centre from to The primaryoutcomes were the Harris hip score HHS and the rates of revision loosening periprostheticfracture and dislocationResults In total patients were included in the final analysis UTA n¼ CTA n¼ The mean followup duration was months range “ months The mean HHS was1Department of Microsurgery Trauma and Hand SurgeryThe First Affiliated Hospital Sun Yatsen UniversityGuangzhou China2Department of Pediatrics The First Affiliated HospitalSun Yatsen University Guangzhou China3Department of Orthopaedics The First AffiliatedHospital Sun Yatsen University Guangzhou China4Department of Orthopaedics The Third People™sHospital of Wuxi Jiangsu Province The Affiliated Hospitalof Jiangnan University Wuxi China5Department of Urology The First Affiliated Hospital SunYatsen University Guangzhou ChinaThese authors contributed equally to this workCorresponding authorsJunxing Ye Department of Orthopeadics The ThirdPeople™s Hospital of Wuxi Jiangsu Province The AffiliatedHospital of Jiangnan University No Xingyuan NorthRoad Liangxi District Wuxi Jiangsu ChinaEmail yejunxing0514163comJintao Zhuang Department of Urology The First AffiliatedHospital Sun Yatsen University No Zhongshan 2ndRoad Yuexiu District Guangzhou ChinaEmail brianzg86163comCreative Commons Non Commercial CC BYNC This is distributed under the terms of the CreativeCommons AttributionNonCommercial License creativecommonslicensesbync40 which permitsnoncommercial use reproduction and distribution of the work without further permission provided the original work is attributedas specified on the SAGE and Access pages ussagepubcomenusnam accessatsage 0cJournal of International Medical Research 06 for UTA and 06 for CTA Significant dissimilarities were detected in therates of revision loosening and periprosthetic fracture between UTA and CTA vs vs and vs respectively A significant difference was also detected inthe probability of revision between the two groupsConclusion Elderly patients with osteoporotic FNFs AOOTA 31BC treated with CTA showgreater improvements in functional outcomes and key orthopaedic complications than thosetreated with UTAKeywordsFemoral neck fracture arthroplasty outcome complication retrospective osteoporosisDate received December accepted July Introduction31BCAOOTAfemoral neck fracturesManagement ofin elderlyFNFspatients is still undergoing considerableresearch12 Uncemented total hip arthroplasty UTA or cemented total hip arthroplasty CTA for displaced FNFs tendsto be a recognised surgical strategy3“Comparisons between UTA and CTA forelderly individuals with an FNF generallyfavour CTA this is primarily attributed tothe exceptional clinical outcomes of CTA interms of relieving pain and improving dailyactivities as well as the higher rate of majororthopaedic complications ie revisionloosening periprosthetic fracture and dislocation associated with UTA4 Howeverrecent studies of UTA in elderly individualshave demonstrated encouraging shorttermclinical outcomes67 Moreover cementedprosthesis syndrome tends to occur morefrequently in CTA than UTA8 Cementedprosthesis syndrome theoretically poses asignificantlifealthough the specific probability of thisthreat has not been calculated89 Hencewhether to utilise CTA for elderly individuals may present the clinician with a dilemma9 The lack of consensus regarding whichtechnique UTA or CTA is preferable fortreating FNFs AOOTA 31BC in elderlyindividuals is related to the remarkableto the patient™sthreatdistinction in clinical outcomes betweenthe two types of implants610Most previousconcentrated medicalstudies have involvedhighlycentresand several brands of prostheses356Additionally shortterm followup is commonplace in these studies To overcomethese drawbacks of previous studies and tocompare the midterm results of the twoprostheses we assessed the clinical outcomesof elderly patients with osteoporotic FNFsAOOTA 31BC treated with initial UTAor CTA with a mean followup of yearsMaterials and methodsStudy populationtheandrequirementEthical approval was obtained from theFirst Affiliated Hospital of Sun YatsenUniversityforinformed consent was waived by theInvestigational Review Board Consecutiveelderly individuals with the principle diagnosis of an FNF AOOTA 31BC whounderwentinitial UTA or CTA from March to March and forwhom detailed information was availablethroughoutidentifiedfrom the orthopaedics department of theFirst Affiliated Hospital Sun YatsenUniversity The manufacturer details offollowup were 0cMao et alTable Manufacturer details of stems and cupsemployed in the arthroplasty proceduresProcedureStemCupCORAIL1Exeter3Exeter3REFLECTIONUncemented2UTA n¼ CTA n¼ 1DePuy Synthes Warsaw IN USA2Smith Nephew London UK3Stryker Corporation Kalamazoo MI USAUTA uncemented total hip arthroplasty CTA cementedtotal hip arthroplastyclosed FNFsthe stems and cups employed in the arthroplasty are shown in Table The surgicalprocedure and postoperative rehabilitationprotocol were described in our previouslypublished study11 The inclusion criteriawereAOOTA 31BCactive and cognitively intact patients ageof 15 years independently mobile priorto the injury and a bone mineral densityTscore of at the femoral neck Themajor exclusion criteria were multiple fractures or contralateral limb fractures pathological FNFs lower limb dyskinesia priorto surgery cancer planned surgery polytrauma severe comorbidities eg thyroiddisorder with calcium and phosphorusmetabolism disorderwithcomplications drug abuse affecting bonehealing or bone metabolism early interruption of followup months and cognitive impairment Clinical and radiographicassessments were performed at and months after surgery and every monthsthereafter The primary outcomes were theHarris hip score HHS and the rates ofrevision loosening periprosthetic fractureand dislocationdiabetesStatistical analysisRevision was defined as partial or completereplacement of the prosthesis12 Looseningof the acetabulum andor stem componentstomographycompared usingas well as dislocation were defined based ona previous description13 Periprostheticconfirmed by Xray orfracture wascomputedexaminationContinuous data ie age bone mineraldensity body mass index followup timeand HHS wereanindependentsamples t test and categoricalvariables ie sex side [leftright] fracturetype comorbidities mechanism of injuryAmerican Society of Anesthesiologists classification and major orthopaedic complications were compared using the chisquaretest or the Mann“Whitney test A Kaplan“Meier survival curve was used to assess theprobability of revision Hazard ratios werecalculated using a Cox proportional hazards model The significance threshold wasset at p The statistical analysis wasperformed using SPSS IBM CorpArmonk NY USAResultsIn total consecutive patientsarthroplasties with an FNF AOOTA31BC who underwentinitial UTA orCTA met our inclusion criteria and wereincluded for analysis Figure The meanfollowup duration was months range“ months The patients™ mean age was 06 years for UTA and 06 for CTA The mean body mass indexfor UTA andwas 06 kgm2 for CTA The patients™baselinesimilarbetween the two groups Table 06 kgm2characteristics werePrimary outcomesImproved functional outcomes were notedin both groups as indicated by the HHSUTA 06 prior to surgery vs 06 at final analysis p CTA 06 prior to surgery vs 06 at final analysis p At the end of followup the HHS was 0cJournal of International Medical ResearchFigure Flow diagram exhibiting methods for identifying patients with FNFs AOOTA 31BC whounderwent an initial UTA or CTAFNFs femoral neck fractures UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysignificantly different between the twoUTA 06 vs CTAgroups 06 p¼ and patients whounderwent CTA had higherfunctionalscores than those who underwent UTANo distinct betweengroup differenceswere observed at any time point before months postoperatively Table No early year postoperative complications were detectedincluding revisionloosening periprosthetic fracture or dislocation The rate of key orthopaedic complications wasfor UTAand for CTA p Table In the UTA group patients underwent revision UTA developed prosthesis loosening developed periprosthetic fracturesand developed prosthesis dislocation In the CTA group patientsunderwent revision UTA developed prosthesis loosening developed periprosthetic fractures and developed prosthesis dislocation The average time interval from the initial surgery torevision UTA was months range “months for UTA and months rangefor CTA p¼ “ monthsSignificant differences in revision looseningand periprosthetic fracture were observedbetween the UTA and CTA groups revision vs p¼ loosening 0cMao et alTable Patient demographics and outcomesVariableSex femalemaleAge yearsBMI kgm2BMDSide leftrightFNFs AOOTA type31B31CComorbiditiesDiabetes mellitusHypertensionCerebrovascular diseaseMechanism of FNFsTraffic accidentFallingTamping accidentASA classificationUTA n¼ 06 06 06 06 HHS prior to surgeryData are presented as n n or mean 06 standard deviationpvalueCTA n¼ 06 06 06 06 UTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty HHS Harris hip score ASA AmericanSociety of Anesthesiologists BMI body mass index BMD bone mineral density FNFs femoral neck fracturesTable Comparison of hip functional scoresMonths postoperativelyFinal followupData are presented as mean 06 standard deviationUTA n¼ 06 06 06 06 06 06 06 06 CTA n¼ 06 06 06 06 06 06 06 06 pvalueStatistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplasty vs p¼ and periprosthetic fracture vs p¼ respectively A significant difference in theprobability of revision was also detectedbetween the groups hazard ratio interval confidence“p¼ Figure No significant difference was found in the rate of prosthesis dislocation between the UTA and CTA groups vs respectively 0cJournal of International Medical ResearchTable Rates of key orthopaedic complicationsComplicationsProsthesis revisionProsthesis looseningPeriprosthetic fractureDislocationUTA n¼ CTA n¼ pvalueData are presented as n Statistically significantUTA uncemented total hip arthroplasty CTA cemented total hip arthroplastyFigure Kaplan“Meier curves showing probability of revision after primary surgery HR was calculatedper the Cox proportional hazards model with age sex American Society of Anesthesiologists classificationbody mass index bone mineral density and femoral neck fracture type as covariates and surgery as the timedependent factorHR hazard ratio CI confidence interval UTA uncemented total hip arthroplasty CTA cemented total hiparthroplastysurgeryProbableDiscussionThis review characterised the outcomes of asolitary brand of a total hip arthroplastyimplant during a mean followup of years in elderly patients with osteoporoticFNFs AOOTA 31BC The data demonstrated that patients treated with CTAshowed better improvements in functionaloutcomes and key orthopaedic complications than those treated with UTAThe current findings are consistent withprevious studies361415 Although the betterfunctional outcomes and lower rates ofrevision loosening periprosthetic fractureand dislocation are apt to favour CTA nosignificant betweengroup differences in theHHS were detected during the initial yearsafterexplanationsinclude the timedependent clinical efficacyof the implants and the properties of theprostheses34616 Whether UTA or CTA ispreferable in elderly patients with a discontroversial6917placed FNF remainsA recent retrospective study involving patients with an FNF AOOTA 31B whounderwent primary unilateral UTA or CTAshowed that the mean HHS was 06 for CTA and 06 for UTAp¼ A singleblinded randomisedcontrolled trial CHANCEtrial involving individuals treated with an uncementedor cemented tapered hydroxyapatitecoatedfemoral stem and a cemented cup demonstratedtaperedhydroxyapatitecoated femoral stem andcementedthatthe 0cMao et alcemented cup provided better functionalresultsthan the uncemented taperedhydroxyapatitecoated femoral stem andcemented cup16ratesrevisionthe bone microstructureIn the present study the Kaplan“Meiersurvival curve demonstrated that at the2year analysis neither group showed evidence of a target event and no significantbetweengroup differences were found inofthelooseningfracture or dislocationperiprostheticNeverthelessit would be interesting toexplore whether the prosthesis materialinfluencesthepeak effect and the duration of the effectand if so what mechanisms affect the bonemicrostructure and whether there is a wayto change the outcome by blocking thiseffect during a 2year followup We currently have one option for prevention oravoidance of adverse events and thesechanges in treatment strategies may play akey role in improving the clinical resultsif the effects of the prosthesis materialitself cannot be blocked18 There is still alack of consensus on standards for prosthesis revision in this context19When assessing the impact of CTA onthe target events we did not observe anincreased incidence of severe orthopaediccomplications other than the complicationsmentioned in this study In one systematicreview the authors presumed that CTA wassuperior to UTA with respect to functionalscores and tolerable orthopaedic complications20 We obtained analogous results interms of hiprelated complications andfunctional scores Multicentre hip arthroplasty data indicate that UTA remains ahighrisk factor for late revision looseningand periprosthetic fractures810 The notabledissimilarities in the results of these variousstudies on hiprelated complications may belargely attributed to the design of the prosthesis prosthesis size and material selection and the surgeon™s experience46affected theThis study has several limitations It hada small sample and its retrospective designis association with some inherent disadvantages We did not stratify the patientsaccording to fracture type or sex In addition the potential comorbidities betweengroups were not well exposed and compared The statistical power used to addressdifferences between the groups was insufficient Differences in the patients™ baselinedata may haveresultsFurthermore our analysis did not determine whether the deaths were instigatedby bone cement The risk of hiprelatedcomplications was not analysed The survivalcurve of other prosthesisrelatedcomplications was estimated using theKaplan“Meier method and competitiverisks ie death could have affected thesurvival of the prosthesis Patients whodied lostrevisionHence the revision rate might have beenunderestimated during this long followupwith a fairly high mortality ratethe opportunity forevidenceIn conclusion the findings described inthe current review uphold an increasingbody ofthat CTA provideshigher functional scores and lower rates ofhiprelated complications than does UTAin elderly patients with osteoporotic displaced FNFs AOOTA 31BC In thiscontext we recommend CTA for the treatment of such FNFs Our findings may beconducive to alleviating continuing debateregarding which prosthesis UTA or CTAis more suitable for the elderly populationA future prospective study may be essentialto confirm whether our conclusion continues to be acceptable as the followup timeincreasesDeclaration of conflicting interestThe authors declare that there is no conflict ofinterest 0cFundingThis research received no specific grant from anyfunding agency in the public commercial ornotforprofit sectorsORCID iDsWeiguang YuGuowei HanReferencesorcid00000001orcid00000003 Chammout G Muren O Laurencikas Eet al More complications with uncementedthan cemented femoral stems in total hipreplacement for displaced femoral neck fractures in the elderly a singleblinded randomized controlled trial with patientsActa Orthop “ Gjertsen JE Lie SA Fevang JM et al Totalhip replacement after femoral neck fracturesin elderly patients results of fracturesreported to the Norwegian ArthroplastyRegister Acta Orthop “ Hailer NP Garellick G and Karrholm JUncemented and cemented primary totalhip arthroplastyin the Swedish HipArthroplasty Registerof operations Acta Orthop “evaluation Makela KT Eskelinen A Paavolainen Pet al Cementless total hip arthroplasty forprimary osteoarthritis in patients aged years and older results ofthe mostcommon cementless designs compared tocemented reference implants in the FinnishArthroplasty Register Acta Orthop “ YliKyyny T Sund R Heinanen M et alCemented or uncemented hemiarthroplastyfor the treatment of femoral neck fracturesActa Orthop “ Yang C Han XL Wang J et al Cementedversus uncemented femoral component totalhip arthroplasty in elderly patients with primary osteoporosisretrospective analysiswith 5year followup J Int Med Res “Journal of International Medical Research Solarino G Zagra L Piazzolla A et alceramiconResults of consecutiveceramic cementless hip arthroplastiesinpatients up to years of age a yearsof followup study J Arthroplasty S232“S237 Hanly RJ Whitehouse SL Lorimer MFet al The outcome of cemented acetabularcomponents in total hip arthroplasty forosteoarthritis defines a proficiency thresholdresults of cases from the AustralianOrthopaedic Association NationalJointReplacement Registry J Arthroplasty “ Imam MA Shehata MSA Elsehili A et alContemporary cemented versus uncementedhemiarthroplasty for the treatment of displaced intracapsular hip fractures a metaanalysis offortytwo thousand fortysixhips Int Orthop “ Jameson SS Baker PN Mason J et al Thedesign of the acetabular component and sizeof the femoral head influence the risk of revision following singlebrand cementedhip replacements a retrospective cohortstudy of mediumterm data from a nationaljoint registry J Bone Joint Surg Br 94B “ Zeng XS Zhan K Zhang LL et alConversion to total hip arthroplasty afterfailed proximal femoral nail antirotationsor dynamic hip screw fixations for stableintertrochanteric femur fractures a retrospective study with a minimum followupof years BMC Musculoskelet Disord Johnson RL Abdel MP Frank RD et alImpact of frailty on outcomes after primaryandarthroplastyJ Arthroplasty “64e5revisiontotalhip Chen KH Tsai SW Wu PK et al Partialcomponentretained twostage reconstruction for chronic infection after uncementedtotal hip arthroplasty results of sixteen casesafter five years of followup Int Orthop “ DeAngelis JP Ademi A Staff I et alCemented versus uncemented hemiarthroplasty for displaced femoral neck fracturesa prospective randomized trial with early 0cMao et alfollowup J Orthop Trauma “ Rolfson O Donahue GS Hallsten M et alPatientreported outcomes in cemented anduncemented total hip replacements Hip Int “ Chammout G Muren O Boden H et alCemented compared to uncemented femoralstems in total hip replacement for displacedfemoral neck fractures in the elderly studyprotocol for a singleblinded randomizedCHANCEtrial BMCcontrolled trialMusculoskelet Disord Liu T Hua X Yu W et al Longtermfollowup outcomes for patients undergoingprimary total hip arthroplasty with uncemented versus cemented femoral components a retrospective observational studywith5year minimum followupJ Orthop Surg Res a Engesaeter LB Espehaug B Lie SA et alDoes cement increase the risk of infection inprimary total hip arthroplasty Revisionrates in cemented and uncementedprimary THAs followed for years inthe Norwegian Arthroplasty Register ActaOrthop “ Schmale GA Lachiewicz PF and Kelley SSEarly failure of revision total hip arthroplasty with cemented precoated femoralcomponents Comparison with uncementedcomponents at to years J Arthroplasty “ Azegami S Gurusamy KS and Parker MJCemented versus uncemented hemiarthroplasty for hip fractures a systematic reviewof randomised controlled trials Hip Int “ 0c'

" the activation of the nfκb pathway plays a crucial role in the progression of breast cancer bca andalso involved in endocrine therapy resistance on the contrary to the canonical nfκb pathway the effect of thenoncanonical nfκb pathway in bca progression remains elusivemethods bca tumor tissues and the corresponding cell lines were examined to determine the correlation betweenrelb and the aggressiveness of bca relb was manipulated in bca cells to examine whether relb promotes cellproliferation and motility by quantitation of apoptosis cell cycle migration and invasion rnaseq was performedto identify the critical relbregulated genes involved in bca metastasis particularly relbregulated mmp1transcription was verified using luciferase reporter and chip assay subsequently the effect of relb on bcaprogression was further validated using bca mice xenograft modelsresults relb uniquely expresses at a high level in aggressive bca tissues particularly in triplenegative breastcancer tnbc relb promotes bca cell proliferation through increasing g1s transition andor decreasing apoptosisby upregulation of cyclin d1 and bcl2 additionally relb enhances cell mobility by activating emt importantlyrelb upregulates bone metastatic protein mmp1 expression through binding to an nfκb enhancer elementlocated at the ²flanking region accordingly in vivo functional validation confirmed that relb deficiency impairstumor growth in nude mice and inhibits lung metastasis in scid micekeywords relb emt mmp1 cancer progression and breast cancer according to the latest statistic data the numbers ofnew cases and deaths of breast cancer bca accountedfor and of total cancers worldwide sincethe past decade bca incidence has been consistently increasing in developing countries such as south america correspondence yxu4696njmueducn tangjhnjmueducn2jiangsu cancer hospital jiangsu institute of cancer research theaffiliated cancer hospital of nanjing medical university baiziting nanjing p r china1department of general surgery the first affiliated hospital with nanjingmedical university guangzhou road nanjing p r chinafull list of author information is available at the end of the asia and africa owing to improved early diagnosisand advanced therapy strategies the current death ratesof bca have appreciatively decreased in the western developed countries including the united states howeverdistantan metastasis associated with endocrine therapy resistance still remains as a large obstacle to successful control of advanced bca [ ] to data bca patientswith distant metastasis at the time of diagnosis appearedto be worse prognosis with a 5year survival rate of since metastasis is the main cause of death ofbca patients the key for improving the bca survival rateis to accurately evaluate the metastatic potential for the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cwang cell communication and signaling page of guidance of clinical implementation of the individualizedtreatment plan current therapeutic strategies for bcamainly consisted of surgical reduction and radiotherapybased locally therapy with anticancer drugs providedsystemic therapy including chemotherapy endocrinetherapy and targeted therapy however the comprehensive treatment strategy is still limited and a large numberof patients eventually developed to more aggressive malignant forms that are resistant to the most of treatments[ ] thus the discovery of new biomarkers for surveillance of the disease progression and novel molecular targets for improving therapeutic efficiency are urgentlyneededthe reoccurrence ofnuclear factorκb nfκb a family of transcriptionfactors regulates immune responses and inflammation it has been welldocumented that nfκb plays a crucial role in bca endocrine therapy resistance in particular nfκb activation is thought to promote bcaprogression toward erindependent phenotypes especially fortriplenegative bcatnbc after hormone treatment [ ] mammals express five nfκb membersincluding nfκb1 p50p105 nfκb2 p52p100 rela p65 relb and crel[ ] based on the components of the signaling cascade the nfκb signaling can be activated via either canonical pathway p50rela or noncanonical pathwayp52relb [ ] the canonical nfκb signaling pathway is regarded as the central regulator of the inflammatory response but it has been extensively studied in thecontext of hormone disorders autoimmunity obesityand cancer [ ] nevertheless the molecular basisfor the activation of the noncanonical nfκb pathway awell as its role in cancer progression have been recentlyreceiving increased attention tissuematerials and methodsbca patient tissue samples and immunohistochemistryihcthe first affiliated hospital with nanjing medical university nanjing china has collected fresh tumor tissuesfrom bca patients prior to treatment and noncancerousbreast tissues the ethics committee of nanjing medicaluniversity has approved the study protocol with writteninformed consents obtained from all the patients participated in this study a total of bca cases including cases with lymphatic metastasis were selected to examine the correlation between the level of relb and bcapathological grade or stage the tissues were fixedparaffinembedded slides theslides weredewaxed by xylene and then ethanol washing off excessliquid the slides were further rehydrated by rinsingwith dh2o for ihc the slides were soaked in bsabuffer for h and then incubated with 400x diluted primary antibodies within bsa buffer at °c overnightthe slides were washed and then incubated with biotinylated secondary antibody at room temperature for min after washing the slides with 1x bps a dabsubstrate kit cell signaling tech usa was used toobserve immunostaining images under a microscopethe intensity of ihc staining was scored as negativescore weak medium and strong totalcell positivity was scored as the percentage of positivecells including no positive cell “ “ and respectively the œhscore was calculated using œˆ‘pi i  for all slides inwhich œpi represented the percentage of positive and œirepresented the staining intensityrelb has been originally identified in bcells and therelbbased noncanonical nfκb signaling has beenrecognized to impair antigen presentation by dcs whichis associated with skin inflammation and excessive immune cell infiltration to various ans in the past decade [“] recently relb has been implicated incancer progression particularly in sex hormonerelatedcancers including bca prostate cancer pca and cervical cancer [“] we have reported that relb ishighly expressed in advanced pca and enhances radioresistance speculating that the activation of noncanonicalnfκb pathway contributes to pca malignancy from androgen receptorto arindependentphenotypes [ ] the present study further demonstrated that relb highly expresses in advanced bcawhich is necessary for sustaining bca malignant progression mechanistically relb promoted bca cell proliferation emt and metastasis through transcriptionalupregulation of several oncogenic proteinsincludingbcl2 cycline d1 and mmp1ardependentlinesthe selected human breast cellcell culture and gene manipulationalllines were purchased from the american type culture collectionatcc usa and grown in the recommended mediaincluding normal breast epithelial cellline mcflines mcf7 and t47d10a erpositive bca celland tnbc cellmdamb231 and bt549relb was ectopically expressed in erpositive cellswith low constitutive relb conversely relb wasknocked out from the tnbc cells using crisprcas9approach briefly several grnas were designed usingthe optimized crispr design tool doublestrand oligo dna for each grna was cloned into theecorisite in pgl3u6 spsgrna vector the constructwas cotransfected with 331sp cas9 plasmid intomdamb231 and bt549 cells singlecell colonieswere selected using puromycin and neomycin andfurther validated by western blots dna was extractedfrom the candidate clones and confirmed by dna sequencing prior to t7en1 digestion 0cwang cell communication and signaling page of cell proliferationcell proliferation was determined using cell countingand colony formation assay respectively bca cells wereseeded into 96well plates at cellswell and treatedwith cck8 reagent dojindo mol tech japan andcell viability was measured as the optical density at nm for colony formation assay bca cells were seededinto 6well plates at cellswell and cultured for “weeks the cells were fixed in paraformaldehyde for min stained with crystal violet beyotime biotech china for h after rinsing three times with1xpbscolonies were photographed andcountedcelltheflow cytometryflow cytometry was used to analyze apoptosis and cellcycle for apoptotic cell qualification × cells wereseeded in 6well plates and cultured for h the cellswere collected and washed three times with cold pbsand then stained with μm annexin vfitc and μmpi dojindo mol tech in a supplied binding buffer for min at room temperature and dark condition apoptotic cells were counted using a bd facscalibur flowcytometer bd biosciences usa and the percentage ofapoptotic cells was calculated by the number of preapoptotic and apoptotic cells divided by the amount oftotal cells for cell cycle analysis the cultured cells weredetached washed and fixed with ethanol overnightat °c the cells were then washed times with coldpbs and treated with μl propidium iodide bd biosciences usa for min the cell cycling phases weredetermined by flow cytometrycell motilitycell movability was analyzed using wound healing assayand transwell assay respectively bca cells were platedinto 6well plates at × cellswell with serumfreedmem for h after rinsing with × pbs three timesthe cultured cells were separated using pipette tips tocreate three parallel wounds the wells were thenwashed several times with pbs to remove floating cellsthe cells were continuatively cultured in the completedmedia the wound closure was monitored at and h using a microscope and the wound surface area wasquantified in addition transwell assay corning usawas used to quantify cell migration and invasion abilitiesfor migration assay the cells were plated into the upperchambers without coated membrane at × cellswellfor invasion assay the chamber inserts were coated with μl serumfree medium mixed with matrigels bdbiosci usa and solidified at °c for h the cellswere placed into the upper chamber at cellswell inboth assays the cells were plated in μl serumfreemedium in the upper chambers and μl mediumsupplemented with fbs was placed into the lowerchambers after culturing the cells for h the invadedcells on the lower surface were fixed in paraformaldehyde stained with crystal violet and counted using amicroscoperna sequencingtotal rna was isolated from bca cells using a totalrna isolation kit invitrogen mrna was converted tocdna libraries and added adapters for sequencing theprocedure of ngs sequencing on illumina hiseq platform was conducted by vazyme biotech co ltd nanjing chinachromatin immunoprecipitation chipa chipit system active motif usa was used toquantify relb binding to the nfκb enhancer elementlocated at the ²flanking region of the human mmp1gene briefly chromatin isolated from bca cells werepulled down using a relb antibody cell signalingusa unprecipitated chromatin was used as input control and chromatin pulled down by an igg antibodyserved as a negative antibody control the pulled downthe enhancer fragment was quantified using a quantitative pcr with the genespecific primerswestern blottingcytosolic and nuclear proteins were extracted from cellsand tumor tissues using a ripa lysis buffer containingpmsf and then quantified using a bca assay kit key“gen biotech china the extracted proteins μg were separated on sdspage gels and thentransferred to pvdf membranes the membranes weresubsequently incubated overnight at °c with theprimary antibodies against relb bcl2 cyclin d1 and βactin santa cruz biotech usa against er ecadherin vimentin snail slug twist cell signalingtech usa thereafter the membranes were washedthree times with tbst buffer and incubated at roomtemperature for h with hrpconjugated secondaryantibody santa cruze biotech the immunoblots werevisualized using an enhanced chemiluminescence detection system biorad usa the intensities of blots werequantified using quantity one software and protein expression was normalized by loading controls such as βactin and gapdhanimal experimentthe effects of relb on tumorigenesis and metastasiswere validated using bca cells bearing mouse xenografttumor experimental models all animal studies wereconducted according to the institutional animal careand use approved by the research committee ofnanjing medical university no iacuc1711030 five 0cwang cell communication and signaling page of weekold female balbc athymic nude mice beijingvital river lab animal tech co ltd china wereused for studying tumor growth and fiveweekold female scid mice nanjing medical university chinawere used for studying tumor metastasis respectivelyfor the tumor growth experiment × bca cellswere subcutaneously implanted into the right axilla ofmice tumor volume was measured using digital calipersevery other day and calculated using a standard formulav × ab2 a and b represent the diagonal tumorlengths the mice were executed when tumor volumereached to mm3 and tumor tissues were removedfor tumor metastasis study bca cells were injectedinto mice through tail vein and assessed for lung metastasis the mice were sacrificed at weeks and the number of metastatic lung nodules was countedusage of tcga databasethe tcga bca dataset was analyzed to assess the association of rela or relb expression with bca occurrenceand the correlation between the mrna level of rela orrelb and ernegative bca patient survival ratestatistical analysisdata were presented as the mean ± standard deviationsd from at least three replicates significant differencesbetween the experimental groups were analyzed by unpaired student™s ttest oneway analysis of varianceanova followed by dunnett™s or bonferroni™s multiple comparison test was performed using prismgraphpad san diego usa statistical significance wasaccepted at p additional materials and methods are in additional fileresultsrelb is correlated with bca aggressivenesswe analyzed the bca cohort in tcga database toexamine whether the expression level of relb is correlated to bca progression and patient survival the statistical analytic data indicated that the mrna levels ofrela and relb in tumor tissues were higher than theirlevels in normal breast tissues particularly the differencein relb levels appeared to be highly associated with bcaprogression fig 1a consistently the expression ofrela and relb was also associated with the overall survival of ernegative bca patients notable compared torela the high level of relb led to lower survival ratesfig 1b to assess the correlation between nfκb andbca normal breast tissues and tumor tissues from theselected bca patients with different stages were analyzedby ihc additional file table s1 compared to thenormal breast tissue control the levels of four membersof the nfκb family increase in bca tumor tissuesespecially in the ernegative phenotype notably the expression of relb is apparently correlated to the aggressiveness of bca fig 1c subsequently we furtherexamined the levels of relb in tnbc tissues vs erpositive bca tissues by western blots as expected thehigh levels of relb were detected in all the tnbc tissues suggesting the relb is inversely related to er inbca fig 1dfurthermore several bca cell lines correspondent tothe different stages of bca progression vs a normalbreast epithelial cell line mcf10a were examined consistently the high constitutive levels of the nfκb members were detected in bca cell lines particularly relbuniquely expressed at high levels in tnbc celllinesfig 2a accordingly the nfκb activity was consistently elevated in bca cells especially in tnbc cellsfig 2b furthermore erpositive mcf7 cells vstriplenegative mdamb231 cells were used to measure cytoplasmic and nuclear levels of the nfκb members as expected all the members highly increased inmdamb231 cells compared to mcf7 cells importantly the nfκb upstream ikkα and its phosphorylatedlevels also heighten in tnbc cells fig 2c the distribution of the nfκb members was further confirmed byconfocal microscope fig 2d taken together these results indicated that the high level of relb is associatedwith aggressiveness of bca suggesting that relb maycontribute to the advanced bcarelb promotes bca cell proliferationto investigate the potential mechanism by which relbregulates downstream gene expression involved in bcaprogression relb was ectopically expressed into mcf7and t47d cells with low levels of constitutive relbfig 3a in parallel relb was knocked out from tnbccells mdamb231 and bt549 using a crisprcas9gene edition system additional file figure s1 fig 3bthe nfκb activity was slightly elevated in the relboverexpressed ernegative cells but significantly reduced in relbknocked out tnbc cells fig 3c and dconsistently the elevated relb in erpositive cells led toincreased cell colony number and the cell proliferationrate fig 3e g and i conversely cell colony formationand the cell proliferation rate significantly decreased inrelbknocked out tnbc cells fig 3f h and jrelb promotes g1s transition and inhibits apoptosisto elucidate the role of relb in bca cell proliferationthe cell cycling process and the cell apoptotic rate wereanalyzed by flow cytometry the enforced expression ofrelb in mcf7 cells resulted in decreasing g1 phase butincreasing s and g2m phases which enhances the acceleration of cell division fig 4a in contrast knockout of relb in mdamb231 cells led to an increasing 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig the correlation between relb and aggressiveness of bca a b tcga database geo accession gse11121 was analyzed to examine thecorrelation between mrna expression profiles of rela and relb and bca occurrence and over survival of patients c bca tumor tissues withdifferent pathological grades and normal breast tissues were screened by ihc using nfκb antibodies the luminal group was characterized aserpositive and her2negative lowgrade genotype her2 group was assessed as a middlegrade genotype with high tumor growth tnbcgroup was determined as a high grade d the levels of relb in tnbc tumors vs erpositive bca tumors were quantified βactinnormalizedimages were plottedfig the nfκb activation in bca cell lines a several corresponding cell lines were selected to confirm the relationship between nfκb proteinsand the aggressiveness of bca cells b the relative nfκb activity was quantified in the bca cell lines c cytoplasmic and nuclear levels of nfκbproteins in erpositive mcf7 and tnbc mdamb231 cells were quantified accordingly the nfκb upstream phosphorylated ikkα levels in thetwo cell lines were measured d the cellular distribution of the nfκb proteins in the two cell lines were further examined using aconfocal microscope 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig determination of relbenhanced bca cell survival and growth a relb was ectopically expressed in erpositive bca cells by stablytransfected a human relb cdna construct b relb was knocked out in tnbc cells using a crisprcas9 gene edition system c d the relative nfκb activity was quantified in relbmanipulated bca cells e f the survival rates in the relbmanipulated bca cells were examined by colonyformation assay gj the proliferation rates in the relbmanipulated bca cells were quantified by cck8 assay mean ± sd was representative ofthree independent experiments carried out in duplication p p show the significances between two groups as indicatedg1 phase but decreasing s phase fig 4b furthermoreapoptosis was examined in the relbmanipulated bcacells although the elevated relb in mcf7 cells unlikelychanged the cell apoptotic rate the depletion of relb inmdamb231 cells obviously increased apoptotic cellrates fig 4c and d consistently the overexpression ofrelb in mcf7 cells resulted in upregulation of cyclind1 but no effect on bcl2 expression fig 4e additionally knock out of relb in mdamb231 cells led to suppression of cyclin d1 and bcl2 suggesting that relbsustains tnbc cell growth via activation of prosurvivaland antiapoptotic pathways fig 4f moreover relb depletion led to increases in p21 and p27 but decreases incmyc cyclin e1 and bclxl additional file table s2additional file figure s2relb enhances bca cell mobility by enhancing emtit has been widely recognized that bca progression isgenerally through the erdependent phenotype to erindependent malignancyimportantly emerging evidence demonstrated the inverse relationship between erand nfκb in bca cells consistently the expression of relb appeared to be inversely correlated to erlevels in the tested cell lines additional file figures3a furthermore er elementsdriven luciferase reporter gene expression construct was constructed toexamine whether relb is able to regulate the er response expectedly the erreporter response was remarkably reduced due to the increased relb in mcf7cells suggesting that relb negatively regulates er signaling additional file figure s3bfurthermore to testify whether relb contributes toestrogendeprived bca progression the effect of relb onthe metastatic ability of bca cells was examined sincethe emt process has been well documented in bca metastasis several emt markers were measured in ourpanel of bca cell lines the elevated relb in erpositivecells led to decreasing ecadherin but increasing snail islug i twist i and vimentin fig 5a in parallel knockout of relb in tnbc cells resulted in decreasing thoseemt markers while ecadherin was not detectable intnbc cells fig 5b accordingly the effect of relb onthe cell motility was analyzed by quantifying cell capacities of wound healing migration and invasion as expected the enforced expression of relb in mcf7 cellssignificantly enhanced the cell capacity for wound recovery compared to a vehicle control fig 5c consistentlythe elevated relb in mcf7 cells also resulted in enhancing cell migration and invasion fig 5e subsequentlyknock out of relb in mdamb231 cells led to decreases in those capacities fig 5d and f these resultssuggest that relb promotes erindependent bca progression mainly through activating emt processrelb upregulates bone metastasis associated proteinmmp1to identify the important relbregulated genes involvedin bca metastasis we applied rnaseq analysis usingtotal rna isolated from relbknocked out mdamb cells vs the parent cells the results indicated thatthe mrna expression profiles of numerous genes werealtered in the relbknocked out cells fig 6a and b importantly multiple genes relating to metastatic signalingpathways were downregulated fig 6c and table among of them mmp1 an activator in bca bone metastasis was highly related to the level of relb toassess the effect of relb on mmp1 expression we verified the expression level of mmp1 increased in relboverexpressed mcf7 cells but decreased in relbknocked out mdamb231 cells suggesting that relbmay directly regulate the mmp1 gene fig 6dto elucidate how relb regulates mmp1 expressionwe identified a putative nfκb element located in the²finking region of the human mmp1 gene for verifying this nfκb element functional response to transcriptional activation by relb a ²flanking region containingthe nfκb enhancer element and the core promoter inthe human mmp1 gene was cloned into a pgl4 vectorto drive the luciferase reporter gene expression compared to the basic vector control the reporter gene response driven by the mmp1 enhancer highly increasedin relboverexpressed mcf7 cells but significantly decreased in relbknocked out mdamb231 cellsfig 6e moreover to confirm that relbmediated transcriptional activation through binding to the nfκb enhancer element located at the ²flanking region of themmp1 gene a 240bp fragment containing the nfκbenhancer element was pulled down using a specific relbantibody and amplified using pcr the amount ofpulleddown dna fragment from mcf7 cell chromatinwas less than the level from mdamb231 cell chromatin fig 6ffurthermore to validate whether the increased mmp1can further promote emt mmp1 was manipulated by 0cwang cell communication and signaling page of fig the effect of relb on cell cycle and apoptosis a b the cell cycling process in relbmanipulated bca cells was analyzed using flowcytometry c d preapoptotic and apoptotic cells in the relbmanipulated bca cells were quantified by flow cytometry e f the levels of bcl2 andcyclin d1 proteins were measured by western blots statistical significance between two groups as described in fig 0cwang cell communication and signaling page of fig the effect of relb on emt a b the emtassociated markers in relbmanipulated bca cells were measured by western blots c d the cellwound healing abilities were analyzed e f the cell a migration and invasion capacities were examined using a transwell assay statisticalsignificance between two groups as described in fig 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig relbmediated transcriptional regulation mrna expression profiles in the relbknocked out mdamb231 cell vs the parent cell wereexamined using a rnaseq platform a the distribution of mrna profiles in the relbdeprived cell was illustrated b cluster analysis of rnaseqdata indicates updownregulated mrna profiles heatmap c kegg pathway enrichment analysis was performed to assess emtmetastasisrelating mrna profiles d the protein level of mmp1 was quantified in the relbmanipulated bca cells e a ²flanking region of the human mmp1gene ™mmp1 containing the nfκb element underline and the core promoter region was cloned in pgl4 vector to drive the luciferasereporter gene expression as illustrated the enhancer activity modulated by relb in bca cells was estimated by normalized reporter responses f asmall dna fragment containing the nfκb element was pulled down from chromatins using a relb antibody and then quantified by pcr iggserves as a negative antibody control statistical significance between two groups as described in fig overexpressing in mcf7 cells but silencing in mdamb231 cells the elevated mmp1 in mcf7 cells led toa slight decrease in ecadherin but increases in snail twist1 and vimentin in contrast the silence of mmp1in mdamb231 cells resulted in decreasing snail twist slug and vimentin regardless of the undetectable of ecadherin in the cells additional file figures4a in addition to determine the functional contribution of mmp1 to emt under the relb regulationmmp1 was either ectopically expressed in relbknockedout mbamb231 cells or wassilenced in relboverexpressed mcf7 cells intriguingly the elevatedmmp1 in relbknocked out mbamb231cells resultedin partially restored the emt proteins while the silenceof mmp1 in relboverexpressed mcf7 cells was ableto abrogate the elevated emt proteins except for no effect on twist expression additionalfile figures4brelbmediated bca progression was validated in micetwo mouse xenograft tumor experimental models wereused to validate the effects of relb on tumor growth andmetastasis in vivo relbknocked out mdamb231 cellsand the parent cells were subcutaneously injected intothe front leg area of nude mice one week after injection tumors were formed and continuously grew toreach the maximum volume mm3 compared toinjection of the control cells tumor formation by therelbknocked out cells was approximately week late month after injectionthe tumor volumes in thecontrol group reached the maximal volume howeverthe tumor growing speed and tumor weight wereremarkably reduced in the group injected with the relbknocked out cellsfig 7ac according to relbknocked out bcl2 and cycline d1 in the tumor tissueswere significantly reduced fig 7d which just reflectedto in vitro results as shown in fig furthermore the reduction in the metastatic capacityof the relbknocked out mdamb231 cells was validated by injecting the cells through tail vents of scidmice six weeks after the injection the tumor occurredon the lung of mice injected with the control cells however the incidence of metastatic lung tumors was dramatically reduced in the relbknocked out groupfig 7e the excised tumor tissues were analyzed byihc analysis using the relative antibodies compared tothe control group tumor aggressiveness stain ki67 wassignificantly reduced in the relbknocked out groupconsistently the levels of mmp1 and emt markerswere also reduced in relbknocked out group fig 7fadditionally tumor images by hematoxylin and eosinhe staining confirmed that the metastasized tumorstable mrna expression profiles in mdamb231 cellsgene namevimrelbkoctrlcdh11zeb1snai1mmp1mmp9mmp28junhsp70dmbt1smad2log2relbkoctrlˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’ˆ’upordowndowndowndowndowndowndowndownupupupunchangedp value500e05500e05500e05500e05vim vimentin cdh11 cadherin zeb1 zinc finger eboxbinding homeobox snai1 snail1 mmp1 matrix metallopeptidase mmp9 matrix metallopeptidase mmp28 matrix metallopeptidase jun jun protooncogene ap1 transcription factor hsp70 heat shock protein dmbt1 deleted in malignant brain tumor tumor suppressor smad2 a member in tgfbsmad signaling family 0cwang cell communication and signaling page of fig see legend on next page 0cwang cell communication and signaling page of see figure on previous pagefig validation of relbenhanced tumor growth and metastasis in mice a relbknocked out mdamb231 and the parent cells weresubcutaneously injected into the right axilla of female nude mice and allowed to form tumors thirty days after bca cell implantation tumortissues were removed out prior to the execution the excised tumor tissues were photographed two out of five mice injected with relbknockedout cells didn™t form the tumor b c tumor growth rates and the excised tumor weights were measured and plotted d total proteins wereextracted from the tumor tissues and immunoblotted with the relating antibodies βactin served as an internal control e female scid mice wereinjected with relbknocked out mdamb231 and the parent cells through the tail vein after weeks the mice were sacrificed and lungmetastasis was examined the excised lungmetastasized tumors indicated by arrows were photographed f the metastatic lung tissues werescreened by ihc with relative antibodies incorporated with ki67 staining the relative images were plotted g he staining was applied toconform the lung metastatic tissues and the numbers of micrometastasis in the two groups were plotted statistical significance between twogroups as described in fig were obviously reduced in the relbknocked out groupcompared to the control group fig 7g it should benoted that the detection of low levels of relb in therelbknocked out group reflected the constitutive mouserelb additionallythe enforced expression relb inmcf7 cells was still insufficient to tumor metastasizeto the lung altogether these results suggest the noncanonical nfκb pathway may serve as a major contributor to the progression of malignant bcadiscussionit has been widely recognized that bca is

case of a 14year old boy with tumorassociated refractory epilepsy Positron emission tomography imaging demonstrated a region with heterogeneous high 11Cmethionine uptake and a region with homogenous low 18Ffluorodeoxyglucose uptake within the tumor Histopathological and genomic analyses confirmed the tumor as BRAF V600Emutated polymorphous lowgrade neuroepithelial tumor of the young PLNTY Within the highmethionineuptake region we observed increased protein levels of Ltype amino acid transporter LAT1 a major transporter of methionine cMyc and constituents of the mitogenactivated protein kinase MAPK pathway We also found that LAT1 expression was linked to the BRAF V600E mutation and subsequent activation of MAPK signaling and cMyc Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 expression in BRAF V600Emutated PLNTY and glioblastoma cells The BRAF inhibitor dabrafenib moderately suppressed cell viability in PLNTY Collectively our results indicate that BRAF V600E mutationactivated MAPK signaling and downstream cMyc induces specific metabolic alterations in PLNTY and may represent an attractive target in the treatment of the diseaseKeywords PLNTY BRAF V600E mutation Methionine PET LAT1IntroductionPediatric lowgrade neuroepithelial tumors PLGNTs encompass a group of central nervous system neoplasms that longterm epilepsyassociated tumors LEATs such as ganglioglioma and dysembryoplastic neuroepithelial tumor DNT PLGNTs have different characteristics than their adult counterparts and includes Correspondence ktate12yokohamacuacjp Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama JapanFull list of author information is available at the end of the are commonly driven by genomic alterations in the Rasmitogenactivated protein kinase MAPK pathway such as mutations in BRAF and NF1 [ ] Recent largescale genomic studies and genomewide methylation analyses allowed a thorough characterization of PLGNTs [] and cIMPACTNOW the Consortium to Inform Molecular and Practical Approaches to CNS Tumor Taxonomy currently classifies P“LGNTs as distinct disease entities [ ] In Huse et a0al described ten cases of polymorphous lowgrade neuroepithelial tumor of the young PLNTY which were histologically characterized by oligodendrogliomalike cellular components with intense CD34 immunopositivity According to previous publications PLNTYs are indolent tumors The Authors Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cTateishi a0et a0al acta neuropathol commun Page of that generally exhibit a benign clinical course and harbor either a BRAF V600E mutation or FGFR2FGFR3 fusion [] Based on its histological and genomic profiles cIMPACTNOW Update recommends PLNTY as a possible future classification for pediatrictype glialglioneuronal tumors However because of their rare etiology only a few PLNTYs have been described to date [ ] and it is unclear how genomic alterations promote the pathogenesis of the disease Herein we present a case of PLNTY with unique metabolic imaging features Using positron emission tomography PET we found regions of heterogeneous high 11Cmethionine uptake and homogenous low 18Ffluorodeoxyglucose FDG uptake within the tumor Activation of the MAPK pathway cMyc and expression of Ltype amino acid transporter LAT1 were increased in the highmethionineuptake area compared with the surrounding cortex lowmethionineuptake Glycolytic metabolites were expressed only weakly in tumor cells Pharmacological and genetic inhibition of the MAPK pathway suppressed cMyc and LAT1 and inhibited tumor cell viability suggesting that MAPK signaling and downstream cMyc activates methionine metabolism and inhibition of this pathway induces therapeutic vulnerability in PLNTYMaterials and a0methodsCell viability analysisAM38 and normal human astrocytes was purchased from JCRB Cell Bank and ScienCell Research Laboratories respectively Tumorsphere lines were cultured in serumfree neural stem cell medium as previously described [] Normal human astrocytes were cultured with astrocyte medium ScienCell To assess cell viability primary cultured cells were dissociated into single cells and seeded into 96well plates at a density of cellswell After a0h dabrafenib Selleck and trametinib Selleck were serially diluted and added to the wells Cell viability was measured using the CellTiterGlo Promega assay at day and the results were indicated as viability of the DMSO controlshRNA cell line generationTo knockdown BRAF 293T cells were transfected with lentiviral vector packaging plasmid DNA containing a0 μg of Human BRAF shRNA TRCN0000381693 GP and a0μg of a0pVSVgRev a0with Lipofectamine„¢ TRCN0000196844 Sigma Aldrich a0μg of a0pHIVThermo Fisher Scientific YMG62 and AM38 cells were infected with lentivirus in polybrene a0μgmL for a0h Two days later the cells were selected with puromycin a0μgmL for a0days and used for experiments GIPZ nonsilencing lentiviral shRNA Control RHS4348 Horizon Discovery was used as a nonsilencing NS controlImmunohistochemistryTumor tissue specimens were fixed in neutral buffered formalin and embedded in paraffin Hematoxylin and eosin staining was performed using standard procedures For immunohistochemical analysis 5µmthick sections were deparaffinized treated with H2O2 in methanol rehydrated and heated for a0min for antigen retrieval After blocking with serum tissue sections were incubated with primary antibodies against CD34 Novus Biologicals LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology phosphoERK Bethyl Laboratories and cMyc Cell Signaling Technology at a0°C overnight The next day sections were washed with PBS incubated with biotinylated secondary antibody for a0 min at room temperature and then incubated with ABC solution PK6101 PK6102 Vector laboratories for a0 min at room temperature Finally the sections were incubated with DAB Dako and counterstained with hematoxylinWestern blottingcOmplete„¢ Mini EDTAfree Protease Inhibitor Cocktail Cells were lysed in RIPA buffer SigmaAldrich with a Roche Fifty micrograms of protein was separated by SDSPAGE gel and transferred to polyvinylidene difluoride membranes Millipore by electroblotting After blocking with or nonfat dry milk in TBST a0mM Tris [pH ] a0 mM NaCl Tween20 membranes were incubated at a0 °C overnight with primary antibodies After washing and incubation with horseradish peroxidaseconjugated secondary antibodies Cell Signaling Technology blots were washed and signals were visualized with chemiluminescent HRP substrate Millipore Primary antibodies against BRAF Gene Tex cMyc Cell Signaling Technology GAPDH Gene Tex LAT1 Cell Signaling Technology phosphoMEK Cell Signaling Technology a0phosphoERK Bethyl Laboratories and Vinculin Novus Biologicals were used for western blottingCase presentationThis study was performed in accordance with declaration of Helsinki and was approved by the Institutional Review Board Yokohama City University [YCU Yokohama Japan] IRB numbers A1711300006 and B190600002 Written informed consent was obtained from the patient and parents A 14year old boy presented with chronic medial temporal lobe epilepsy for a year Magnetic resonance imaging MRI indicated 0cTateishi a0et a0al acta neuropathol commun Page of See figure on next pageFig Characteristics of a patient with PLNTY a T2weighted left T1weighted middle and contrastenhanced right MR images b Computed tomography CT left 18FfluorodeoxyglucosePETCT middle and 11CmethioninePETCT right images c Video electroencephalography indicating ictal onset in the left temporal lobe with spread to the contralateral temporal lobe d PETCT and MRI merged intraoperative navigation image left and surgical image right showing the highmethionineuptake region and surrounding abnormal lesion on MRIhypointensity on T2weighted images and hyperintensity on T1weighted images with a cystic component in the left temporal lobe Contrastenhanced MRI showed no significant enhancement in the lesion Fig a01a while computed tomography revealed heavy calcification FDGPET showed lower FDG uptake in the tumor while 11CmethioninePET demonstrated increased methionine uptake in the same lesion SUVmax tumornormal tissue ratio Fig a01b Videoelectroencephalographic EEG monitoring indicated ictal onset in the left temporal lobe with subsequent spread to the contralateral temporal lobe Fig a01c We speculated that this abnormal lesion was a LEAT Since we considered this tumor to be completely resectable the patient underwent craniotomy and resection of the neoplasm including the highmethionineuptake region Fig a01d To achieve epileptic control electrocorticography was performed intraoperatively After removal of the highmethionineuptake and T2 hyperintense lesions the surrounding tissue was resected until interictal epileptiform discharge could no longer be detected by electrocorticography The patient became epilepsyfree after lesion removal and MRI indicated complete remission a0months after the surgeryTissue samples of the highmethionineuptake region and surrounding cortex low methionine uptake were collected Hematoxylin and eosin staining indicated diffusely infiltrating growth patterns and presence of oligodendroglialike cellular components Fig a02a Astrocytic and highgrade features were absent with a Ki67 index of less than Chicken wirelike branching capillaries and microcalcification were also found in region Despite lower cellularity oligodendroglialike cells were present in the surrounding tissue Immunohistochemistry revealed extensive CD34 expression and peripherally associated ramified neural elements in the tumor cells Fig a0 2a Targeted DNA sequencing identified a BRAF V600E mutation in the tumor without recurrent mutations in IDH1 IDH2 TERT promoter FGFR1 H3F3A or HIST3H1B Fig a0 2b Chromosome 1p19q codeletion was absent Fig a02c The above histological and genetic features fulfilled the diagnostic criteria for PLNTYTo assess the mechanisms underlying the methionineFDG uptake mismatch indicated by PET we compared the expression of LAT1 glucose transporter GLUT and hexokinase2 HK2 between tissue regions and Notably LAT1 which is a major methionine transporter was more highly expressed in than in Fig a0 3a In contrast GLUT1 and HK2 which is correlated with FDG uptake and lactate dehydrogenase A LDHA expression were weak in either region Additional file a0 Fig a0 S1 LAT1 expression is mediated by cMyc activation and BRAF V600E mutation activates the MAPK pathway and downstream cMyc [ ] Therefore we hypothesized that BRAF V600E mutation promotes LAT1 expression through MAPK signaling and consequent cMyc activation a0 in PLNTY Levels of phosphoMEK phosphoERK and cMyc were higher in tissue region than in Fig a03a suggesting activation of the MAPK pathway and cMyc within the highmethionineuptake lesion To verify whether the BRAF V600E mutation can induce the expression of LAT1 we exposed primary cultured YMG83 PLNTY cells to a BRAF inhibitor dabrafenib As expected the expression of phosphoMEK phosphoERK cMyc and LAT1 was suppressed after dabrafenib treatment in YMG83 cells Fig a0 3b Notably BRAF inhibitor dabrafenibtreated YMG83 cells had lower cell viability compared to normal human astrocytes Fig a03c To confirm the reproducibility of these molecular features we used patientderived YMG62 cells epithelioid glioblastoma with the BRAF V600E mutation which exhibited high 11Cmethionine uptake by PET imaging Additional file a0 Fig a0 S2 and AM38 glioblastoma cells BRAF V600E mutant We found that dabrafenib and a MEK inhibitor trametinib inhibited the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03d and 3e Similarly BRAF knockdown suppressed the expression of proteins in the MAPK pathway as well as cMyc and LAT1 Fig a03f Collectively these findings indicated that activation of the MAPK pathway by the BRAF V600E mutation deregulates cMyc and promotes LAT1 expression This oncogenic signaling pathway increases methionine metabolism and tumor maintenance in PLNTYDiscussionThirty cases of PLNTY have been described to date with the first ten reported by Huse et a0al in [ ] BRAF V600E mutation was seen in of the patients and BRAF fusion in patient These BRAF alterations were mutually exclusive with other genomic events including FGFR3TACC3 fusion FGFR3 amplification FGFR2CTNNA3 fusion FGFR2INA fusion 0cTateishi a0et a0al acta neuropathol commun Page of 0cTateishi a0et a0al acta neuropathol commun Page of Fig Histopathologic and genomic features of a patient with PLNTY a Hematoxylin and eosin HE staining top and CD34 immunohistochemistry bottom in the highmethionineuptake and lowmethionineuptake region within tumor tissue Bars μm b Sanger sequencing for detection of BRAF V600E arrow left and IDH1 R132H arrow right mutations c Fluorescence in situ hybridization for detection of 1p311q25 left and 19q1319p13 right chromosomal deletionsFGFR2 KIAA1598 fusion FGFR2 rearrangement and NTRK2 disruption suggesting that the vast majority of PLNTYs are induced by BRAF mutation or FGFR fusion and subsequent MAPK activation Therefore targeting MAPK signaling may become a potential therapeutic strategy in PLNTY Indeed BRAF V600Emutated PLNTY cells were relatively vulnerable to dabrafenib and trametinib in the present study Thus targeted molecular therapy for the MAPK pathway may be particularly useful in PLNTY located in surgically unresectable regions In addition Koh et a0 al reported that the BRAF V600E mutation contributes to the intrinsic epileptogenicity in pediatric brain tumors and that inhibition of BRAF suppressed epileptic seizures [] Thus BRAFMEK inhibitors could exert antiepileptic as well as antitumor effects in PLNTYPET imaging revealed a region with increased methionine uptake and low FDG uptake within tumor tissue in our patient Consistent with this finding previous case reports demonstrated increased methionine uptake but only mild FDG uptake in patients with BRAF V600Emutated PLNTY [ ] Thus excessive 0cTateishi a0et a0al acta neuropathol commun Page of Fig Activating the MAPK pathway induces LAT1 expression in a patient with PLNTY a Immunohistochemistry of indicated proteins in the highmethionineuptake and lowmethionineuptake regions within tumor tissue Bars μm b Western blot analysis of phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG83 PLNTY left cells treated with DMSO and μM BRAF inhibitor BRAFi dabrafenib for h GAPDH loading control c Relative cell viability of dabrafenibtreated left and trametinibtreated right YMG83 cells and immortalized normal human astrocytes NHA P DMSO versus dabrafenib left and trametinib right d Western blot analysis for indicated proteins in YMG62 epithelioid glioblastoma left and AM38 glioblastoma right cells treated with DMSO μM BRAF inhibitor BRAFi dabrafenib and μM MEK inhibitor MEKi trametinib for h GAPDH loading control e Western blot analysis of BRAF phosphoMEK phosphoERK cMyc and LAT1 proteins in YMG62 left and AM38 right cells treated with DMSO and dabrafenib at indicated concentrations Vinculin loading control f Western blot analysis for indicated proteins in nonsilencing NS and BRAF and transduced YMG62 and AM38 cells GAPDH loading controlmethionine uptake and low FDG uptake may be imaging features specific to PLNTY A preclinical study has demonstrated that high uptake of 18FFDG was correlated with increased Glut1 and HK2 expression in human cancers [] Although the diagnostic accuracy is insufficient FDGPET imaging is useful to differentiate highgrade from lowgrade gliomas [] In the present case low FDG uptake and weak expression of Glut1 HK2 and LDHA were observed in tumor tissue suggesting low glycolytic activity in PLNTY On the other hand due to a high signaltonoise ratio 11Cmethionine PET imaging is practical for brain tumors [ ] Several PET imaging studies have demonstrated that methionine uptake was higher in 0cTateishi a0et a0al acta neuropathol commun Page of highgrade adult gliomas than in lowergrade gliomas [ ] In epileptogenic brain tumors however all gangliogliomas and “ of DNT had increased methionine uptake although these tumors are classified as WHO grade I [ ] implying that methionine uptake may be irrespective of tumor grade in LEATsPrevious studies have reported that methionine uptake was correlated with LAT1 in gliomas [ ] LAT1 plays a major role in the transport of neutral essential amino acids including methionine and is driven by several cancerrelated genes such as MYC [] It has been demonstrated that cMyc which is partly mediated by the MAPK pathway regulates LAT1 expression and MEK inhibitor suppresses LAT1 SLC7A5 transcription [ ] thereby indicating a role of the MAPK pathway and cMyc in the regulation of LAT1 Since RASMAPK pathwayassociated genomic alterations are common in LEATs [] and that the BRAF V600E mutation has been identified in “ and of gangliogliomas and DNTs respectively [ ] there is a possibility that the BRAF V600E mutation and MAPK pathwayrelated genomic alterations may activate methionine metabolism in LEATs To investigate this hypothesis we evaluated the protein expression of LAT1 and the molecules that are involved in the MAPK pathway As expected levels of phosphoMEK phosphoERK cMyc and LAT1 were higher in the highmethionineuptake area than in the lowmethionineuptake area We also found that genetic andor pharmacological BRAF inhibition suppressed MAPK pathway activation and attenuated LAT1 expression in BRAF V600EmutatedPLNTY cells and glioblastoma cell lines These findings support the hypothesis that the BRAF V600E mutation may upregulate LAT1 and methionine metabolism through cMyc activation for cell survival In addition to LAT1 methionine uptake was correlated with microvascular density MVD in gliomas [] PLNTYs are considered benign brain neoplasms proposed as WHO grade I however in the present case a chicken wirelike MVD which is one of the histopathological characteristics of oligodendroglioma was also observed in the highmethionineuptake tissue region Intriguingly methionine uptake has been reported to be relatively higher in oligodendrogliomas than in astrocytomas [] Thus PLNTY which has an oligodendrogliomalike microvascular structure might show unique metabolic imaging features Further studies are warranted to validate this hypothesis Nonetheless our data indicated that the BRAF V600E mutation induced MAPK pathway activation and downstream cMyc promoted LAT1 expression and methionine metabolism with little effect on glycolytic pathway activation These findings may explain the unique metabolic imaging features of FDGmethionine mismatch in PLNTYSupplementary informationSupplementary information accompanies this paper at https doi101186s4047 Additional file a0 a0Figure S1 Low glycolysis activation in a patient with PLNTY Immunohistochemistry for glucose transporter hexokinase and lactate dehydrogenase A in the highmethionineuptake upper and lowmethionineuptake lower region within tumor tissue A Bars μm Figure S2 Images of the patient™s glioblastoma with the BRAF V600E mutation Contrastenhanced magnetic resonance left and 11Cmethionine positron emission tomography right images of the YMG62 patientAcknowledgementsWe thank Mrs Emi Hirata and Yasuko Tanaka YCU for technical and administrative assistance We also would like to thank Editage wwweditagecom for English language editingAuthors™ contributionsKT led the study collected samples designed experiments performed experiments interpreted data and wrote the manuscript JS TH and YM performed experiments NI HM provided tumor samples and associated clinical details TO RM and DU interrupted PET and MRI studies NU and SY performed the histological classification of tumor samples TY designed experiments and interpreted data All authors read and approved the final manuscriptFundingThis work was supported by GrantAid for Scientific Research 19K09488 Princess Takamatsu Cancer Research Fund Takeda Science Foundation SGH Cancer foundation Yokohama Foundation for Advancement of Medical Science and BristolMyers Squibb FoundationCompeting interestsThe authors declare that they have no competing interestsAuthor details Department of Neurosurgery Graduate School of Medicine Yokohama City University Fukuura Kanazawa Yokohama Japan Department of Pathology Yokohama City University Hospital Yokohama Japan Department of Radiology Graduate School of Medicine Yokohama City University Yokohama Japan Departmento of Radiology Division of Nuclear Medicine National Center for Global Health and Medicine Tokyo Japan Received June Accepted August References Borbely K Nyary I Toth M Ericson K Gulyas B Optimization of semiquantification in metabolic PET studies with 18Ffluorodeoxyglucose and 11Cmethionine in the determination of malignancy of gliomas J Neurol Sci “ https doi101016jjns200602015 Chappe C Padovani L Scavarda D Forest F NanniMetellus I Loundou A Mercurio S Fina F Lena G Colin C et al Dysembryoplastic 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"Sleep disturbance is an issue reported by caregivers Waking at night is a feature of dementia and byproxy sleep disturbance among caregivers is reported to be high Little is known about the characteristics ofdementia caregivers™ sleep and the factors that may influence sleep disruptionThe purpose of this study was to investigate the sleep characteristics and disturbances of Australian caregivers of aperson living with dementia In addition it evaluated the psychological wellbeing of caregivers by evaluatingassociations between mood and sleep in this populationMethods This study used a crosssectional descriptive correlation design Participants were recruited with theassistance of Alzheimer™s Australia Dementia Australia and targeted social media advertising In total adultprimary informal caregivers of people with dementia participated completing a questionnaire on demographiccharacteristics the Depression Anxiety and Stress Scale DASS21 and the Pittsburgh Sleep Quality Index PSQIResults In this study of caregivers were female who had been caring for someone living with dementia onaverage for years of participants had two or more comorbidities namely cardiovascular disease osteoarthritisand diabetes of participants were poor sleepers with with difficulty initiating sleep and reporting havingdifficulty maintaining sleep Overall psychological distress was common with high levels of moderate to severedepression anxiety and stress Global PSQI scores were significantly positively associated with depression and anxietywith the strongest correlation seen with stress scores Depression scores were also moderately associated with daytimedysfunction Stress was identified as a significant predictor of overall sleep qualityConclusions Sleep problems are common within the population of dementia caregivers Due to the nature andduration of caregiving and the progression of dementia of the care recipient there is the potential for a decline in thecaregivers™ mental and physical health Caregivers of those living with dementia are more likely to have comorbiditiesdepression anxiety and stress Sleep quality is correlated with emotional distress in dementia caregivers although thedirection of this association is unclear Therefore sleep and psychological wellbeing may be intertwined withimprovements in one aspect resulting in a positive impact in the otherKeywords Carers Caregivers Sleep Mood Psychological wellbeing Dementia Correspondence aislingsmythecueduau1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA AustraliaFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cSmyth BMC Geriatrics Page of BackgroundDementia is an inclusive term used to describe a numberof neurological conditions resulting in cognitive impairment and can include Alzheimer™s Disease senile dementia frontotemporal dementia vascular dementia LewyBody dementia Korsakoff syndrome alcohol related braininjury and younger onset dementia [“] Globally thenumber of individuals with a formal dementia diagnosisrose from million in to million in []A further million new cases of dementia worldwideare predicted each year [] as populations around theglobe continue to live longer [] These figures could bevery much under estimated as it is widely accepted thatbetween and of people affected have no formaldementia diagnosis [] This growing epidemic effects notonly the individuals living with dementia but also theirfamilies caring for them the communities they live in andthe health care systems they rely uponFamily or friends are often key informal caregivers forpeople living with dementia [ ] Globally over billion hours ofinformal care is provided to peopleliving with dementia [ ] Whilst providing this carecan be highly rewarding it has also been described as achronic stressor with caregivers reporting low qualityand quantity of sleep [] and high levels of stress anddepression [] In fact sleep disruption is prevalentamongst dementia caregivers with over of caregiversexperiencing sleep disturbances []The National Sleep Foundation recommend that olderadults ‰¥ years require seven to h sleep for optimalphysical and psychological wellbeing [] Yet numerousstudies have illustrated that dementia caregivers sleepsignificantly less than that with estimates reporting mostcaregivers sleep less than h per night [] Not only arecaregivers sleeping less than they should the sleep theydo get is of significantly lower quality than their noncaregiver counterparts []Poor sleep is associated with a myriad of negative physical and psychological outcomes including hypertensionobesity mood disorders and dementia [] A disruptedsleep pattern is also recognised as a significant factor inpredicting caregiver strain [ ] and perhaps more importantly in predicting placing an individual into long termcare [] Enabling people living with dementia to stay athome rather than transfer to longterm care is the optimaloutcome for many families However this cannot be to thedetriment of the caregiver™s own physical andor psychological wellbeing Therefore in order to support the personliving with dementia PLWD to remain in the communitymaintaining caregiver health is vital Given the pivotal rolesleep has in a myriad of physiological processes it is essential to optimise and preserve caregivers sleepDespite the important role sleep plays in dementiacaregiver health it remains an understudied populationparticularly within the Australian context In fact arecent report by Carers Australia identified noAustralian studies on sleep disruption in dementia caregivers Only sixteen international studies were identifiedwhich subjectively measured sleep in the dementiacaregivers population with the majority of studies notreporting on the causes or consequences of disturbedsleep []In order to address this paucity of Australian data thepurpose of this study was to elucidate the sleep characteristics and disturbances of Australian caregivers ofPLWD Furthermore this study will determine whetherthere is a relationship between sleep and psychologicalwellbeing among caregivers of communitydwellingpeople living with dementia Lastly we will aim to identify significant predictors of poor sleep which in turncould offer a therapeutic target of poor sleep in dementia caregiversMethodsRecruitmentThis study used a crosssectional descriptive correlationdesign One hundred and four informal caregiversof people with dementia living in the community wereenrolled in the study Participants were invited via anumber of anisations including Alzheimer™s AustraliaDementia Australia and targeted social media advertising Mail outs were conducted and online questionnairesdistributed The inclusion criterion required the participant be an adult years or older primary informalcaregiver of a communitydwelling person living withdementia Power analysis was undertaken to computeminimum number of sample size required A samplesize of at least participants is necessary to detect amedium to large effect with power assuming atwotailed ttestEthicsThis study was granted ethical approval by Edith CowanUniversity Human Research Ethics Committee No Informed written consent was obtained from allparticipants prior to participation Participants receivedno incentive for taking partMaterialsThe survey collected demographic characteristics including gender age body mass index BMI as well as information about preexisting medical history caregivinghistory and use of respite The survey also included the questions from the Depression Anxiety and StressScale DASS21 [] and the Pittsburgh Sleep QualityIndex PSQI []The DASS21 is a selfreport questionnaire assessinglevels of caregiver stress anxiety and depression over the 0cSmyth BMC Geriatrics Page of previous seven [] day period The results provide anindication of the individual™s perception of their experience of depression anxiety and stress The DASS21 isoften used as a screen for high levels of distress whenthe depression andor anxiety scores are high and as ascreen for the presence of a significant life event orproblem if the stress score is elevated DASS scorescorrespond to ranges of severity normal mild moderate severe extremely severe for depression anxiety andstress individually []The PSQI [] is a selfreport questionnaire assessinglevels of perceived quality and patterns of sleep over theprevious month across seven components subjectivesleep quality sleep latency sleep duration habitual sleepefficiency sleep disturbances use of sleep medicationand daytime dysfunction Each component has a potential score of three with a higher score indicating poorersleep related performance The total score for the sevencomponents creates a global score range to wherea score greater or equal to five ‰¥ indicates that theperson is a œpoor sleeper with severe difficulties in atleast two of the seven components or moderate difficulties in three or more components []Presence of comorbidities was determined by participantshaving one or more chronic conditions As per WorldHealth anisation WHO chronic conditions wereidentified as those requiring ˜ongoing management over aperiod of years or decades™ covering a wide range of healthproblems such as heart disease diabetes asthma immunodeficiency disorder depression and schizophreniaStatistical analysesData analysis was undertaken using IBM SPSS Version and GraphPad Prism Descriptive statistics weregenerated for demographic characteristics DASS21 andPSQI scales Spearmans rho r correlation analysis wasused to assess the relationship between categorical components of DASS21 depression anxiety and stress andboth PSQI global scores and individual componentscores subjective sleep quality sleep latency sleep duration sleep efficiency sleep disturbances use of sleepingmedication and daytime dysfunction For logistic regression all variables were categorical age in years genderBMI length of care in months comorbidities DASS21component scores An r of was considered a mediumcorrelation and an r of was considered a large correlation [] Nonpaired ttests were used to determine ifthese values were statistically significant Results wereconsidered statistically significant if p Multivariatestepwise regression analysis was undertaken to determinethe predictive factors of global PSQI scores and identify variables significantly associated with sleep quality Independentvariables included in multivariate stepwise regression analyses were age gender BMI length of care comorbiditiesand DASS21 subscale scores Subjects with missing data oneither PSQI or DASS21 were excluded from inclusion incorrelation and regression analysesResultsCharacteristics of the caregiversOne hundred and four n surveys were completedin either hardcopy n or online n Table As the surveys were widely distributed by service providers and links made available online the response ratecannot be determinedParticipating caregivers were predominantly femalen with a mean age of years Range “years The average BMI for respondents was kgm2range “ kgm2 Table which lies within theoverweight category While half of the participants n had a BMI within the healthy weight range“ twenty two participants were classified as underweight BMI less than and participants were classified as overweight The averagelength of time in the caregiving role in this study was months years ranging from months to yearsOnly n of participants reported using formalrespite services and all were female n of participants reported no comorbidities of participants n reported one comorbidity and n reported two or more comorbidities Themost commonly reported comorbidities were cardiovascular disorders n mainly hypertension and hypercholesterolemia bone and joint disorders n mainlyTable Demographic of participantsCharacteristicGendern or meanˆ’ SDMaleFemaleAllAgeBMI kgm2Caregiver role MonthsComorbiditiesNoneOneTwo TotalUse of RespiteYesNoTotal99a94b96c ± ± ± a n missingb n missingc n missing 0cSmyth BMC Geriatrics Page of osteoporosis and arthritis and endocrine disorders n mainly prediabetes diabetes and thyroid dysfunctionTable Characteristics of Caregivers™ sleepMeasuren Subjective Sleep qualityThe majority of participants n reported aglobal sleep score equal or greater than which indicated they had clinically significant sleep issues in thepreceding month with a mean global score of ± Table The highest scoring individual subcomponentsincluded sleep latency ± subjective sleep quality ± and sleep disturbances ± n of participants had a sleep latency period greater than min indicative of issues initiating sleep Table ofparticipants had sleep efficiencies less than suggestingissues with maintaining sleep while in bed of participants had taken sleep medication at least once a weekover the previous month Participants were invited to addany additional relevant comments to their PSQI sleep assessment Forty participants provided additional information regarding their sleeping issues and identified issueswhich fell broadly into three themes Sleep disruption dueto caregivers physical needs such as pain and restless legs caregivers emotional distress such as stress anxietyand worrying and responding to care recipientneeds Depression anxiety and stress in caregivers of participants reported mild depression scores of participants reported mild anxiety scores and of participants reported mild stress scores Over athird of respondents reported moderate to extremelysevere stress levels n and depression n and more than a quarter n of respondents reported moderate to extremely severe anxietylevels Table In the bivariate analyses numerous subcomponents ofthe PSQI scale were significantly associated with measures of depression anxiety and stress Table Theglobal PSQI score is significantly positively associatedwith depression r anxiety r and stressscores r Stress scores also significantly correlatedwith other PSQI subcomponents including subjectivesleep quality r sleep latency r and daytimedysfunction r Depression scores were moderatelyassociated with daytime dysfunction r Global ScoreSubjective Sleep QualityVery GoodFairly GoodFairly BadVery BadSleep Latency‰¤ mins“ min“ min minsSleep duration h“ h“ h hSleep Efficiency‰¥ ““ Sleep DisturbancesDaytime DysfunctionFrequency of SleepingMedicationNeverOnce per weekTwice per weekThree per week PSQI ComponentMean ˆ’ SD ± ± ± ± ± ± ± ± Predictors of Sleep quality in caregiversTo understand the relationship between the predictorsand global PSQI scores multivariate stepwise regressionanalyses were conducted to assess variables significantlyassociated with sleep quality The dependent variablewas the global PSQI score and the independent variableswere age gender BMI use of respite length of carecomorbidities and scores on the subscales of DASS21scale Depression Anxiety and Stress Stress was theonly significant covariate of global PSQI scores Stressscores could statistically significantly predict PSQIGlobal scores accounting for of variance Thestandardised coefficient was which was statisticallysignificant p as was the overall model F p All other variables were excluded from themodel due to nonsignificance Forward stepwise regression 0cSmyth BMC Geriatrics Page of Table Depression anxiety and stress in caregivers DASS21Clinical classificationTotalDepression n Anxiety n Stress n NormalMildModerateSevereExtremely Severe analyses were then undertaken with global PSQI scores asthe dependent variable and stress scores as a predictorwhile adjusting for the following confounders gender agecomorbidities of the caregiver and length of care Whilethese confounders were not statistically significant theywere retained within the model to adjust the effects ofstress on PSQI After adjusting for these factors that modelremains statistically significant F p andaccounts for of the adjusted variance in sleep scoresFor every one unit increase in stress scores PSQI Globalscores increases by a score of t p Thisregression model confirms that stress is a key significantcovariate of selfreported sleep issuesDiscussionA recent Australian report highlighted a significant gapin the literature around Australian caregivers of a PLWD[] Our study provides a comprehensive overview ofAustralian dementia caregiverssleep characteristicsassociations between psychological wellbeing and sleepand highlights a predictive role for stress in sleep qualityAll previous international studies identified an averagePSQI in dementia caregiver studies [] highlightingthe prevalence of the issue As expected and consistentwith previous studies [] Australian caregivers ofPLWD have a high prevalence of poor sleep with ofparticipants classified as poor sleepers The PSQI globalscore ± was higher than those found in somedementia caregiver studies [ ] but comparable withTable Correlation between PSQI subcomponent scores andDASS21 subcomponent score in dementia caregiversPSQI componentDASS21DepressionDASS21AnxietyDASS21StressPSQI Global ScoreSubjective sleep qualitySleep latencySleep durationSleep efficiencySleep disturbancesUse of sleeping medicationDaytime dysfunctionp p ˆ’ˆ’ˆ’others [] The PSQI subcomponents which greatestcontributed to the overall score were sleep latency timetaken to fall asleep sleep quality overall subjectivequality and sleep disturbances Sleep latency and sleepdisturbances have previously been identified as the mostcommon contributors to sleep quality in caregivers ofPLWD in a recent systematic review [] Given thedearth of Australian specific data this study identifiedimportant depth of detail around caregiver™ssleepcharacteristics of caregivers took longer than therecommended min to fall asleep only of caregivers slept more than h of caregivers had sleepefficiency lower that the recommended and ofcaregivers used sleep aiding medication in the previousmonth Taken togetherfindingspresent a novel and ominous overview of the poor sleephealth conditions that Australian dementia caregiversexperiencethese descriptiveCaregivers of community dwelling people living withdementia reported poor sleep quality and high levels ofdepression anxiety and stress This study found that poorsleep was correlated with subjective feelings of depressionanxiety and stress which were in keeping with previousliterature around caregiving and psychological distressMore than half of the caregivers surveyed reported symptoms of depression and stress and respectivelyand reported anxiety These findings are in keepingwith that of a recent study of dementia caregivers in ruralVictoria where of caregivers reported depression orstress and reported anxiety []Previous work has identified an association betweendepression and poor sleep among dementia caregivers[ ] Reduced quality of sleep and depression arehigher among caregivers of people living with dementia[] Furthermore caregivers of people living withdementia who were also depressed experience a greatervariation in sleep patterns []This study reveals sleep scores were significantly associated with measures of depression anxiety and stressThese findings illustrate the interplay between sleepquality and quantity and psychological wellbeing incaregivers providing care for an individual living withdementia Further exploration of predictive factors incaregiverssleep quality identified stress as a keypredictor of poor overall sleep quality whilst adjustingfor age gender comorbidities and length of care This isa novel finding and represents a potential therapeutictarget to improve sleep quality in dementia caregiversDespite the high prevalence of selfidentified poorsleep overnight respite was only used by of participants which is in keeping with previous research thatcites of Australian caregivers have never usedrespite services [] Access to respite care remains oneof the major means of easing caregiver burden and is 0cSmyth BMC Geriatrics Page of frequently identified as needed by caregivers yet remainsunderutilised Whilst a high proportion of dementiacaregivers report a need for respite services there isinsufficient awareness of and access to respite servicesfor caregivers [] Respite service availability can beinvaluable with caregivers reporting lower stress levelsand improved health after use [] Sleep disturbanceshave also been shown to be partially reversed for caregiversduring periods of respite care [] perhaps due to reversalof hyper stressed state Respite services have been cited asan important measure to allow caregivers time to attend totheir own health imperative to supporting the caregiver tocontinue in providing care for their loved one []A recent Australian parliamentary inquiry into sleephealth identified sleep as a foundation of positive healthand wellbeing alongside diet and exercise [] Furthermore the report urged government to prioritise sleephealth as a national priority given its pivotal role inmaintaining health It is clear that caregivers of PLWDhave suboptimal sleep which is associated with poorerpsychological wellbeing and potential increased risk ofdeveloping chronic health conditions such as diabetesand cardiovascular disease [] Interestingly cardiovascular disease and endocrine disease such as diabeteswere the most prevalent comorbidities in our studypopulation Previous studies have also identified highblood pressure diabetes and arthritis as the most prevalent chronic disease in the dementia caregiver groupwhich is in keeping with our findings []Managing sleep and its associated mediators will havedirect impact on both the caregiver™s own health as wellas the caregiver care recipient relationship It is imperative to provide educational support around sleep considerpractical interventions such as overnight respite and toaddress stress management interventions for caregivers inturn reinstating and preserving sleep of this critical population of informal caregiversLimitationsA limitation of this study was the small sample size despitenumerous attempts to recruit participants and involvementof national anisations Although over participantsprovided demographic details only participants completed all questionnaires and were included in statisticalanalyses Challenges related to recruiting caregivers andparticularly caregivers of people living with dementia hasbeen described in numerous publications [ ]Caregivers of people with dementia living in the community are more likely to be female and this was representedin our population Furthermore caregivers who are underthe greatest stress may also be those least likely to participate However this remains one of the larger studies ofAustralian dementia caregivers sleep Another limitationwithin this study was that no data was collected regardingthe severity of dementia or behavioural disturbances whichmay impact sleep of the caregiverConclusionSleep problems are widespread within the population ofdementia caregivers Given that the majority of peopleliving with dementia are reliant on family caregiversminimising health and psychologicalimpact on caregivers should be of major concern Furthermore sleepquality is correlated with emotional distress in dementiacaregivers so improving the sleep of caregivers may inturn improve their psychological wellbeing Converselystress is a significant predictor of poor sleep so managing stress may have a positive impact on sleepDementia caregivers are often older with coexistingmorbidities and high levels of psychological distress Withprolonged caregiving and the progression of dementia ofthe care recipient there is the potential for a concurrent decline in the caregivers mental and physical health In orderto support the caregiver in their role it is of the upmost importance that we promote and maximises their health andwellbeing By managing and minimising the negative factorsassociated with caregiving we can enhance caregiving satisfaction and gratification In turn this can ensure optimalcaregivercare recipient relationship supporting the personliving with dementia to remain at home as long as possibleAbbreviationsDASS21 Depression Anxiety and Stress Scale21 PLWD PersonPeopleliving with dementia PSQI Pittsburgh Sleep Quality Index WHO WorldHealth anisationAcknowledgementsThe authors acknowledge the statistical advice of Dr Mark Jenkins and DrPeter PalamaraAuthors™ contributionsAS was involved in study design data collection data interpretation andmanuscript preparation LW supported study design data interpretation andediting of the manuscript CV supported study design and manuscriptpreparation EQ advised on use of psychological tools interpretation of theirdata and manuscript preparation LE was involved in data collection andmanuscript preparation All authors have read and approved the manuscriptFundingThis research was supported by an Edith Cowan University ECU Early CareerResearcher Grant awarded to Dr Aisling Smyth ECU had no role in thedesign data collection and data interpretation of the studyAvailability of data and materialsThe datasets used andor analysed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateThis study was granted ethical approval by Edith Cowan University HumanResearch Ethics Committee No Informed written consent wasobtained and participants received no incentive for taking partConsent for publicationNot applicableCompeting interestsThe authors state that they have no competing interests 0cSmyth BMC Geriatrics Page of Cohen J Statistical power analysis for the behavioral sciences 2nd edHillsdale NJ Erlbaum Wilcox S King AC Sleep complaints in older women who are familycaregivers J Gerontol B Psychol Sci Soc Sci 1999543P189“Simpson C Carter P Dementia behavioural and psychiatric symptomseffect on caregiver's sleep J Clin Nurs ““Kochar J Fredman L Stone KL Cauley JA Sleep problems in elderlywomen caregivers depend on the level of depressive symptoms resultsof the caregiver“study of osteoporotic fractures J Am Geriatr Soc “Lee D Heo S Yoon S Chang D Lee S Rhee H Sleep disturbances andpredictive factors in caregivers of patients with mild cognitive impairmentand dementia J Clin Neurol “Johnson E Respite reconsidered a discussion of key issues and futuredirections for carer respite Sydney Carers Australia Phillipson L Johnson K Cridland E Hall D Neville C Fielding E et alKnowledge helpseeking and efficacy to find respite services an exploratorystudy in helpseeking carers of people with dementia in the context ofaged care reforms BMC Geriatr “ O™connell B Hawkins M Ostaszkiewicz J Millar L Carers™ perspectives ofrespite care in Australia an evaluative study Contemp Nurse “Lee D Man K Lindesay J Effect of institutional respite care on the Sleepof people with dementia and their primary caregivers J Am Geriatr Soc“ Parliament of the Commonwealth of Australia Bedtime Reading inquiry intoSleep health awareness in Australia Canberra Commonwealth of Australia [Available from httpsparlinfoaphgovauparlInfodownloadcommitteesreportrep024220toc_pdfBedtimeReadingpdffileTypeapplication2Fpdf] Accessed Mar Moon H DilworthAnderson P Baby boomer caregiver and dementiacaregiving findings from the National Study of caregiving Age Ageing“ Bristow M Cook R Erzinclioglu S Hodges J Stress distress and mucosalimmunity in carers of a partner with frontotemporal dementia Aging MentHealth “ McConaghy R Caltabiano ML Caring for a person with dementia exploringrelationships between perceived burden depression coping and wellbeing Nurs Health Sci “Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsAuthor details1School of Nursing Midwifery Edith Cowan University JoondalupDrive Joondalup WA Australia 2School of Arts and HumanitiesPsychological Services Centre Edith Cowan University Joondalup AustraliaReceived April Accepted August ReferencesAlzheimer's Association Types of dementia [Available from httpswwwalzalzheimersdementiawhatisdementiatypesofdementia]Ozcare Types of Dementia The six most common forms of dementia [Available from httpswwwozcareaudementiacareunderstandingdementiatypesofdementia]Dementia Australia Types of dementia [Available from httpswwwdementiaauinformationaboutdementiatypesofdementia]Global Burden of Disease Dementia Collaborators Global regional andnational burden of Alzheimer's disease and other dementias “ asystematic analysis for the Global Burden of Disease Study LancetNeurol “Alzheimer's Disease International The Global Voice on Dementia2020 Availablefrom httpswwwalzcoukresearchstatistics] Accessed Mar James BD Bennett DA Causes and patterns of dementia an update inthe era of redefining Alzheimer's disease Annu Rev Public Health “Ahern S Cronin J Woods N Brady NM O'Regan NA Trawley S et alDementia in older people admitted to hospital an analysis of length of stayand associated costs Int J Geriatr Psychiatr 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recommendations finalreport Sleep Health “Jay S Vincent G Kovac K Dorrian J Thomas M Reynolds A Ferguson SReport Reducing Sleep Disruption in Carers Australia Carers Australia httpswwwsleephealthfoundationaufilesResearch_GrantsSpecial_reportsReport_Carers_Australia_FINALv2pdf Accessed Mar2020 Worley SL The extraordinary importance of Sleep the detrimental effects ofinadequate Sleep on health and public safety drive an explosion of Sleepresearch P T “ Rowe MA McCrae CS Campbell JM Benito AP Cheng J Sleep patterndifferences between older adult dementia caregivers and older adultnoncaregivers using objective and subjective measures J Clin Sleep Med“Schulz R Belle SH Czaja SJ McGinnis KA Stevens A Zhang S Longtermcare placement of dementia patients and caregiver health and wellbeingJAMA “ Gaugler JE Yu F Krichbaum K Wyman JF Predictors of nursing homeadmission for persons with dementia Med Care “Lovibond SH Lovibond PF Manual for the Depression Anxiety Stress ScalesSydney Smyth C The 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Neurologic Manifestations of Systemic Disease D Lapides Section EditorNeurologic Manifestationsof Systemic Disease SleepDisordersEric M Davis MD1Chintan Ramani MBBS1Mark Quigg MD MSc2Address1Division of Pulmonary and Critical Care Department of Medicine University ofVirginia Charlottesville VA USAEmail emd9bvirginiaedu2Department of Neurology University of Virginia Charlottesville VA USA Springer ScienceBusiness Media LLC part of Springer Nature This is part of the Topical Collection on Neurologic Manifestations of Systemic DiseaseKeywords Sleep disorders I Sleep manifestations of systemic diseases I Sleep impacts on health I Sleep apnea IInsomniaAbstractPurpose of review Sleep is intimately involved in overall health and wellbeing We provide acomprehensive report on the interplay between systemic diseases and sleep to optimizethe outcomes of systemic disordersRecent findings Spanning the categories of endocrinologic disorders metabolictoxicdisturbances renal cardiovascular pulmonary gastrointestinal infectious diseases autoimmune disorders malignancy and critical illness the review highlights the prevalentcoexisting pathology of sleep across the spectrum of systemic disorders Although it is rarethat treating a sleep symptom can cure disease attention to sleep may improve quality oflife and may mitigate or improve the underlying disorder Recent controversies inassessing the cardiovascular relationship with sleep have called into question some ofthe benefits of treating comorbid sleep disorders thereby highlighting the need for anongoing rigorous investigation into how sleep interplays with systemic diseasesSummary Systemic diseases often have sleep manifestations and this report will help theclinician identify key risk factors linking sleep disorders to systemic diseases so as tooptimize the overall care of the patient 0c Page of IntroductionCurr Treat Options Neurol All Earth™s species maintain a solar 24h cycle of rest andactivity and disrupting the cycle affects adaptation andhomeostasis Sleep™s quotidian œnormalness meansthat analogous to fish not knowing about water until itis dry sleep is not commonly thought about until it isdisruptedFor example about of the adult populationcomplain of transient insomnia and about experience chronic insomnia that disrupts daytime function[] Patients with chronic insomnia experience less workproductivity more absenteeism more accidents andmore hospitalizations leading to direct treatment costsof approximately 60B annually [] Considering thepotential widespread reach of comorbid sleep disordersevaluating sleep in the neurological patient is importantThis review will introduce the accepted anizationof sleep disorders review important features in historytaking and evaluation and survey the systemic diseasesthat have important comorbidities with particular sleepdisordersGeneral considerationsClassification of sleep disordersAn abridged listing of sleep disorders from the American Academy of SleepMedicine Table provides an overview of the current classification []Insomnia is a chronic dissatisfaction with sleep duration and quality that isassociated with daytime dysfunction Although pharmacologic treatment isoften pursued for chronic insomnia management outcomes are often betteraddressing underlying factors with the early use of cognitivebehavioral therapyfor insomnia CBTi []Sleeprelated breathing disorders involve dysfunction of the respiratory systemduring sleep usually resulting in daytime hypersomnia Obstructive sleepapnea OSA central sleep apnea CSA and respiratory effort related arousalsare classified under this category Treatment options including continuouspositive airway pressure CPAP positional therapy mandibular advancementdevices healthy weight loss and even a novel cranial nerve stimulator whichprotrudes the tongue forward during sleep [4cid129cid129]Central hypersomnias are defined as a primary dysregulation of sleep resultingfrom dysfunction of the central nervous system that causes daytimehypersomnia Often treatment addresses the underlying cause and may includeuse of strategic napping and wakepromoting medicationsCircadian disorders consist of various lesions or external disruptions of thecircadian timing system that desynchronize the brain™s clock from the externalsolar lightdark cycle resulting in hypersomnia or insomnia in a clockdependent fashion Treatment of circadian rhythm disorders involves adjustinglife around the patient™s desired sleep time or augmenting factors that entrainthe body™s clockParasomnias represent disorders of faulty inhibition of waking behaviors thatarise inappropriately during sleep and are divided into those that occur duringnonREM sleep REM sleep or state transitions REM sleep behavior disorder is aparasomnia characterized by loss of muscle atonia during REM sleep thatusually occurs in patients with neurodegenerative disorders It is often treatedeffectively addressing other sleep disturbances and treating with clonazepam ormelatonin [] 0cCurr Treat Options Neurol Page of Table Abridged classification of the AASM sleep disordersInsomniaChronic insomnia disorderShortterm insomnia disorderExcessive time in bedShort sleeperSleeprelated breathing disordersObstructive sleep apneaCentral sleep apneaSleeprelated hypoventilation disordersSleeprelated hypoxemia disordersCentral disorders of hypersomnolenceNarcolepsy types and Idiopathic hypersomniaKleineLevin syndromeHypersomnia due to medical disorder medication substance psychiatric disorderInsufficient sleep syndromeCircadian rhythm sleepwake disordersDelayedAdvancedIrregularNon hShift workJet lagParasomniasNREM relatedArousal disordersConfusional arousalsSleepwalkingSleep terrorsSleeprelated eating disorderREM relatedREM sleep behavior disorderRecurrent isolated sleep paralysisNightmare disorderOtherExploding head syndromeSleeprelated hallucinationsEnuresisSleep talkingSleeprelated movement disorders 0cCurr Treat Options Neurol Page of Table ContinuedRestless legs syndromePeriodic limb movement disorderLeg crampsBruxismRhythmic movement disorderBenign sleep myoclonus of infancyPropriospinal myoclonus at sleep onsetNormal variantsSleep historySleeprelated movement disorders consist of fragmentary often repetitive bodymovements that can disrupt sleep or sometimes worse disturb the sleep of bedpartners Periodic limb movement disorder PLMD and restless legs syndromeRLS both fall under this category and are treated with repletion of iron storesand consideration of dopaminergic agonists []A sleep history helps a patient disclose sleep findings and helps the physiciananize it into categories of hypersomnia sleep habits and scheduling sleepcharacteristics environmental issues and sleep interrupters Table The Epworth Sleepiness Scale quantifies the degree of hypersomnia []Most adults require “ h of daily sleep [] and prefer it anized into eithera monophasic nocturnal schedule or in a biphasic pattern augmented with anafternoon œsiesta The sleep pattern characterizes the presence and severity ofsleeponset insomnia sleep maintenance insomnia or terminal insomnia insomnia distributed within the last half of the sleep period œCatchup sleep aphenomenon of prolonged sleep on a free day is a classic sign of sleepdeprivation Habitual earlyphase advances œmorning larks latephase delays œnight owls or a chaotic irregular schedule can be a sign of circadiandisorders One also must inquire about common sleep disruptors including legmovements snoring witnessed apneas and environmental factorsDiagnostic testing modalitiesSleep diaryPolysomnographyThe sleep diary often available through standardized forms or evenwebsites or smartphone apps consists of “ weeks of selfreported sleeptimesThe overnight polysomnography PSG is the goldstandard measurementof sleep architecture respiratory disorders such as OSA and parasomniasIn the case of OSA the unattended home sleep study has had an 0cCurr Treat Options Neurol Page of Table A categorical sleep historyHypersomniaEpworth Sleepiness Scale Considering the last weeks how likely would you fall asleep while doing each task not at all points slight moderate severe Normal ‰¤ pointsSitting and readingWatching TVSitting inactive in public lecture church ¦Car passenger for an hourLying down to rest in the afternoonSitting conversationSitting quietly alone after lunchDriving stopped in trafficSchedulesleep timeWorkday bedtime and out of bedtimeWeekday bedtime and out of bedtimeWhat is your estimated sleep latency If min what are you doing in bed before you fall asleepHow often do you awaken at night and whyDo you need an alarm clock to awaken in the morningHow many days of the week do you nap and for how longEnvironmentDo you have a bedroomDo you have a bedpartner TV Mobile phone or other electronicsWhat are you doing right before bedtimeHow much caffeine coffeeteasoda popenergy drinks and alcohol do you consume and when is the latest intakeInterruptersDo you have leg pain or restlessnessDo you have chronic pain that prevents or interrupts sleepDo you have daytime hallucinations or dreams severe or lucid nightmares sleep paralysis or cataplexyDo you snore or have witnessed apneasMultiple sleep latency testincreasing role as a diagnostic testing alternative to the traditional inlabPSG Concerns of other sleep disorders or those that may be presentcomorbidly with probable OSA require inlab PSG that can measure sleeparchitecture and sleepassociated movementsThe multiple sleep latency test MSLT consists of a series of daytime napsfrom which sleep onset is calculated The test in combination with PSGperformed the night before is the gold standard in measuringhypersomnia especially in the evaluation of narcolepsy 0c Page of ActigraphyPersonal devicesCurr Treat Options Neurol Wrist actigraphy provides measurements of longterm patterns of rest andactivity as proxies for sleep and wakefulness Such patterns can help tocorroborate histories of sleep duration and timingPopular smartphones and other ambulatory devices with physiologicalmonitoring capabilities may transform the evaluation of sleep However arecent comparison of different brands of activity trackers found that sleepwake measurements varied widely in comparison with sleep diaries orstandard PSG [] The overall conclusion is that at the beginning of wearable devices are not ready for reliable quantification of sleep acrossindividuals Although serial recordings confined to a single individual mayhold some value these measurements have yet to be validatedSleep comorbidities with systemic diseasesEndocrine disordersThyroid diseaseConsidering the various sleep disorders and diagnostic tools afforded by a goodsleep history and sleep testing understanding the relationship between sleepdisorders and systemic diseases has farreaching implications in optimizing thecare of the patient The following sections will address sleep manifestations ofvarious neurological disorders arising from systemic disease based on an systemAlmost half of the patients with hypothyroidism report at least one sleep complaint such as restless sleep choking hypersomnia or fatigue [] OSA is presentin approximately [] A unique mechanism of airway restriction in hypothyroidism is myxedematous mucoprotein deposition in the airway™s soft tissuesand dilator muscles even though myxedema can be absent [] Larger goiters canalso cause OSA by external compression of the airway []On the other side of the thyroid spectrum hyperthyroidism is most closelyassociated with insomnia occurring in of patients [] Arousaldisorders”specifically sleep walking”also occur especially in the setting ofthyrotoxicosis [] proposed to arise from frequent arousals and impairmentof attaining slowwave sleep as the direct result of thyroid hormoneBeyond the treatment of the specific sleep disorder sleep problems usuallyremit following appropriate treatment of the underlying thyroid disorder []Type diabetes mellitusSleep disorders affect high proportions of those with type diabetes mellitusDM surveys of patients with DM compared with those of controls show a 0cCurr Treat Options Neurol Page of nearly 2fold propensity for insomnia fourfold higher use of sedativehypnoticsand a 10fold higher rate of hypersomnolence [] OSA is highly prevalent inDM and many are undiagnosed [] Contributors to a multifactorial series ofsleep disruptors include periodic limb movements and restless legs syndromeRLS diabetic neuropathy and fluctuations in blood glucose []DM presents an excellent model by which to demonstrate the reciprocaleffects of sleep disruption on the primary disease First sleep disturbances affectthe regulation of the neuroendocrine control of appetite Sleep deprivationpromotes overeating through hyperactivity of orexin system [] and activatesthe hypothalamicpituitaryadrenal system to increase cortisol secretionresulting in impaired glucose tolerance [ ] These multiple mechanismssupport clinical observations that untreated OSA may be reason for the ineffective treatment of DM and that accordingly treatment with CPAP leads toimprovements in glycemic control in some patients []Sex hormones and gender affect the distribution and susceptibility to a varietyof sleep disorders Men on the basis of relative airway collapsibility haveapproximately a twofold increased risk of OSA compared with women “ in males and “ in females [] A potential side effect in thetreatment of hypoandrogenism is the facilitation of OSA given the impacttestosterone has on upper airway collapsibility []Testosterone levels may affect the propensity for chronic insomnia Menwith hypoandrogenism demonstrate reduced sleep efficiency increased nighttime awakenings and reduced deep sleep compared with the normaltestosteronelevel controls although it is not clear whether these features improve with testosterone therapy [] Women experience higher rates of chronicinsomnia risk ratio of for women versus men which becomes even morepronounced in the elderly [] Despite sleeping longer overall sleep quality isoften lower in women than men []The distribution of sleep disorders in women varies with reproductivelifespan Younger women are more susceptible to restless legs syndromeRLS mainly on the basis of mensesassociated irondeficiency During pregnancy women are at significantly increased risk for the development of RLSwith an overall prevalence exceeding of all pregnant patients [] Treatment of RLS in pregnancy involves iron supplementation with a goal ferritinlevel mcgl Often oral iron repletion is adequate although there arereports of intravenous iron therapy in severe cases of pregnancyrelated RLSand irondeficiency [] Pregnancy is also associated with an increased prevalence of OSA up to of pregnant patients during the third trimester whichis associated with increased risks of complications including gestational hypertension gestational DM and preeclampsia []Although not a particular systemic neurological disease pharmacological effectson sleep form an important aspect of neurological sleep medicine since manymedications that are used by neurologists may affect sleep Table showscommon medications that provoke insomnia hypersomnolence respiratorysuppression parasomnias and RLSperiodic limb movement disorderSex hormonesMedications 0c Page of Curr Treat Options Neurol Table Medication classes and specific examples that can cause sleep disturbancesInsomniaCentral nervous system stimulants methylphenidate amphetamines modafinilCaffeineAntidepressantsSelective serotonergic reuptake inhibitors fluoxetine sertralineSelective norepinephrine reuptake inhibitors venlafaxine duloxetineSecondary tricyclic antidepressants desipramine nortriptylineCardiovascularBeta2 agonists albuterolVasopressors epinephrine dopamineCorticosteroidsSympathetic amines phentermineHypersomniaBenzodiazepines alprazolam diazepamNonbenzodiazepine receptor agonists zolpidem eszopicloneOpioidsH1 antihistamines diphenhydramineAntiepileptic agents phenytoin levetiracetamAntidepressantsSelective serotonergic reuptake inhibitors paroxetine sertralineTertiary tricyclic antidepressants amitriptylineTypical and atypical antipsychotics haloperidol olanzapineDopaminergic agonists ropinirole carbidopalevodopaAnticholinergic medicationsCentrally acting α agonists clonidine dexmedetomidineRespiratory suppressionOpioids oxycodone morphineBenzodiazepines diazepam clonazepamAlcoholPhenobarbitalParasomniasAntidepressants clomipramine fluoxetine citalopramNonbenzodiazepine receptor agonists zolpidemCaffeineAlcohol withdrawalRestless legs syndrome and periodic limb movementsSelective serotonergic reuptake inhibitors fluoxetine mirtazapineAntipsychotics haloperidol risperidoneTricyclic antidepressants amitriptyline clomipramine 0cCurr Treat Options Neurol Page of Renal diseaseInfectious diseasesSleep disturbances are highly prevalent in patients with chronic kidney diseaseCKD spanning the broad spectrum of sleep disorders including hypersomniainsomnia sleeprelated breathing and RLSThe prevalence of OSA in CKD ranges from to rates that are notexplained solely by overlapping comorbidities common to both OSA and CKD[] The cooccurrence of both CKD and OSA is associated with increasedcardiovascular events and allcause mortality [“] Usually OSA develops inpatients with CKD independent of underlying renal dysfunction but someevidence shows that CKD can cause or exacerbate OSA and central sleep apneaProposed mechanisms for this causal relationship include uremic neuropathyaltered chemosensitivity and hypervolemia [] Accordingly renal replacement therapy and fluid removal [] may improve obstructive or central sleepapnea Conversely treatment of sleep apnea with PAP may improve renalfunction in those with borderline renal impairment []RLS is a common and debilitating symptom in patients with CKD occurringin up to of patients on hemodialysis compared with that in approximately of the general population [] Although RLS symptoms generally follow acircadian rhythmicity with increased symptoms occurring at night RLS symptoms can occur during the long periods of daytime inactivity during hemodialysis [] Treatment is primarily focused on ensuring adequate iron stores thenconsidering medical therapy as per routine care of RLSSleep disorders and infectious diseases have few specific associations In general acute infection is associated with mild encephalopathy that masquerades ashypersomnolence and fatigue Proinflammatory cytokines are implicated inthe development of these constitutional symptoms Some infections howeverdirectly affect regulatory centers of the sleepwake systemEncephalitis lethargica is a historical pandemic cause of hypersomnolence ofrenewed interest since this review is being written in the middle of the COVID pandemic Also known as Von Economo™s encephalitis it occurred inassociation with the Spanish flu pandemic of [] An estimated millionwere affected worldwide The most common subtype the somnolentophthalmoplegic form developed after flulike symptoms of fever and malaiseand consisted of subsequent ophthalmoplegia accompanied by long periods ofhypersomnia Despite the appearance of deep sleep patients could be easilyawoken and sometimes maintained memories of activities that had transpiredaround them while œasleep This state of acute akinetic psuedosomnulencecould be followed by the development of chronic postencephaliticparkinsonismThe pandemic associated with the severe acute respiratory syndrome coronavirus SARSCoV2 ie COVID19 occurring during the writing of thisreview features evolving literature The first reports centered on respiratorysymptoms Although the involvement of the nervous system now appearsprevalent [] sleep disorders have yet to be specifically reported Howeverthe psychological responses to social distancing change in schedules and otherfeatures of an active pandemic have caused a wave of anxiety and depressionwhich in turn have been associated with poor sleep quality For example a 0c Page of Curr Treat Options Neurol survey of Chinese health care workers showed prevalences of depressionat anxiety at and insomnia at []Postinfectious or postvaccination narcolepsy is rare but is important in developing overall hypotheses in the etiology of idiopathic narcolepsy In certainvaccinations in Europe for the H1N1 pandemic caused narcolepsy at a risk of in pediatric patients [] Fortunately the risk of postvaccinationnarcolepsy appeared confined to specific vaccine formulations The incidenthowever has led to ongoing research in the immunological etiology ofnarcolepsyAfrican trypanosomiasis or sleeping sickness remains important in the developing world It is a parasitic infection spread by the tsetse fly that is endemic insubSaharan Africa The first symptoms include fever headaches and lymphadenopathy Once the parasite enters the central nervous system disorderedfragmented sleep ensues often with inversion of the circadian sleepwake cycleThe World Health anization outlines treatment with a regimen of antiparasitic medications once symptoms have started []Nonalcoholic fatty liver disease NAFLD consists of idiopathic hepatic steatosiswith a prevalence of to of the general population with increasedfrequency in individuals with obesity or DM [] Given these coassociationsOSA is common Untreated OSA may exacerbate liver injury because of oxidative stress and systemic inflammation [] and is a risk in conversion fromNAFLD to liver fibrosis [] Trials with CPAP have shown inconsistent resultsin markers of liver injury following treatment of OSA []The symptoms of gastroesophageal reflux disease GERD worsen during sleepparticularly if sleep occurs soon after a meal [] The lower esophageal sphincter that normally prevents reflux may be compromised by the increase inthoracic pressure in the setting of the upper airway obstruction [] Patientswith symptoms of GERD should be screened for OSA and conversely interruption of sleep in absence of OSA may improve with treatment with a protonpump inhibitor PPI [] or by simply elevating the head of the bedInflammatory bowel disease IBD has bilateral interactions with sleep []Given the relationship between sleep deprivationfragmentation on cytokineregulation and immune dysfunction it is hypothesized that poor sleep qualityworsens overall symptoms of IBD [ ] Additionally the proinflammatorystate disrupts the circadian rhythm [] Subjective and objective measurementsof sleep quality and timing should be considered in patients with IBD particularly in those who have frequent inflammatory flares despite otherwise adequate management An algorithmic approach to sleep assessment in IBD patients has been proposed by Canakis et al []Systemic lupus erythematosus and rheumatoid arthritis serve as the prototypical diseases of this group of disorders with a prevalence of sleep disturbancesof greater than [] The mechanisms of sleep disturbances as well as thereciprocal relationship in the contribution of poor sleep to worse autoimmunestatus are thought to be similar to those described above with IBD [ ] Thespecific sleep disorders prevalent in this group are OSA and periodic limbGastrointestinal systemAutoimmune disorders 0cCurr Treat Options Neurol Page of Pulmonarymovement disorder PLMD both with greater than prevalence [ ]As seen above hypersomnolence and activitylimiting fatigue arise from specificsleep disorders pain and medication side effects well as the primary effects ofthe primary proinflammatory status [ ] Often treating the underlyingautoimmune disorder improves associated fatigue However if sleepiness persists then evaluating for a comorbid sleep disorder such as obstructive sleepapnea is indicatedOne syndrome with possible autoimmune origins is chronic fatigue syndromeSleep disturbances insomnia and unrefreshing sleep are common symptoms yetpatients rarely report relief despite appropriate identification and treatment ofcomorbid sleep disorders [] Cognitivebehavioral therapy CBT and gradedexercise therapy are commonly pursued treatment approaches []Obstructive lung diseases most commonly asthma chronic obstructive pulmonary disease COPD and less common disorders such as cystic fibrosisCF or bronchiolitis obliterans may affect nocturnal ventilation OSA andCOPD often overlap given shared body habitus and other mutual risk factorsestimates of comorbid OSA and COPD range from to [] Patients withsevere COPD treated with nocturnal noninvasive ventilation NIPPV a moreadvanced form of positive airway pressure experience an absolute risk reduction of of the risk of hospital readmission or death at months compared with those treated with standard care and without NIPPV [64cid129]Insomnia is another common complaint among patients with COPD Circadian bronchial constriction may cause nocturnal wheezing dyspnea or othersymptoms of asthma prompting the patient to awaken [] In addition thehyperadrenergic response to beta agonist inhalers used in treatment for acutedyspnea impairs sleep onset see Table The growing success in treatments for CF patients means that sleep disordersarising from their intrinsic obstructive lung disease are now coming to theattention of caregivers Many factors contribute to sleep disruption includingchronic cough frequent infections abdominal discomfort reflux frequentstools medication side effects and psychological disease [] In addition tosleep disruption patients with CF are susceptible to hypoventilation thatworsens with disease progression Use of NIPPV in highrisk patients withhypercapnia has been shown to improve physiologic parameters and at timescan positively impact symptoms particularly in patients who have severedisease while awaiting lung transplant []Restrictive lung diseases defined by a reduced total lung capacity includethose with parenchymal damage such as idiopathic fibrosis hypersensitivitypneumonitis or other interstitial pneumonias Alternatively lung parenchymais normal in restrictive diseases such as obesity hypoventilation syndromehemidiaphragm paresis or neuromuscular disorders muscular dystrophiesamyotrophic lateral sclerosis Restrictive lung disease patients as seen abovewith obstructive disease patients are susceptible to nocturnal hypoventilationsubsequent CO2 retention and compensatory sleep fragmentation Use ofNIPPV in patients with severe restrictive lung disease spanning obesityhypoventilation syndrome to muscular dystrophies and ALS has had positiveimpacts on survival and quality of life [ ] 0c Page of CardiacCurr Treat Options Neurol Over of patients with congestive heart failure CHF have comorbid OSAmainly on the basis of mutual risk factors of DM hypertension obesity andolder age [ ] In addition insomnia in those with CHF may arise from avariety of factors including diuretic medications and subsequent nocturiapositional heart failure symptoms increased adrenergic status or psychosocialfactors [] Treatments addressing comorbid OSA and insomnia improve sleepquality but demonstrate mixed results in terms of longterm cardiovascularoutcomes [ ]Patients with acute myocardial infarction AMI experience both acute andchronic sleep disorders Due to the circadian variability of adrenergic hormonesand cardiac and systemic vasculature [] the timings of AMI sudden cardiacdeath and arrhythmia occur with increased frequency at night [] Cardiacischemia may present a series of nocturnal symptoms including paroxysmaldyspnea chest pain agitation or insomnia Surviving patients are at risk forchronic sleep disorders such as insomnia and sleepdisordered breathing withor without the cooccurrence of anxiety or depression []Retrospective longitudinal data demonstrate that those with OSA and whoare adherent with CPAP experience improved cardiovascular morbidity andmortality over nonadherent patients [] However these findings have notbeen clearly supported by prospective randomized trials The Sleep ApneacardioVascular Endpoints Trial SAVE Trial has called into question the causallink between the treatment of OSA and cardiovascular outcomes With a meanfollowup of years those randomized to PAP experienced no significantimprovements in study endpoints of death from cardiovascular causes AMIstroke and hospitalization for unstable angina CHF or transient ischemicattack compared with controls [78cid129cid129] Because of possible insufficient CPAPuse and because of the lack of main indications for CPAP treatment such assevere sleepiness interpretation of the findings of this large trial remainscontroversial In practice these authors often pursue CPAP treatment for patients with OSA and cardiovascular risk factors even in the absence of sleepiness at least for a trial period to assess adherence to treatment and to determineif there are subjective and objective improvements to sleep qualityWith a prevalence range of “ OSA is common in patients with atrialfibrillation and other arrhythmias [] Accordingly the Sleep Heart HealthStudy showed a two to fivefold higher risk of arrhythmia in patients with severeOSA compared with that in controls [] Retrospective series show that inpatients with atrial fibrillation and untreated OSA the risk of atrial fibrillationrecurrence following cardioversion is compared with in patients whoare adherent to CPAP [] However a prospective randomized control trialcalled retrospective findings into question [] Similar in design to the SAVETrial patients with atrial fibrillation were randomized to CPAP versus usualtherapy from a cohort in which sleepiness was specifically excluded This smalltrial total assessed the primary outcome of time to arrhythmia recurrenceBoth arms had recurrence rates of Although the trial showed that CPAPitself provides no specific benefit to those with atrial fibrillation the outcomesfor treatment of those with both disorders remain unclearAlthough the above studies centered on associations between cardiac diseaseand OSA patients with CHF AMI and atrial fibrillation experience high rates of 0cCurr Treat Options Neurol Page of CancerCritical illnesscentral sleep apnea CSA as well exceeding in patients with mild symptomatic CHF as an example [] CheyneStokes respiration a cyclical form ofCSA results when circulatory impairment perturbs the normal responsivenessin respiratory control resulting in alterations in œthe loop gain in modulatingchanges in carbon dioxide and oxygen levels in the bloodstream [] analogous to overly aggressive adjustments to a thermostat in response to changingtemperature The presence of CSA has been considered a marker of increasedmortality in patients with CHF although aims to resolve the treatment of CSAwith CPAP or more advanced modalities have not clearly demonstrated animprovement in cardiovascular outcomes []Estimates of the prevalence of sleep disturbances across cancer patients range widelyfrom to [ ] Insomnia is the most common disorder with prevalencelevels ranging from to [ ] Patients with cancer who undergo PSGhave shorter total sleep times longer times in bed low sleep efficiency andproportionately less deep sleep than controls [] Insomnia in patients with canceris driven by a multitude of factors including preexisting socioeconomic andpsychiatric disorders fatigue age RLS pain and medication effects [ ]Treatment follows that for the general population Although sedativehypnoticsare most commonly prescribed no evidence exists for specific pharmacologicinterventions for sleep disturbances in this population [] Cognitivebehavioraltherapy is currently the recommended firstline treatment for chronic insomnia[] Because the rarity of trained psychologists makes finding a provider difficult insome circumstances the electronic delivery of cognitivebehavioral therapy hasbeen sought as an alternative to facetoface therapy [ ]The bilateral interactions between sleep and critical illness form a rapidlychanging area of investigation which is made particularly challenging giventhe difficulties in measuring sleep in critically ill patients [ ] Lack ofsleep”or its encephalopathic analog”may affect outcomes in critical illnessesFor example a lack of scorable REM sleep correlates with longer ventilatorweaning time compared with controls with intact REM [] Failure rates onnoninvasive ventilation are impacted by sleep continuity [] Delirium acommon neurobehavioral syndrome seen in upwards of of patients inthe ICU [ ] is associated with significantly worse outcomes i

endometrial cancer ec is a common malignancy of the female reproductive system circular rnas circrnas were demonstrated to exert critical roles in cancers including ec this study aimed to investigate the effects of hsa_circrna_0001776 circ_0001776 on ecmethods realtime quantitative pcr rtqpcr was used to measure circ_0001776 microrna182 mir182 and leucinerich repeats and immunoglobulinlike domains lrig2 expression the diagnostic and prognostic values of circ_0001776 were identified by receiver operating characteristic roc curve analysis and survival analysis respectively rnase r digestion was used to characterize circ_0001776 and the localization of circ_0001776 was evaluated by cell fractionation assay then cell counting kit8 cck8 colony formation and flow cytometry analysis were used to detect cell proliferation and apoptosis respectively the realtime glycolytic rate ecar and lactate production were measured by extracellular flux analysis and a lactate assay kit respectively bioinformatics analysis and dualluciferase reporter assay were used to determine the interaction among circ_0001776 mir182 and lrig2 the protein expression of lrig2 was determined by western blot moreover circ_0001776 overexpression vector was used to upregulate circ_0001776 expression in an animal tumor modelresults circ_0001776 and lrig2 were downregulated while mir182 was upregulated in ec tissues and cells low expression of circ_0001776 was correlated with the 5year survival rate of ec patients upregulated circ_0001776 markedly attenuated cell proliferation and glycolysis and enhanced cell apoptosis besides circ_0001776 sponged mir182 to regulate lrig2 expression circ_0001776 could suppress ec progression by mir182lrig2 axis furthermore we also found that circ_0001776 significantly inhibited tumor growth in vivo our results confirmed that circ_0001776 inhibited ec tumorigenesis and progression via mir182lrig2 axis providing a potential therapeutic target for eckeywords endometrial cancer hsa_circrna_0001776 mir182 lrig2correspondence xgiaee163com department of obstetrics weifang people™s hospital weifang shandong chinafull list of author information is available at the end of the the authors this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons licence and indicate if changes were made the images or other third party material in this are included in the ™s creative commons licence unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cjia a0et a0al cancer cell int page of highlights circ_0001776 was lowly expressed in endometrial cancer tissues and cells circ_0001776 overexpression inhibited endometrial cancer cell proliferation and glycolysis and promoted cell apoptosis circ_0001776 upregulated expression lrig2 through targeting mir182 circ_0001776 suppressed endometrial cancer progression via mir182lrig2 axis endometrial cancer ec mainly occurred in postmenopausal women is one of the most common malignancies of the female reproductive system with the incidence of about in worldwide ec is conventionally classified into type i estrogendependent ec and type ii estrogennondependent ec according to molecular genetic features and clinicopathological features and grades and are regarded as œtype i while grade is regarded as œtype ii [ ] the prognosis of œtype i ec patients was relatively favorable while œtype ii ec was always accompanied by the poor outcomes however the early diagnosis of ec was extremely difficult due to the complex uterus endocrine function and the unadvanced technology of anatomy thus it is meaningful to explore the potential targets for ec diagnosis and treatmentcircular rnas circrnas are a class of abundant endogenous conserved noncoding rnas which have a circular structure lacking ² poly a tails and ² caps [“] altered expression of circrnas was found in numerous cancers such as hsa_circ_0001313 in colon cancer circbanp in lung cancer hsa_circ_0072995 in breast cancer several circrnas were confirmed to be the ideal biomarkers for the diagnosis treatment and prognosis of various human cancers [ ] for example hsa_circ_0052112 could regulate breast cancer tumorigenesis through facilitating cell metastasis additionally hsa_circrna_0001776 circ_0001776 was downregulated in ec tissues however the regulatory effects of circ_0001776 in ec remain largely unknown and the underlying mechanisms need further understandingmicrornas mirnas are a group of small singlestranded noncoding rnas with the length of “ nucleotides which can be involved in the pathological and physical processes including cell survival proliferation and metastasis in various tumors [“] recent studies showed that circrnas could sponge mirnas to exert the regulatory effects for instance repeats circ_0044516 could target mir29a3p to facilitate cell metastasis in prostate cancer microrna182 mir was confirmed to aberrantly express in ec in this research circinteractome showed that mir182 might be a target of circ_0001776 we aimed to explore the functional effects of circ_0001776 and mir182 on ec tumorigenesisleucinerich immunoglobulinlike domains lrig2 is a member of lrig protein family which harbored a single transmembrane domain [ ] lrig2 was demonstrated to play a suppressive role in ec the molecular mechanism of lrig2 in ec remains unclear in this study we predicted that lrig2 contained the potential binding site of mir182 thus we aimed to explore the functional role of lrig2 in ec mechanically we investigated the relationship among circ_0001776 mir182 and lrig2 and their effects on regulating the tumorigenesis and progression of ecand material and a0methodstissues sampleshuman normal endometrial tissue samples were collected from healthy volunteers while the tumorous tissues were obtained from endometrial cancer patients who underwent surgery at weifang people™s hospital the clinicopathologic features of these patients were presented in table a0 all participants signed written table correlation between a0 circ_0001776 expression with a0clinicopathologic features of a0ecparametersncirc_0001776 expressionhigh n low n page years histological stage g1 g2 g3lymph node metastasis yes nofigo stage i ii iii iver expression positive negativepr expression positive negative p p 0cjia a0et a0al cancer cell int page of informed consent all experiments in this study were approved by the human research ethics committee of weifang people™s hospitalcell linesrl952 ishikawa hec1a hec1b and an3ca cells were human endometrial cancer cell line while heec cells were normal human endometrial epithelial cells rl952 hec1a and hec1b cells purchased from american type culture collection manassas va usa and heec ishikawa and an3ca cells obtained from shanghai zeye biological technology co ltd shanghai china were used in this study dulbecco™s modified eagle™s medium dmem gibco rockville md usa containing streptomycinpenicillin uml invitrogen carlsbad ca usa and fetal bovine serum fbs invitrogen was used to incubate the cells in a humidified chamber while the cell culture condition was co2 at ˚ccell transfectionrl952 and ishikawa cells were selected for the following experiments circ_0001776 overexpression vector circ_0001776 and empty vector vector mir182 mimics mir182 and the control mirnc mir182 inhibitor antimir182 and the corresponding control antinc and small interfering rna sirna targeting lrig2 silrig2 and the control group sinc genepharma shanghai china were transfected into rl952 and ishikawa cells by lipofectamine reagent invitrogenreal‘time quantitative polymerase chain reaction rt‘qpcrtotal rna was extracted from nontumorous tissues and tumor tissues as well as endometrial cancer cells and heec cells using trizol reagent invitrogen and a0µg of total rna was reversetranscribed using superscript iii rt invitrogen the specific gene amplified transcript level was measured by a realtime quantitative pcr system u6 and glyceraldehyde 3phosphate dehydrogenase gapdh were used to normalize the expression of hk2 mir182 circ_0001776 and lrig2 respectively the specific primer sequences were as follows circ_0001776 tca aac ctc gac aag gtg ct sense and cct tag aac acc cgg aag gt antisense mir182 gag aac agc agg tcc agc at sense and ctt cct cag cac aga ccg ag antisense lrig2 cag tgc ata gct gga ggg agtc sense and tac aat gat gag aag ctg att ggc tgca antisense hk2 caa agt gac agt ggg tgt gg sense and gcc agg tcc ttc act gtc tcantisense u6 ctc gct tcg gca gcaca sense and aac gct tca cga att tgc gt antisense gapdh aag gct gag aat ggg aaa c sense and ttc agg gac ttg tca tac ttc antisense the relative expression of circ_0001776 mir182 and lrig2 was assessed by the ˆ’ˆ†ˆ†ct methodrnase r digestion ug total rna isolated from rl952 and ishikawa cells were incubated with units of rnase r epicenter biotechnologies shanghai china for a0min at a0°c after the incubation of total rna and rnase r the expression of circ_0001776 was determined through rtqpcrsubcellular localizationthe localization of circ_0001776 was evaluated by cytoplasmic nuclear rna purification kit nen biotek corp belmont ma usa briefly cells were lysed by lysis buffer j and then centrifuged subsequently the cytoplasmic rna and nuclear rna were treated with buffer sk and anhydrous ethanol respectively then the nuclear rna and cytoplasmic rna were eluted using the spin column finally the expression of circ_0001776 in cytoplasmic and nucleus fractions was detected by rtqpcrcell counting kit‘ cck‘ assaythe proliferation ability of rl952 and ishikawa cells was assessed by cck8 assay × a0cells in a0μl cell suspension were seeded into the 96well plates and cultured for a0h then cells were treated with a0μl cck8 solution dojindo tokyo japan and the absorbance was detected at and a0h after transfection at a0nm respectivelycolony formation assaythe selected rl952 and ishikawa cells were plated into the 6well plates after incubation for a0weeks cells were fixed with paraformaldehyde stained with crystal violet and counted using a microscope biorad laboratories inc hercules ca usacell apoptosis assaythe apoptotic cells were detected by flow cytometry analysis using an annexin vfluorescein isothiocyanate fitc apoptosis detection kit bd pharmingen san diego ca usa in brief cells were treated with trypsin gibco and then centrifuged and removed the supernatant subsequently cells were washed and resuspended with the binding buffer then fitc and propidium iodide pi were added in dark for a0min finally cell apoptosis was assessed by a facscalibur flow cytometer bd pharmingen 0cjia a0et a0al cancer cell int page of determination of a0the a0real‘time glycolytic rate ecarin this study we used the seahorse extracellular flux analyzer xf96 seahorse bioscience north billerica ma usa to monitor cell metabolic alternation in a0vitro after cell transfection cells with a density of × per well were added into the xf96well plate and incubated overnight then cells were starved with the nonserum medium for a0h for measurement of ecar cells were cultured with unbuffered medium following sequentially injected glucose oligomycin om and 2deoxyglucose 2dg ecar detection was noted as mphmindetermination of a0lactateafter the transfection of rl952 and ishikawa cells cells were treated with nonserum medium and starved for a0h then the cell culture medium was collected to detect the lactate production using a lactate assay kit keygen nanjing china lactate production was normalized on the basis of the total protein concentrationcarrying the widetype dual‘luciferase reporter assayplasmids circ_0001776 circ_0001776 wt or mutanttype circ_0001776 circ_0001776 mut were cotransfected with mir182 mimics or mirnc in ec cells while wildtype sequence of lrig2 ²utr lrig2 wt or mutant sequence of lrig2 ²utr lrig2 mut plus mir182 mimics or mirnc were cotransfected into rl952 and ishikawa cells after transfection the relative luciferase activity was detected by the dualluciferase reporter assay system promega madison wi usawestern blottotal protein was isolated from the ec tissues and normal tissues as well as rl952 and ishikawa cells by ripa lysis buffer beyotime shanghai china briefly the total protein was separated using “ odium dodecyl sulfate“polyacrylamide gel electrophoresis and then the proteins were transferred onto the polyvinylidene difluoride membranes sigma billerica ma usa subsequently the membranes were blocked using nonfat milk and incubated with the primary antibodies against lrig2 ab157492 abcam cambridge uk hk2 ab104836 abcam and gapdh ab8245 abcam overnight at a0°c then the blots were incubated with goat antirabbit ab6721 abcam or antimouse ab205719 abcam for a0h finally the protein signals were calculated by an electrochemiluminescent system perkinelmer life science waltham ma usamouse xenotransplantation a0 weeks old balbc mice were purchased from the beijing laboratory animal center beijing china mice were randomly divided into two groups vector and circ_0001776 rl952 cells stably transfected with circ_0001776 overexpression vector and empty vector were injected into the right flank of the mice after a0days tumor volume was detected every a0days after the mice euthanasia on day tumor weight was measured all the in a0 vivo experiments were approved by weifang people™s hospital experimental animal ethics committeestatistical analysisall statistical analyses were performed using spss software data were expressed as the mean ± the standard deviations sd the interaction between variables was analyzed by pearson correlation analysis student™s ttest and oneway anova analysis were performed for comparisons the diagnosed value was evaluated by the receiver operating characteristic roc curve analysis and the area under the curve auc meant no diagnostic value a value of p was considered as a statistically significant differencein ec resultscirc_0001776 was a0downregulated in a0endometrial cancertissues and cells circ_0001776 expression was measured using rtqpcr as shown in fig a0 1a circ_0001776 was lowly expressed in ec tissues in comparison to that in normal tissues compared with the grades in and g1 g2 ec tissues circ_0001776 had a lower expression level in grade g3 ec tissues fig a01b besides we constructed the roc curve to explore the potential value of circ_0001776 as described in fig a01c circ_0001776 was confirmed to have the diagnosis value of auc p more importantly we observed that the low expression of circ_0008285 was closely related to the poor survival of ec patients after surgery fig a01d furthermore circ_0001776 expression was decreased in rl952 ishikawa hec1a hec1b and an3ca cells relative to that in heec cells fig a01e all these data demonstrated that circ_0001776 was decreased in ec which might be a biomarker in ec progressioncirc_0001776 overexpression suppressed proliferation and a0facilitated apoptosis of a0ec cellsto elucidate whether circ_0001776 played a key role in ec tumorigenesis the function assays were performed firstly we confirmed that circ_0001776 was indeed circrna which was resistant to rnase r digestion in both 0cjia a0et a0al cancer cell int page of fig circ_0001776 was downregulated in endometrial cancer a rtqpcr was used to detect the expression of circ_0001776 in ec tissues b the expression of circ_0001776 in g1 g2 and g3 ec tissues was measured by rtqpcr c the roc of circ_0001776 as a biomarker was evaluated d survival was analyzed and compared between patients with high and low levels of circ_0001776 using kaplan“meier analysis e the expression of circ_0001776 in ec cells was assessed by rtqpcr p rl952 and ishikawa cells fig a02a b then we detected the subcellular localization of circ_0001776 in ec cells the results showed that circ_0001776 was mostly located in the cytoplasm of ec cells fig a02c d as expected the expression of circ_0001776 was significantly increased in rl952 ishikawa and heec cells transfected with circ_0001776 fig a0 2e f and additional file a0 fig s1a cck8 assay indicated that circ_0001776 overexpression markedly inhibited cell proliferation in rl952 and ishikawa cells compared with the control group fig a02g h similarly we observed the reduction of cell colonies number in rl952 and ishikawa cells transfected with circ_0001776 fig a0 2i additionally we measured the effects of circ_0001776 overexpression on cell apoptosis the data suggested that overexpression of circ_0001776 significantly induced cell apoptosis in ec cells fig a02j moreover we detected the protein levels of cleavedcasp3total casp3 and cleavedcasp9total casp9 in ec cells transfected with vector or circ_0001776 the data showed that circ_0001776 overexpression increased the protein expression of cleavedcasp3 and cleavedcasp9 in ec cells additional file a0 fig s2a b however circ_0001776 overexpression showed no significant effects on cell proliferation the number of colonies and apoptosis in heec cells additional file a0 fig s1b“d these results demonstrated that circ_0001776 was a suppressive circrna in ec tumorigenesiscirc_0001776 overexpression inhibited the a0glycolytic metabolism in a0ec cellsnext we explored whether overexpression of circ_0001776 could directly affect glycolytic metabolism in ec cells through detecting extracellular acidification rate ecar and lactate2 production circ_0001776 upregulation significantly reduced ecar in rl952 and ishikawa cells fig a03a b overexpression of circ_0001776 did not change ecar level in heec cells additional file a0 fig s1e moreover lactic acid production was markedly decreased by circ_0001776 overexpression in rl952 and ishikawa cells fig a0 3c we further detected the mrna and protein expression of hk2 and the data showed that circ_0001776 upregulation significantly suppressed hk expression fig a03d e overall our data suggested that circ_0001776 could regulate glycolytic metabolism in ec cellslevel circ_0001776 sponged mir‘circinteractome showed that mir182 contained the binding sites of circ_0001776 fig a0 4a moreover the relative luciferase activity was significantly decreased in rl952 and ishikawa cells cotransfected with circ_0001776 wt and mir182 mimic compared with the control group while there was no significant change of the luciferase activity in ec cells cotransfected with 0cjia a0et a0al cancer cell int page of fig circ_0001776 overexpression suppressed proliferation and facilitated apoptosis of ec cells a b circ_0001776 resistance to rnase r was detected by rtqpcr c d rtqpcr was used to assess the levels of cytoplasmic control transcript gapdh nuclear control transcript u6 and circ_0001776 in nuclear and cytoplasmic fractions e f the overexpression efficiency of circ_0001776 was evaluated by rtqpcr g“i cell proliferation in ec cells transfected with circ_0001776 or vector was detected by cck8 assay and colony formation assays j cell apoptosis in ec cells transfected with circ_0001776 or vector was measured by flow cytometry analysis p circ_0001776 mut and mir182 mimics fig a04b c as shown in fig a0 4d mir182 was highly expressed in ec tumor tissues relative to that in normal tissues besides the expression of mir182 was inversely correlated with circ_0001776 level in ec tissues fig a0 4e consistently mir182 level was also facilitated in rl952 and ishikawa cells compared with heec cells fig a04f moreover overexpression of circ_0001776 could significantly repress mir182 expression in ec cells fig a0 4g these data revealed that circ_0001776 could serve as a competing endogenous rna to sponge mir182circ_0001776 overexpression inhibited ec progression by a0sponging mir‘to further investigate whether circ_0001776 exerted its suppressive functions on tumor through mir182 rescue experiments were performed using mir182 mimics in ec cells with circ_0001776 overexpression rtqpcr demonstrated that mir182 expression was dramatically increased in ec cells transfected with mir182 mimics fig a0 5a b subsequently we performed the functional experiments in ec cells cck8 assay indicated that the inhibitory effect of circ_0001776 overexpression on cell proliferation was reversed by upregulating mir182 in both rl952 and ishikawa cells fig a05c d we also discovered that mir182 overexpression significantly rescued the reduction of colonies induced by circ_0001776 overexpression in ec cells fig a0 5e f flow cytometry analysis indicated that mir182 upregulation markedly reduced circ_0001776 overexpressioninduced cell apoptosis in both rl952 and ishikawa cells fig a05g h furthermore we evaluated the glycolytic metabolism in ec cells the data suggested that circ_0001776 upregulation triggered a decrease of glycolytic metabolism which was reversed by mir182 overexpression fig a05i j consistent with the results in glycolytic metabolism the decrease of lactate production in rl952 and ishikawa cells transfected with circ_0001776 was attenuated by upregulating mir182 fig a05k l taken together circ_0001776 could inhibit cell proliferation and glycolytic metabolism and promote cell apoptosis by targeting mir182 in ec cells 0cjia a0et a0al cancer cell int page of fig circ_0001776 overexpression inhibited the glycolytic metabolism in ec cells a b the change of ecar level with different treatment in rl952 and ishikawa cells was determined after transfecting with control or circ_0001776 overexpression plasmid c the relative lactic acid level in rl952 and ishikawa cells transfected with control or circ_0001776 overexpression plasmid was examined d e the mrna and protein expression of hk2 were detected by rtqpcr and western blot p fig circ_0001776 sponged mir182 a circinteractome predicted that mir182 harbored the binding sites of circ_0001776 b c the interaction between circ_0001776 and mir182 was evaluated by the dualluciferase reporter assay d the expression of mir182 in ec tissues was detected by rtqpcr e the relationship between circ_0001776 and mir182 was analyzed by kaplan“meier analysis r ˆ’ p f the expression of mir182 in ec cells and heec cells was examined by rtqpcr g the expression of mir182 in ec cells transfected with vector or circ_0001776 was detected by rtqpcr p 0cjia a0et a0al cancer cell int page of fig circ_0001776 overexpression inhibited ec progression by sponging mir182 a b rtqpcr was used to measure mir182 expression in rl952 and ishikawa cells transfected with mirnc or mir142 c d cell proliferation was measured by cck8 assay after transfection e f cell colony formation assay was used to detect the number of colonies after transfection in ec cells g h flow cytometry was performed to examine cell apoptosis in rl952 and ishikawa cells after transfection i j ecar was determined in ec cells after transfection k l the lactic acid level was measured in ec cells after transfection p lrig2 was a0a a0direct target of a0mir‘to investigate the potential molecular mechanism of mir182 in ec we screened the potential target gene of mir182 via targetscan as shown in fig a06a lrig2 harbored the binding sites of mir182 the luciferase activity was significantly decreased in ec cells cotransfected with lrig2 wt and mir182 mimics compared with the control group while the luciferase activity had no significant change in rl952 and ishikawa cells cotransfected with lrig2 mut and mir182 mimics relative to the corresponding control group fig a06b c moreover rtqpcr and western bolt assays showed that the mrna and protein expression of lrig2 were dramatically attenuated in tumor tissues relative to that in normal tissues fig a0 6d e furthermore lrig2 expression was negatively related to mir182 level fig a06f and positively correlated with circ_0001776 expression fig a06gmoreover the expression of mir182 was significantly decreased in ec cells transfected with antimir182 fig a06h we found that the protein expression of lrig2 was downregulated in ec cells compared with that in heec cells fig a06i as described in fig a06j downregulation of mir182 could increase lrig2 protein expression in ec cells besides overexpression of circ_0001776 facilitated the protein expression of lrig2 while mir182 overexpression reversed the promotion effect in both rl952 and ishikawa cells fig a06k l overall mir182 directly targeted lrig2 and circ_0001776 could sponge mir182 to regulate lrig2 expression in ec cellslrig2 knockdown reversed the a0effects of a0mir‘ downregulation on a0ec cellswe detected the knockdown efficiency of lrig2 in ec cells and observed that transfection of silrig2 could significantly decrease the protein expression of lrig2 in ec cells fig a0 7a as shown in fig a0 7b c the suppressive effect of mir182 downregulation on cell proliferation was blocked by lrig2 deletion in ec cells analogously the colonies were decreased by downregulating mir182 while lrig2 deletion significantly increased the number of colonies in both rl952 and ishikawa cells fig a0 7d e mir182 deletion induced 0cjia a0et a0al cancer cell int page of fig lrig2 was a direct target of mir182 a the binding site of lrig2 and mir182 was predicted by targetscan b c the relative luciferase activity in ec cells was detected by dualluciferase reporter assay d the mrna expression of lrig2 in ec tissues was examined by rtqpcr e western blot was used to detect the protein expression of lrig2 in ec tissues f the negative relationship between mir182 and lrig2 was discovered in ec tissues r ˆ’ p g the positive relationship between circ_0001776 and lrig2 was observed in ec tissues r p h lrig2 expression was determined after ec cells transfected with antinc or antimir182 i the protein expression of lrig2 in ec cells was detected by western blot j lrig2 expression in ec cells transfected with antinc or antimir182 was measured by western blot k l lrig2 expression in ec cells transfected with vector circ_0001776 circ_0001776 mirnc or circ_0001776 mir182 was examined by western blot p cell apoptosis which was restrained by lrig2 downregulation in ec cells fig a07f g moreover the glycolytic metabolism was markedly suppressed in ec cells transfected with antimir182 while lrig2 deletion could reverse the inhibitory effect of mir182 deletion on glycolytic metabolism fig a07h i consistently mir182 deletion reduced the lactate production in ec cells while lirg2 knockdown significantly rescued the reduction of lactate production fig a0 7j k these results indicated that lrig2 downregulation could block the effects of mir182 deletion on cell proliferation apoptosis and glycolytic metabolism in ec cellscirc_0001776 inhibited tumor growth in a0vivoto determine the effects of circ_0001776 on tumor growth in a0 vivo rl952 cells stably transfected with circ_0001776 or vector were injected into the right flank of the mice the result suggested that circ_0001776 overexpression suppressed tumor growth in a0vivo fig a08a b furthermore tumor weight in the circ_0001776 group was significantly lower than that in the control group 0cjia a0et a0al cancer cell int page of fig lrig2 knockdown reversed the effects of mir182 downregulation on ec progression a the expression of lrig2 in ec cells transfected with sinc or silrig2 was determined by western blot b c cell proliferation was measured in ec cells transfected with antinc antimir182 antimir182 sinc and antimir182 silrig2 by cck8 assay d e the number of colonies was assessed in ec cells after transfection f g the apoptotic rate of ec cells after transfection was detected by flow cytometry h i the change of ecra level in ec cells after transfection was determined j k the lactic acid level in ec cells was examined p fig circ_0001776 inhibited tumor growth in vivo a“c the tumor volume and weight were measured in vivo d e the expression of circ_0001776 mir182 and lrig2 in the tumor tissues from balbc mice was detected using rtqpcr and western blot respectively p 0cjia a0et a0al cancer cell int page of fig a0 8c besides we discovered that circ_0001776 expression was upregulated while mir182 was downregulated by circ_0001776 overexpression fig a0 8d moreover the protein expression of lrig2 was significantly facilitated in the tissues extracted from the mice in circ_0001776 group compared with the control group fig a0 8e these data confirmed the suppressive role of circ_0001776 in ec in a0vivodiscussionthe malignant endometrial tumor is the most common female cancer among all reproductive system diseases accompanying with increasingly younger women diagnosed with ec thus it is urgent to develop the novel methods for the diagnosis treatment and prognosis for ec and find out the regulators in the tumorigenesis of ec recently accumulating evidence indicated that circrnas were closely involved in the regulation of the biological processes in human cancers including ec [ ] for instance hsa_circ_0000190 was demonstrated as a novel biomarker for gastric cancer diagnosis which was significantly associated with lymphatic metastasis tumor diameter and distal metastasis in gastric cancer besides circ8073 was confirmed to be a crucial regulator in cell proliferation of endometrial epithelial cells and further targeted mir449a to regulate the pi3kaktmtor pathway in endometrial receptivity development in this research we found a reduction of circ_0001776 expression in ec tissues and cells low expression of circ_0001776 was demonstrated to be related to the grade of ec and the g3 ec existed lower expression than that in g1 g2 ec patients the data in our study were consistent with the results from ye and his colleagues besides we also observed that ec patients with low circ_0001776 expression had a poor survival rate additionally the functional effects of circ_0001776 were explored by performing functional assays as expected overexpression of circ_0001776 significantly inhibited cell growth and glycolytic metabolism and facilitated cell apoptosis in ecseveral researches reported that glycolytic metabolism was a key hallmark in human cancers the activation of glycolysis and the increased lactic acid production were observed in multiple cancer cells which led to the energy metabolism alteration and were correlated with the prognosis of cancer patients a recent study confirmed that the glycolytic activity was positively related to ec cell proliferation through activating the mapk and ampkmtors6 pathways thus we detected the effect of circ_0001776 on glycolytic metabolism in ec cells our data suggested that circ_0001776 overexp

ulcerative colitis is a type of ‚ammatory bowel disease thatcan potentially lead to cancer e age of onset of colitis istypically “ years old and it can seriously threaten thequality of life of patients e immunopathogenesis andimmunosuppressive treatment of colitis are currently theresearch topics of significant interest e research goals areto diagnose and treat colitis in order to prevent exacerbationof the disease e drugs used to treat colitis in the clinicoften have adverse eï¬ects after their longterm applicationanother crucial area of colitis research is focused on thediscovery of functional foods that can prevent colitis withoutside eï¬ects natural plants including aegle marmeloslinn also have the intervention eï¬ect on colitis a recentstudy has shown that the intestinal flora is closely related tocolitis and that the intestinal flora participates in the mucosalimmune response bacteria are an important promoter of‚ammatory bowel disease e symptoms of colitis can bealleviated by regulating the intestinal flora preventing floralimbalance and increasing the number of probiotics yak yoghurt is a natural fermented food that is rich innutrients and is common in the minority areas of theqinghai tibet plateau previous research has suggested thatyak yoghurt exerts various physiological activities such asantioxidationimmunitycholesterolreductionand 0cevidencebased complementary and alternative medicineenhancement e qinghai tibet plateau has a specificclimate and unique environment for the fermentation of yakyoghurt additionally the availability of yak milk and specialtibetan fermentation utensils eg certain fermentationmicroanisms can make the flavor and quality of yakyoghurt diï¬er greatly from ordinary fermented milk aprior study on the intestinal physiological activity of lacticacid bacterial species in yak yoghurt showed that the lacticacidproducing bacteria isolated from yak yoghurt hadantioxidant and constipation preventing eï¬ects in this studythe potential eï¬ects of lactobacillusplantarum ys4 lpys4 on oxazolidoneinduced colitiswere investigated for the first time e findings provide apossible foundation for further development of lpys4especially its application in functional food or medicineratio � solution massnormal group were daub treated with ml of ethanolwith those in the remaining four groups being daub treated with ml of oxazolidonesolvent � after treatment for days the mice wereanesthetized en the blunt head of a silicone tube wasinserted into the intestinal tract from the anus of the mouse at adepth of cm e mice in normal group were administeredwith ml of ethanol solution while those in theremaining four groups were administered with ml of oxazolidone solution mass ratio � solvent � ethanoltwenty seconds later the catheters were removed and the micewere lifted up by their tails for half a minute on the last dayof treatment day all the mice were sacrificed by decapitation and their plasma samples and colon tissues were collected e length and weight of the colon were documentedexperiment animal materials materials and methodsand reagentsexperimental strain the strain was isolated from yak yoghurt in the yushu area of qinghai province china by ourteam it was named lpys4 and stored in china center fortype culture collection cctcc wuhan china nom2016750 e negative control strain lb was purchasedfrom the cctcc no ab fifty male balbc mice weeks old were purchasedfrom the experimental animal center of chongqingmedical university certificate no syxk yu “oxazolone was purchased from sigmaaldrich co llcusa il2 il10 et1 sp ss and vip serum cytokine kitswere purchased from biolegend inc usa gsh sodmpo and mda kits were purchased from nanjing jiancheng bioengineering institute nanjing china trizol reagent oligodt18 rnase dntp mlv primer bca proteinquantitative kit aps temed sdspage pvdf membrane first antibody and second antibody were purchasedfrom ermo fisher scientific inc usa instruments and equipment imark microplate readerwas purchased from biorad usa steponeplus pcrinstrument was purchased from ermo fisher scientificinc usa tanon chemiluminescence imager waspurchased from tanon science and technology co ltdchina sas v91 statistical software package was purchasedfrom sas institute inc usalb animal grouping and intervention a total of balbcmale mice were assigned to five groups model groupnormal group lactobacillus bulgaricustreatmentgroup and highdose lpys4h and lowdoses lpys4l treatment groups and there were mice in each groupe mice in lb lpys4h and lpys4l groups were fedwith — — and — cfukg ml livingbacteria physiological saline of each corresponding straindaily by oral gavage for consecutive days and normal andmodel groups were fed with ml physiological salineafter days of treatment the abdomens of all mice wereshaved with an area of cm — cm e mouse abdomens in detection of endothelin1 et1 substance p spsomatostatin ss and vasoactive intestinal peptide vipconcentrations in serum samples e whole blood samplesof mice were allowed to clot at room temperature for h andthen centrifuged at rpmmin for min after collecting the serum samples the concentrations of et1 sssp and vip were detected using commercial kits determination of interleukin2 il2 and interleukin10il10 levels in serum samples e mouse serum sampleswere prepared according to section en the serumlevels of il2 and il10 cytokines were assessed usingcommercial kits determination of glutathione gsh malondialdehydemda myeloperoxidase mpo and superoxide dismutasesod activities in colon tissues a mixture of g colontissue and ml normal saline was prepared at weightratio after homogenizing the mixture the activities of gshmda mpo and sod in colon tissues were evaluated usingcommercial kits pathological observation of he staining e lesionsite — — cm of colon was cut with a scalpel etrimmed tissue and corresponding label were placed in neutral formalin solution for h e colon tissue wasdehydrated embedded sliced dewaxed stained and thendehydrated transparent and sealed finally the pathologicalstate of colon tissue was observed under a microscopebx43 olympus tokyo japan qpcr assay total rna was isolated using rnazol andthen diluted to the final concentration of μgμl for cnasynthesis μl of the diluted rna extract was taken andcdna was prepared using a reverse transcriptase kit enthe cdna template μl was added into μl of sybrgreen pcr master mix and μl of forward primer andreverse primer each table qpcr amplification wascarried out for cycles under the reaction conditions of95oc s °c s °c s and °c s followed by 0cevidencebased complementary and alternative medicineckitinosenosnnossequencegene nametable sequences of primers used in this studyforward ²agagagatcgggttcaca3²reverse ²cacagaactgagggtaca3²forward ²tcgtccaacttctgggctctt3²reverse ²ccttctcttcctcccctctcttc3²forward ²tcagccatcacagtgttccc3²reverse ²atagcccgcatagcgtatcag3²forward ²catagcccaggtaaagcacaat3²reverse ²gaacactccagaatcgtcaactcforward ²tcagggactacgctgcgaaag3²reverse ²aagagctggcagaccgactca3²forward ²tgcaccaccaactgcttag3²reverse ²gatgcagggatgatgttc3²Î´ctdetection gene δctgapdh measured according to the following equation ˆ’δct �cycle under the reaction conditions of °c s and°c s gapdh was used as internal control for thisdetermination and the relative mrna expression was²gapdhscf western blotting mg of tissue samples was mixedwith μl of pmsf and ml of ripa and then homogenized at rmin 4oc for min protein quantificationwas conducted using the bca protein quantitative kit andthe protein samples were diluted to μgml en thediluted protein and sample buï¬er were mixed at heatedat 100oc for min and icebathed for min subsequentlyacrylamide starting buï¬er resolving buï¬er temed aps and diï¬erentiated water were mixed in specific proportions in order to prepare sdspage separation glue andconcentration glue e prestained samples and proteinladder were placed into the sample hole of the rubber sheetrespectively and then the proteincontaining sdspageglue was subjected to vertical gel electrophoresis for minafter activation with methanol for min the pvdf wereblocked with skimmed milk in — tbst solution for hen the blocked pvdf membranes were rinsed with — tbst followed by incubation with the primary antibodyat °c for h after washing with — tbst for times thesecondary antibody was incubated at °c for h lastly theprotein bands were visualized by supersignal west picoplus chemiluminescent substrate and the images werecaptured using a chemiluminescence imager multiple comparisons were conducted using onewayanova followed by tukey™s test statistical analysis e average value of three experimental results was determined and the statistical softwaresas was used to analyze whether there was a significantdiï¬erence between each group at the level of p longest in normal group ± cm while being the results eï¬ect of lpys4 on colon parameters e experimentalresults demonstrated that the length of mouse colon was thepared to those in the remaining four groups in contrast thethe ratio of colon weightlength was the highest in normalpgml and the lowest concentrations were found for sslpys4h group and these eï¬ects were better than those of eï¬ect of lpys4 on the serum contents of et1 sp ss andvip in mice it can be seen from figure that the serumml was decreased in normal mice compared to that of theremaining four groups e colitis model mice exhibited theopposite results in which the highest concentrations wereserum concentrations of ss and vip in colitis mice andmarkedly decrease those of et1 and sp more importantlythe eï¬ects were better after treatment with lpys4hshortest in the colitis model group ± cm similarlygroup ± while being the lowest in the colitis modelgroup ± figure it was found that lpys4 couldsignificantly p attenuate the decline in colon lengthand weightlength ratio induced by colitis ± cm and± for lpys4l group ± cm and ± forlb ± cm and ± concentrations of ss ± pgml and vip± pgml in normal mice were increased comlevel of et1 ± pgml and sp ± pgobserved for et1 ± pgml and sp ± ± pgml and vip ± pgml interestingly lpys4 could significantly p improve the± ± ± and ± pglb ± ± ± and ±± pgml in colitis model mice was signifigroups p following the treatment with lpys4 anhigher in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml and the serum levels of il10 werelower in lpys4h ± pgml and lpys4ltreated mice ± pgml than in lbtreated mice± pgml p gsh ± μmolmg and sod ± μmolhighest while those of mpo ± mumg and mda± nmolmg were the lowest among the five± μmolmg and sod ± μmolgprotwere significantly reduced p while those of mpo± mumg and mda ± nmolmg wereremarkably increased p in the model group mice eï¬ect of lpys4 on the serum concentrations of il2 andil10 in mice it can be seen from figure that the serumcontentand il10± pgml eï¬ect of lpys4 on the activities of mpo sod gsh andmda in mouse colon as shown in figure the activities ofincrease in il2 and a decrease in il10 cytokine level wereobserved notably the serum levels of il2 were markedlyml and the eï¬ects of lpys4 were better than those ofgroups after induced by oxazolidone the levels of gshcantly reduced and raised compared to the remaining fourgprot in the colon tissue of the normal group were the pgmlofil2 0cevidencebased complementary and alternative medicinebacmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgfigure ac colon length and colon weightcolon length of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4land mda levels p such eï¬ect was stronger comparedto lbtreated mice ± μmolmg ± μmolgprot ± mumg and ± nmolmg more± μmolgprot in colon tissue and markedly decreased those of mpo ± mumg and mda± nmolmg when compared to colitis model groupand the goblet cells were increased compared with the modelgroup among them lpys4h had the most obvious eï¬ecton improving colon tissue which indicated that lpys4 couldreduce the colon injury caused by dss and the high efficiencyeï¬ect was also enhanced with the increase of concentrationlpys4 could markedly attenuate the decline in gsh andsod levels and prevented colitisinduced increase in mpo± μmolmgspecifically treatment with lpys4h significantly increasedthesodlevelsandof pathological observation as shown in figure in thenormal group the epithelial cells of colon mucosa were intactthe ‚ammatory cells were normal without ltration andthe goblet cells were arranged orderly without congestionand edema in the model group the epithelial cells of colontissue were obviously damagedthe intestinal wall wasthickened and edema ‚ammatory cell ltration andgoblet cells were reduced after treatment with lb and lpys4 congestion edema and cell ltration were alleviated eï¬ect of lpys4 on mrna and protein expression inmouse colon as shown in figures and colitis inductioncould lead to the upregulated mrna and protein expressionof inos in mouse colon but downregulated the relativeexpression of ckit enos nnos and scf treatment withlpys4h could increase the relative expression of nnosenos ckit and scf and decrease that of inos in the colontissues of colitis mice such eï¬ects were stronger than thoseof lpys4l or lb treatment group discussione ratio of colon weightlength is employed as a vitalstandard for assessing colitis in vivo e colon length ofnormalmodellblpys4llpys4hadccb0020406080100normalmodellblpys4llpys4hcolon length cmcolon lengthadccb00100200300400500normalmodellblpys4llpys4hcolon weightcolon lengthcolon weightcolon length 0cevidencebased complementary and alternative medicineabfigure a et1 b ss c sp and d vip serum levels of each group of mice data are presented as the mean± standard deviationa“d diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4lmice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentrationof lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgdcfigure a il2 and b il10 serum levels of each group of mice data are presented as the mean± standard deviation a“e diï¬erentletters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent test lpys4l mice treatedwith a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a high concentration oflactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgbadabbc0050100150200250normalmodellblpys4llpys4het1 level pgmlet1aedcb00100200300400500600700normalmodellblpys4llpys4hss level pgmlssvipdabbc00100200300400500600700normalmodellblpys4llpys4hsp level pgmlspaedcb00100200300400500600700normalmodellblpys4llpys4hvip level pgmlvipadccb050100150200250300normalmodellblpys4llpys4hil2 level pgmlil2eabcd02004006008001000normalmodellblpys4llpys4hil10 level pgmlil10 0cevidencebased complementary and alternative medicineabfigure a mpo b no c gsh and d mda colon tissue levels of each group of mice data are presented as the mean± standarddeviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly significantly diï¬erent testlpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4h mice treated with a highconcentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillus bulgaricus — cfukgcdcolitis mice was shorter on average than that of control miceand the ratio of colon weightlength was lower in colitis micethan in control mice howeverit appeared thattreatment with lpys4 could attenuate the decline in colonlength and weightlength ratio induced by colitisa prior study has shown that vasoconstriction of theendothelin can lead to colonic mucosa erosion and ulceration which in turn can exacerbate the progression of colitis ss however can reduce gastrointestinal ‚ammationby suppressing the production of gastric acid and othergastrointestinal fluids us a decrease in ss level can inducethe secretion of gastrointestinal fluids thus aggravatingcolitis excessive accumulation of sp can induce colitisbut after antagonizing it has been shown to relieve colitis invivo vip inhibits no production by regulating thetranscriptional activity of inos in the colon tissue thusprotecting the intestinal mucosa besides vip can also ‚uence certain immune aspects of colitis in this studylpys4 inhibited colitis by downregulating the levels of etand sp and upregulating those of ss and vipil2 is an eï¬ector cytokine produced by cells whichhas been closely associated with colitis cells regulate the‚ammatory response that causes colitis and il2 is involved in the suppression of ‚ammatory process andseverity reduction of colitis by ‚uencing cells il is another cytokine produced by treg cells with immunoinhibitory eï¬ects which plays a significant role in thedevelopment of colitis ‚ammatory bowel diseaseibd is a chronic refractory intestinal ‚ammatory diseasemainly including ulcerative colitis uc and crohn™s diseasecd although the etiology of ibd is still unclear theimbalance between and has been recognized as themain cause of mucosal damage in ibd under the action ofdiï¬erentiation factor il10 regulatory t cells derived fromintestinal mucosa associated lymphoid tissue can correct deviation by secreting high level of il10 andmedium level of tgfβ so as to achieve the purpose oftreating ‚ammatory bowel disease to a certain extent it was observed that lpys4 could increase the level of il2thus regulating immunity and alleviating colitis and lpeabcd00100200300400normalmodellblpys4llpys4hmpo level mumgmpoadccb00100200300400normalmodellblpys4llpys4hsod level μmolgprotsodaedcb0020406080100120normalmodellblpys4llpys4hsgh level μmolmggshdabbc000510152025normalmodellblpys4llpys4hmda level nmolmgmda 0cevidencebased complementary and alternative medicinefigure observation of colon pathology in mice by he stainingys4 could inhibit the colitis and reduce the secretion of ile aggregation of neutrophils began to decline afterintestinal ‚ammation and a large amount of them enteredinto the circulation and migrated to tissues at the sametime free radicals such as reactive oxygen species and reactive nitrogen species gathered in large quantities which inturn led to damage and toxicity of colon tissue and furtheraggravated colitis after colitis the levels of sod andgsh were reduced in colon tissue while those of mda andmpo were elevated our findings also indicated that colitiscould lead to decrease in gsh and sod levels as well asincrease in mda and mpo levels [ ] in addition lpys4 attenuated colitis by inhibiting the transcriptional responses to oxidative stressnos can be divided into nnos enos and inos it hasbeen reported that no produced by enos plays a key rolein response to colonic tissue damage and excessive nogenerated by inos promotes colitis damage enoscontrols the production of no to keep the colonic tissue ina normal state which plays an important role in reducingcolitisinduced colonic injury e presence of excessiveno aggravates colon damage nnos can also controlthe level of no in tissue and protect the tissue from beingdamaged by excessive no in this study lpys4upregulated the expression of enos and nnos in the colonand downregulated that ofthereby attenuatingcolitisinosulcerative colitis not only shows hematochezia anddiarrhea but also presents colonic motility disorders it hasbeen proved that interstitial cells of cajal icc are related tocolonic motility dysfunction and directly participate in theprogression of colitis as a specific marker of gastrointestinalicc ckit is a transmembrane glycoprotein specificallyexpressed on icc cell membrane ckit gene located onchromosome 4q1213 belongs to protooncogene and itsproduct is tyrosine kinase type iii as a receptor of scfckit can regulate the proliferation and diï¬erentiation ofhematopoietic stem cells through a series of signalingpathways [ ] scf exerts a direct eï¬ect on ‚ammatorybowel disease by regulating the function and number oficc scf can interact with its ligand ckit and the dysregulation of scfkit signaling pathway may decrease theproliferation and diï¬erentiation of icc thus exacerbatingcolitis [ ] e abnormal expression of scfckitsignaling pathway can also change the physiologicalfunction of icc weaken gastrointestinal motility andaggravate intestinal dysfunction in the present studylpys4 could inhibit colitis by regulating the expressionlevels of scf and ckiticc autophagy regulation has become a new target forthe treatment of intestinal motility disorder in ulcerativecolitis because the drug treatment of colitis often hasside eï¬ects and once stopped it is easy to relapse ereforethe use of natural harmless substances through the regulation of icc prevention and treatment of colitis canmaintain longterm health a study has shown thataurantii fructusimmaturus and atractylodis macrocephalae rhizoma can inhibit the autophagy of cajal stromalcells induced by glutamate which may play an inhibitoryrole in colitis meanwhile there is also a study showingthat lactic acid bacteria can regulate icc thus regulatingnormallblpys4llpys4hmodel 0cevidencebased complementary and alternative medicineacbdthe mean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestlyfigure eï¬ect of lpys4 on a nnos b enos c inos d ckit and e scf mrna expression in mouse colon data are presented asesignificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgintestinal function and protecting the intestine isstudy also confirmed that the lpys4 can regulate the scfckitsignaling pathway and the scfckit signalingpathway is an important icc regulatory pathway erefore the lpys4 may also inhibit the colon by regulating iccaedcb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupnnosadccb001020304050normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupscfaedcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineacbdmean± standard deviation a“e diï¬erent letters mean values are significantly diï¬erent p according to tukey™s honestly sigfigure af eï¬ect of lpys4 on nnos enos inos ckit and scf protein expression in mouse colon data are presented as thenificantly diï¬erent test lpys4l mice treated with a low concentration of lactobacillus plantarum ys4 — cfukg lpys4hmice treated with a high concentration of lactobacillus plantarum ys4 — cfukg and lb mice treated with lactobacillusbulgaricus — cfukgef conclusionin this study oxazolone was used to induce colitis in balbcmice and the inhibitory eï¬ects of lpys4 on colitis weredetected rough the observation of colon tissues and serum samples of mice it was found that lpys4 treatmentcould alleviate colitis by restoring the levels of ‚ammatoryindicators closer to those measured in healthy control miceis work suggests that lpys4 is superiorquality lactic acidbacteria with a potential role in colitis treatment and provides a foundation for further research and developmentabbreviationslpys4 lactobacillus plantarum ys4lbqpcr quantitative polymerase chain reactionhelactobacillus bulgaricushematoxylineosinnormalmodellblpys4llpys4hckitenosinosnnosscfβactinadcbcb0010203040normalmodellblpys4llpys4hrelative to multiple of model groupnnosaedcb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupenoseabcd00020406081012normalmodellblpys4llpys4hrelative to multiple of model groupinosadccb0010203040506070normalmodellblpys4llpys4hrelative to multiple of model groupscfadccb00102030405060normalmodellblpys4llpys4hrelative to multiple of model groupckit 0cevidencebased complementary and alternative medicineendothelin1substance psomatostatinvasoactive intestinal peptideinterleukin2interleukin10et1spssvipil2il10mpo myeloperoxidasesodgshmda malondialdehydeenosnnos neuronal nitric oxide synthaseinducible nitric oxide synthaseinosscfstem cell factorsuperoxide dismutaseglutathioneendothelial nitric oxide synthasedata availabilityno data were used to support this studyconflicts of intereste authors of this manuscript state that they do not haveconflicts of interest to declareauthors™ contributionsruokun yi and fang tan contributed equally to this workruokun yi and fang tan performed the majority of theexperiments and wrote the manuscript huayi suo wenfengli xianrong zhou and jianfei mu contributed to the dataanalysis xin zhao and peng xie designed and supervised thestudy and read the final manuscriptacknowledgmentsis research was funded by national key rd program ofchina 2018yfd0502300 children™s research institute ofnational center for schooling development programmeand chongqing university of education csdp19fs01103theand technology research program ofchongqing municipal education commission kjzdk201901601 and research project of chongqing universityof education ky2015tbzc chinasciencereferences r k yi f tan w liao et al œisolation and identification oflactobacillus plantarum hfy05 from natural fermented yakyogurt and its eï¬ect on alcoholic liver injury in mice microanisms vol no p j liu f tan x h liu et al 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a

"The occurrence of PLC is extremely rare in liver carcinoma. Herein we report the case of a patient with PLC after liver transplantation due to liver carcinoma. PLC was confirmed by clinical manifestations imaging studies and cytologic examination of exfoliated cells in the pleural effusion. Liver carcinoma Liver transplantation Metastasis Pulmonary lymphangitic carcinomatosis Background Primary liver carcinoma is a malignancy originating from hepatocytes and/or intrahepatic biliary epithelial cells. In China there are more than 90 million carriers of the hepatitis B virus (HBV) accounting for 40% to 45% of HBV carriers worldwide. The high prevalence of HBV in China is the underlying reason why liver carcinoma is the malignancy with the highest morbidity and mortality rates in China. Currently resection and liver transplantation are major strategies for the treatment of liver carcinoma. For patients with hepatic cirrhosis liver transplantation can cure both the cancer and liver cirrhosis. However liver carcinoma may recur or metastasize after resection or liver transplantation mainly via the hematogenous route. Although lymphatic metastasis can occur metastasis is usually found in the hepatic hilus upper abdomen and retroperitoneal lymph nodes [1]. Pulmonary lymphangitic carcinomatosis (PLC) is a special manifestation of metastatic cancer in the lymphatic vessels of the lung that is characterized by diffuse or focal growth. Most PLC cases originate from adenocarcinomas. PLC is rare in liver carcinoma patients. To the best of our knowledge no studies reported to date have described PLC after liver transplantation. Case presentation A 45-year-old man was admitted to our hospital with a complaint of repeated episodes of abdominal distension. He was diagnosed with HBV-induced hepatic cirrhosis and liver carcinoma (T3N0M0). He underwent liver transplantation without any metastasis before the operation. Pathological analysis identified a tumor (12 cm?—?8 cm?—?10 cm) in the right lobe of the liver within which the cancer cells were arranged in nests and pleomorphism was seen. These findings together with the results of immunohistochemistry demonstrated features of mixed liver carcinoma: ?-fetoprotein (+) hepatocytes (+) CD34 (+) CD19 (+) CD10 (focal +) synaptophysin (-) chromogranin A (-) and cytokeratin (pan +) (). The function of the graft liver was favorable. FK506 was used alone for antirejection therapy. Immunohistochemical staining of mixed liver carcinoma tissue specimens. (A) Cancer cells were arranged in nests and showed atypia. The interstitium was rich in sinusoids and invasive growth was noted. (B) Image showing cytokeratin 19 (CK19) (+). (C) Image showing CK7 (+). All three images are stained with hematoxylin and eosin and were scanned at 100— original magnification. Two months later the patient developed a dry cough of unknown etiology and his condition deteriorated 1 week later. Expectoration was occasionally present accompanied by chest tightness shortness of breath and hypoxemia (75 mmHg partial pressure of oxygen). Fever and chills were absent and the patient™s white blood cell count neutrophil count and inflammatory factors were normal. His sputum culture was negative. Lung computed tomography (CT) suggested infectious lesions in the lung which were characterized by interstitial changes. Right-sided pleural effusion and segmental atelectasis in the lower lobe of the right lung were noted. Several enlarged lymph nodes were identified in the mediastinum (A). Thoracentesis was immediately performed and approximately 2000 ml of light yellow fluid was collected. The patient™s chest tightness and shortness of breath improved significantly. Posttransplantation interstitial pneumonia was considered at first. FK506 was discontinued and methylprednisolone (40 mg every 12 hours) caspofungin sulfamethoxazole (SMZ) and aminophylline were administered. Computed tomography scans of the lungs. (A) Soon after the appearance of the patient™s respiratory symptoms a computed tomography (CT) scan revealed septal thickening of the peribronchovascular interstitium pleural effusion segmental atelectasis in the right lower lobe of the lung and several enlarged lymph nodes in the mediastinum. (B) Discontinuation of anti-infection therapy and 5 days after thoracentesis extensive involvement of the parenchyma with septal thickening was evident with reticulonodular densities in all lung fields. Five days later a lung CT scan showed reexpansion of the right lung and diffuse exudate in the interstitium. Multiple nodules were found in both lungs (B). Pulmonary function tests showed severe obstructive ventilatory dysfunction and moderate reduction in carbon monoxide diffusion capacity. Examination of exfoliated cells in the pleural effusion showed cancer cells (). Positron emission tomography (PET)-CT indicated multiple nodules and patchy or cloudy shadows with high density in both lungs (maximal standardized uptake value (SUV) approximately 6.27). Several enlarged lymph nodes were found in the mediastinum hepatic hilus and retroperitoneum (maximal SUV approximately 8.39). Moreover lesions with increased density were found in the left third rib the right upper femur and the left acetabulum which were accompanied by an increase in fluorodeoxyglucose. The patient was diagnosed with PLC after liver transplantation due to liver carcinoma. Cancer cells among the exfoliated cells in the pleural effusion are shown. All slides are stained with hematoxylin and eosin and were photographed under light microscope at 400 — original magnification. The treatment with steroid and aminophylline continued to improve the status of the patient™s interstitial lesions. Although antirejection therapy was stopped rejection did not occur and the function of the graft liver was stable. Oral capecitabine was administered but was not effective. The patient experienced increasing chest tightness and shortness of breath and he died as a result of respiratory failure 1 month later. Discussion PLC was first described by Troisier in 1873. About 30% to 40% of malignancies may present with metastasis to the lung and PLC accounts for approximately 6% to 8% of metastatic cancer in the lung. Most PLCs originate from adenocarcinomas and they are most often due to lung cancer followed by breast cancer and gastric cancer [23]. Patients with renal cancer cervical cancer thyroid cancer and melanoma rarely develop PLC [4-6]. The pathologic features of PLC include infiltration of cancer cells and interstitial edema in and around lymphatic vessels as well as infiltration of inflammatory cells caused by lymph node metastasis in the lung. The metastatic cancer in the mediastinal and pulmonary hilar lymph nodes may obstruct lymphatic drainage resulting in retrograde migration of cancer cells into terminal lung tissues via lymphatic vessels or anterograde migration of cancer cells in the pleura into the pulmonary hilar lymph nodes through intrapulmonary lymph vessels. In addition a cancer embolus may form in the terminal vessels of the lung due to hematogenous metastasis which can invade the surrounding lymphatic vessels. Thus hilar and mediastinal lymph node metastasis may be present or absent in PLC depending on the route of metastasis of the primary cancer. Extrahepatic metastasis of liver carcinoma is mostly found in the lung adrenal gland bone and central nervous system. Hematogenous spread is thought to be the most common extrahepatic metastatic route [78]. "

tetrastigma hemsleyanum diels et gilg t hemsleyanum mostly known as œsan ye qing is a kind of folk plant because of its slow growth it usually takes “ years to meet the requirements of commercial medicinal materials so it is a precious perennial medicinal resource it mainly grows in the eastern central southern and south western provinces of china such as zhejiang jiangsu guangxi fujian and yunnan provinces peng and wang t hemsleyanum is known worldwide as sources of phytotherapeutics which have been used for the treatment of conditions related to inflammatory and immune response and been recorded based on clinical trials or the use of animal models xu as an edible plant the leaves of t hemsleyanum consumed as a functional tea or dietary supplement for its health benefits such as improving the immune system of the body sun while the aerial parts of t hemsleyanum developed as potential new traditional chinese medicine tcm preparations guo corresponding author ningbo research institute of zhejiang university ningbo zhejiang people™s republic of china email address px4142163com x peng 101016jjep2020113247 received may received in revised form july accepted august ofethnopharmacology2642021113247availableonline12august2020037887412020elsevierbvallrightsreserved 0ct ji abbreviations t hemsleyanum tetrastigma hemsleyanum diels et gilg tcm uplcesiqtofmsms ultra high performance liquid traditional chinese medicine chromatography tandem triple quadrupole time of flight mass spectrometry minimum inhibitory concentration glutathione malondialdehyde nuclear factorκb 5hydroxytryptamine norepinephrine dopamine prostaglandin e2 lipopolysaccharide tumor necrosis factoralpha interleukin1 beta interleukin mic gsh mda nfκb 5ht ne da pge2 mapk mitogenactivated protein kinase lps celegans caenorhabditis elegans tnfα il1 il6 il12p40 interleukin subunit p40 stnfr1 soluble tnf receptors il10 il1 il4 inos tlr4 md2 myd88 myeloid differentiation protein jnk gpt got alp sod interleukin interleukin interleukin inducible no synthase tolllike receptor myeloid differentiation factor2 cjun nterminal kinase glutamicpyruvic transaminase glutamicoxalacetic transaminase alkaline phosphatase superoxide dismutase and activities antiinflammatory the root tubers of t hemsleyanum are extensively used either alone or in combination with other herbal medicines in tcm clinics for the treatment of children with fever convulsion pneumonia asthma rheumatism hepatitis menstrual disorders scrofula and pharynx pain sun chen and guo therefore it was called as œnatural plant antibiotic according to its wide spectrum of prominent bactericidal in february t hemsleyanum was awarded as the new œeight famous kinds of tcm in zhejiang province meant that it has become a key object of industrialization development of zhejiang™s dominant large varieties of medicinal materials in covid19 broke out and has caused more than deaths in china and infection cases have been reported in more than countries hua shi xuan fei mixture approval number of zhejiang medicine z20200026000 which composed of t hemsleyanum has been approved by zhejiang provincial drug administration for clinical treatment of covid19 futhermore the modern pharmacological studies had shown that t hemsleyanum also had effects of antiinflammatory ji antioxidant hossain antivirus ding antitumor lin antipyretic yang and wang antihepatic injury ma et al immunomodulatory xu antibacterial chen hypoglycemic ru 2018ab etc numerous reports have demonstrated that the biological activities of t hemsleyanum are attributed to its many chemical components fu wang has reported isolated alkaloids from the aerial parts of t hemsleyanum wang ru extracted a novel polysaccharide tdgp3 from is mainly alanine aminotransferase aspartate aminotransferase hyaluronan laminin total bilirubin total protein interferongamma immunoglobulin a secretory immunoglobulin a epithelialmesenchymal transition alt ast ha ln tbili tp ifnÎ iga siga emt mmps matrix metalloproteinase timps matrixmetallo proteinase cytc cat gshpx glutathione peroxidase tregs tgf cox2 foxp3 pdl taoc ccl4 cef hvj vsv a f s1 s2 pef cff eaf baf cytochrome c catalase regulatory t cells transforming growth factor beta cyclooxygenase forkheadwinged helix transcription factor gene population doubling time total antioxidant capacity carbon tetrachloride chicken embryo fibroblast hemagglutinating virus of japan vesicular stomatitis virus alkalicontaining extract of t hemsleyanum ketonecontaining extract of t hemsleyanum crude extract of t hemsleyanum crude extract of t hemsleyanum petroleum ether extractions of t hemsleyanum ethanol extract chloroform extractions of t hemsleyanum ethanol extract ethyl acetate extractions of t hemsleyanum ethanol extract nbutanol extractions of t hemsleyanum ethanol extract t hemsleyanum with a molecular weight of — da by enzymolysisultrasonic assisted extraction method ru 2019ab large amounts of flavonoids were found in leaves aerial parts and root tubers of t hemsleyanum xu 2014ab deng yu in addition t hemsleyanum also contains a variety of functional components such as anic acids hu phenolic acids liu minerals fan amino acids fu etc in recent years wild resources of t hemsleyanum have been overexploited and now are on the verge of extinction due to its multiple medicinal values coupled with the strict requirements of the growing environments in it was listed in the preferentially protected crop germplasm resources of zhejiang province based on our team™s preliminary research peng peng 2016ab li we comprehensively summarized and analyzed the domestic and overseas research progress on traditional uses the bioactive components of t hemsleyanum pharmacological activities toxicology with the aim of providing guidance for indepth research and reference for its development and utilization materials and methods the available information about the traditional uses phytochemicals and pharmacological properties of t hemsleyanum was searched via web of science google scholar pubmed science direct china national knowledge infrastructure cnki and springer search using chinese or english as the retrieval languages the keywords used include t hemsleyanum root tubers of t hemsleyanum radix tetrastigma ofethnopharmacology26420211132472 0ct ji traditional uses phytochemistry bioactive components pharmacological activities toxicology and other related words all references were from experimental studies and published prior to april were reviewed all chemical structures were drawn using chemdraw pro software heatclearing were botanical characteristics t hemsleyanum is a perennial grass climbing vine with longitudinal ribs glabrous or sparsely pilose it is usually grown in a cool and humid environment and the main soil type is yellow soil or yellow brown soil with rich humus the optimum ph is between and the root tubers are thick spindle shaped or elliptical and single or several are connected into a string of beads generally “ cm long and “ cm in diameter fig the epidermis of the root tubers is tan and most of them are smooth a few of them have folds and lenticel like protuberances some of them have depressions in which there are residual tan roots hard and brittle with a flat and rough section the stem of t hemsleyanum is thin and weak with longitudinal rhombus rooting on the lower node palmate compound leaves alternate leaflets are lanceolate oblong or ovate lanceolate the leaflets are “ cm long and “ cm wide with a tapered tip and a wedgeshaped or round base the flowers of t hemsleyanum are small yellow green and ovate the flowering stage of t hemsleyanum ranges from april to june and the fruit phase is normally from august to november when the flower withered it will form a small green round fruit with the size of millet when it is mature the fruit will turn from green to red the berries are spherical and soft spherical traditional uses t hemsleyanum belonging to the family vitaceae was firstly recorded in ben cao gang mu ming dynasty ad the aliases of sanyeqing include shi hou zi shi bao zi shi lao shu lan shan hu lei dan zi po shi zhu tu jing wan sou jia feng san ye dui golden wire hanging gourd golden bell golden wire hanging potato etc the root tubers or whole grass of t hemsleyanum traditionally and ethnically used as a medicine for a long time it has been recorded in multiple hemsleyanum ancient books of tcm such as zhi wu ming shi tu kao qing dynasty wu jiangxi herbal medicine common folk herbal medicine in zhejiang all of these ancient works described the effects of toxicityremoving t dyspnearelieving promoting blood circulation and pain relief thus it can be applied to cure febrile convulsion pneumonia bronchitis pharyngitis sore throat acute and chronic hepatitis rheumatic arthralgia viral meningitis bruise eczema insect and snake bite poor joint flexure and extension irregular menstruation of women national compilation team of chinese herbal medicine in the tcm culture the properties of t hemsleyanum was described as bitter and acrid in taste cool in nature which recorded in dictionaries of traditional chinese medicine and zhong hua ben cao shanghai science and technology press the channel tropism was lung heart liver and kidney meridians decocting with water or mashing for external application are the traditional possess methods of t hemsleyanum considering its extensive traditional effects many prescriptions containing t hemsleyanum have been passed down from generation to generation and have been well supported and clarified by modern pharmacological studies excitingly it has reported that jinlian disinfection drink containing san ye qing combined with interferon can treat covid19 he jinqi tablet made up of san ye qing astragalus and ginsenoside was used to treat cases of malignant tumor cases were completely relieved cases were partially relieved the total effective rate was wei moreover zhonggan mixture including san ye qing could improve the quality of life and prolong the survival time of patients with stage iii primary liver cancer jiang and gong in addition it has been used in the treatment of common gynecological diseases such as blood avalanche and leucorrhea gao and it also has a good effect on measles complicated with pneumonia anal fissure chronic bronchitis and mosquito bites ji chemical compounds of themsleyanum the chemical constituents of t hemsleyanum have been widely investigated sun sun zeng xu 2014ab fu fan chen ding 2015a fig the aerial part a root tuber b and raw herb c of t hemsleyanum ofethnopharmacology26420211132473 0ct ji b ding a total of one hundred and fortytwo compounds have been isolated and identified from t hemsleyanum until now the information about compound name molecular weight compound formula detection method analysis sample is summarized in table flavonoids and their glycosides modern phytochemical studies have indicated that flavonoids are the representative and predominated class of constituents isolated from t hemsleyanum lin zhang table to date fiftyone flavonoids and their glycosides have been extracted and identified from t hemsleyanum in this series compounds quercetin orientin vitexin isorhamnetin apigenin and kaempferol are the main types of skeleton some of their analogues can be identified from hydroxy moiety on c3² and c4™ on the b ring of flavonoid aglycone at present many modern analytical techniques have been used for qualitative and quantitative analysis of flavonoids among them ultra high performance liquid chromatography tandem triple quadrupole time of flight mass spectrometry uplcesiqtofms has become a powerful tool for identifying the complicated compounds due to its higher mass accuracy and resolution our team used uplcesiqtofms to identify chemical constituents from the aerial part of t hemsleyanum including flavonoids such as isoorientin quercetin kaempferol vitexin isovitexin kaempferol3glucoside etc sun according to the report liu total flavonoids of t hemsleyanum could protect the aged mice from acute lung injury through inhibiting the phosphorylation of mitogenactivated protein kinase mapk and nuclear factorκb nfκb in lung tissue moreover the flavonoids of t hemsleyanum had the activity of antilung cancer wei luteolin a flavonoid found in t hemsleyanum acted as an anticancer agent against various types of human malignancies such as lung breast glioblastoma prostate colon and pancreatic cancers muhammad it is certain that t hemsleyanum flavonoids give a new vision for researchers to explore clinical anticancer drugs polysaccharide saccharide is another important active ingredient extracted from t hemsleyanum shao polysaccharide has great potential in clinical application because of its unique pharmacological activity however due to the complex structure of polysaccharide it is difficult and special to determine and synthesize their structures guo table the prescriptions and traditional uses of t hemsleyanum in china prescriptions name qingteng fengshi qufengshi yaojiu main composition jiu traditional use t hemsleyanum parabarium chunianum tsiang zanthoxylum nitidum roxb dc t hemsleyanum deeringia amaranthoides lam merr blumea aromatica wall dc t hemsleyanum deeringia amaranthoides lam merr zanthoxylum nitidum roxb dc panax notoginseng burk fh chen t hemsleyanum gypsum lonicera japonica thunb houttuynia cordata thunb ophiopogon japonicus linn f kergawl t hemsleyanum t hemsleyanum lysimachia christinae hance imperata cylindrica citrus reticulata blanco t hemsleyanum ginsenoside astragalus propinquus schischkin t hemsleyanum nepeta cataria l lonicera japonica thunb saposhnikovia divaricata trucz schischk huatuo fengtongbao capsule sanyeqing gypsum decoction sanyeqing power zhonggan mixture jinqi tablet hua shi xuan fei mixture extracted the polysaccharides from roots of t hemsleyanum rtp1 rtp2 and rtp3 were successively found by protein precipitation and purification moreover further study indicated rtp31 was high purity polysaccharide with a molecular weight of kda and it is mainly composed of kinds of monosaccharides arabinose galacturonic acid galactose and fructose the proportion is and respectively ru 2018ab extracted a polysaccharide thp from t hemsleyanum with the average molecular weight estimated as kda the results of study on the composition of polysaccharide showed that it was mainly composed of rhamnose arabinose mannose glucose galactose with the molar ratio of in ru 2019ab successfully extracted polysaccharide thdp3 from t hemsleyanum with molecular weight of kda which consists of rhamnose arabinose mannose glucose and galactose with molar ratio of moreover tdgp3 mainly consists of †’4αdgalap1†’ †’4dgalp1†’ and †’4αdglcp1†’ residues as backbones and dmanp1†’ †’36dmanp1†’ and αdaraf1†’residues as branches phenolic acids phenolic acids refer to aromatic carboxylic acids with multiple phenolic groups substituted on one benzene ring as a secondary metabolite phenolic acids are widely found in many natural plants and have antiinflammatory antioxidant and lipid lowering effects twenty three phenolic acids no52“ table have been reported in the aerial parts of t hemsleyanum such as caffeic acid chlorogenic acid 1ogalloyldglucose protocatechol glucoside epigallocatechin 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid and 5pcoumaroylquinic acid there were twentyone phenolic acids in the root tuber of t hemsleyanum some of which were the same as aerial parts alkaloids alkaloids are a group of basic anic compounds containing nitrogen that exist in nature alkaloids are stored in small quantities in t hemsleyanum and the bioactivity investigations of those alkaloids are still rather rare wang fu extracted the aerial parts of t hemsleyanum with ethanol and then isolated ten alkaloids for the first time including seven indole alkaloids an amide a maleimide and treatment of joint pain wind cold dampness arthralgia treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis treatment of arthralgia syndrome rheumarthritis rheumatoid arthritis scapulohumeral periarthritis joint pain muscular constricture treatment of infantile hyperpyretic convulsion treatment of blood avalanche leucorrhea treatment of liver cancer treatment of malignant tumor treatment of covid19 usage oral administration “ ml once times a day oral administration ml once times a day oral administration capsules once times a day references ministerial standard ministerial standard ministerial standard one dose a day decoct twice in water and take it “ times after mixing oral administration oral administration ml once times a day oral administration capsules once times a day oral administration ml once times a day xu gao jiang and gong wei zhejiang provincial drug administration ofethnopharmacology26420211132474 0ct ji detection mode analysis parts of sample reference aerial part root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber root tuber aerial part root tuber root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part root tuber aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber sun sun zeng sun sun sun zeng zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun zeng sun zeng sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun xu 2014b sun zeng sun zeng sun zeng zeng sun sun sun sun xu 2014b sun xu 2014b sun zeng sun xu 2014b sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun fu sun sun xu 2014b fan xu 2014b fan sun continued on next page uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms table chemical constituents isolated from the different parts of t hemsleyanum name flavonoids and their glycosides quercetin quercitrin quercetin3oglucoside quercetin3orutinoside quercetin3galactoside quercetin3oxylosylglucoside quercetin3oxylosylglucose7orhamnoside orientin orientin2²²orhamnoside isoorientin isoorientin2²²orhamnoside isoorientin cid0 ²²oxyloside vitexin vitexin2²²orhamnoside vitexin2²²oglucoside vitexin2²²oarabinoside isovitexin isovitexin2²²orhamnoside isovitexin2²²oxyloside isorhamnetin isorhamnetin3rutinoside isorhamnetin3pyranoarabinose7glucosylrhamnoside apigenin apigenin7rhamnoside apigenin8cxylosyl6cglucoside apigenin6cαlarabinose8cdglucose eriodictyol eriodictyolohexoside i eriodictyolohexoside ii luteolin luteolin6 8dichexoside catechin catechin glucopyranoside isomer epicatechin kaempferide kaempferol kaempferol3glucoside kaempferol3rutinoside kaempferol3sambubioside kaempferol3oneohesperidin kaempferol3orhamnoside kaempferol7orhamnose3oglucoside kaempferol3robinoside7rhamnoside kaempferol3rutinoside kaempferol3ocarfuran7orhamnosyl glucoside daidzein biochanin a procyanidin dimmer procyanidin b1 procyanidin b2 procyanidin trimer phenolic acids and derivatives gallic acid protocatechuic acid caffeic acid dihydroxybenzoic acid hexoside 1caffeoylquinic acid 3caffeoylquinic acid 4caffeoylquinic acid 5caffeoylquinic acid 1pcoumaroylquinic acid 4pcoumaroylquinic acid 5pcoumaroylquinic acid phydroxybenzaldehyde pcoumaric acid ferulic acid hexoside salicylic acid chlorogenic acid neochlorogenic acid cryptochlorogenic acid protocatechualdehyde uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr13cnmr ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms ofethnopharmacology26420211132475 0ct ji table continued name salicin2benzoate trihydroxycinnamoylquinic acid isomer protocatechuic acid hexoside apiosylglucosyl 4hydroxybenzoate 1ogalloyldglucose protocatechol glucoside epigallocatechin vanillic acid1ofuran celery glucosyl ester protocatechuic acid1ofuran celery glucosyl ester methoxyphenol1ofuran glycosyloglucoside 2methoxy4methylbenzene1ofuracresyl glucoside oxyresveratrol dicaffeoylquinic acid 4hydroxycinnamic acid alkaloids indole indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid scid0 trolline fatty acids trihydroxy octadecadienoic acid trihydroxy octadecenoic acid dihydroxy octadecenoic acid 9hydroxy1012octadecadienoic acid 9hydroxy octadecatrienoic acid hydroxyoctadecenoic acid hydroxyoctadecatrienoic acid dihydroxyoctadecatrienoic acid dihydroartemisinin ethyl ether trihydroxy octadecadienoic acid isomer hydroxyoxooctadecatrienoic acid octadecenedioic acid dimeester stearic acid linolenic acid linoleic acid palmitic acid oleic acid anic acids and derivatives malic acid quinic acid citric acid azelaic acid oxalic acid galactonic acid gallic acid succinic acid fumaric acid propanoic acid terpenoids and steroids sitosterol daucosterol campesterol stigmasterol 6obenzoyl daucosterol ergosterol taraxerone taraxerol αamyrine pteroside z ganoderic acid h 3epipapyriferic acid oleanic acid saponins ginsenoside rh1 detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms 1hnmr lcms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms nmr uv ms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms gcms tcl hnmr cnmr ms gcms gcms ir hnmr eims ir hnmr ms ir hnmr ms ir hnmr ms ir eims uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms hnmr cnmr ms analysis parts of sample root tuber root tuber root tuber root tuber aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber root tuber root tuber aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts aerial parts root tuber aerial part root tuber aerial part root tuber root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part aerial part aerial part aerial part root tuber root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part root tuber aerial part aerial part aerial part aerial part aerial part root tuber aerial part root tuber root tuber root tuber root tuber root tuber root tuber aerial part aerial part aerial part aerial part root tuber root tuber root tuber root tuber reference sun sun sun sun sun sun zeng sun xu 2014b zeng zeng zeng zeng xu 2014b xu 2014b chen fu fu fu fu fu fu fu fu fu fu sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun sun chen ding sun sun guo ru ru ru ru sun sun sun ding uplcesiqtofmsms root tuber sun continued on next page ofethnopharmacology26420211132476 0ct ji table continued name ginsenoside rh2 vinaginsenoside r1 amino acid and derivatives phenylalanine pyroglutamic acid glutimic acid hexose tryptophan lglutamic acid detection mode uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms uplcesiqtofmsms analysis parts of sample root tuber root tuber root tuber aerial part aerial part aerial part aerial part aerial part reference sun sun sun sun sun sun sun respectively a carboline by comparing with the spectral data of known compounds the alkaloids were indole3carboxylic acid indole3propanoic acid 5hydroxyindole3carboxaldehyde 5hydroxyindole3carboxylic acid 6hydroxy3 4dihydro1oxocarboline hippophamide 4hydroxycinnamide pyrrole3propanoic acid and scid0 trolline the chemical structures were shown in fig identified as indole anic acids and derivatives the biologically essential anic acids have been isolated and characterized from t hemsleyanum as well ten anic acids and seventeen fatty acids were identified from the aerial parts and root tuber of t hemsleyanum most of which were found in the aerial parts except stearic acid propanoic acid and dihydroxy octadecenoic acid all the anic acids and fatty acids are listed in no112“ and no95“ of table respectively terpenoids and steroids terpenoids and steroids are other kinds of secondary metabolites of t hemsleyanum thirteen of these compounds have been isolated and identified no122“ table liu yang liu isolated and identified αamyrine sitosterol ergosterol taraxerone taraxerol from the aerial part of t hemsleyanum in addition daucosterol campesterol stigmasterol 6obenzoyldaucosterol pteroside z ganoderic acid h 3epipapyriferic acid and oleanic acid were successively separated tuber roots of t hemsleyanum liu and yang from the inanic elements the mineral elements of tcm are indispensable supplements to the bioactive components which are closely related to the efficacy toxicity and side effects of tcm wu wu et al demonstrated that t hemsleyanum contained twentyseven different mineral elements namely li be na mg al k ca v cr mn fe co ni cu zn ga as se rb ag cd cs ba hg ti pb u moreover ca cu ni ba al k have higher loading values which are the characteristic elements of t hemsleyanum wang wang has indicated that the contents of fe mn zn and cu in three populations of t hemsleyanum cultivated in different environments were “ “ “ “ mg kgcid0 respectively pharmacology the ethnomedical uses of t hemsleyanum have stimulated various pharmacological studies on it the extracts and isolated compounds from t hemsleyanum showed a variety of bioactivities such as antiviral antibacterial antioxidant antipyretic analgesic hepatoprotective immunoregulatory and antitumor activity the detailed pharmacological activities of t hemsleyanum were presented in table and summarized as follows antiviral activity according to yang™s literatures yang the nitrogenous alkalicontaining extract a ketonecontaining extract f crude extract s1 and crude extract s2 of t hemsleyanum had different antiviral effect on mice and chicken embryo fibroblast cef infected with hemagglutinating virus of japan hvj influenza virus pr6 vesicular stomatitis virus vsv specifically s2 strongly inhibited the proliferation of influenza virus pr6 with at the concentration of mgml and mgml s1 has obvious antiviral effect on hvj at the concentrations of mgml and mgml both f and s1 displayed a strong suppressive effect on the plaque formation of vsv in vivo a f s1 s2 have different degrees of antiviral activity when the concentration of a was gkg the protective rate was up to and that of s1 gkg was however the author did not give the sample preparation method ding had demonstrated compounds quercetin3orutinoside kaempferol kaempferol3glucoside quercitrin quercetin kaempferol3orutinoside procyanidin dimmer and epicatechin which were isolated from t hemsleyanum were positively related to the activity of t hemsleyanum against h1n1 influenza virus the ethyl acetate extracts of t hemsleyanum have been shown to obviously restrain the secretion of hbsag and hbeag released by hbv with the ic50 values of “ mgl however the specific mechanism of action needs to be further confirmed yang and wu wang had proved that the nbutanol and ethyl acetate extraction of t hemsleyanum had antiviral activity against rsv and were superior to ribavirin with the ec50 values of mgl wang moreover the t hemsleyanum extracts had different degrees of inhibition to different hiv1 strains the ec50 values were between μgml and μgml and the

Large cell neuroendocrine carcinoma Descending colon Colonic neuroendocrine neoplasm Colonic adenocarcinoma Neuroendocrine neoplasms are most often found in the small intestine rectum appendix and stomach The colon excluding the appendix and the cecum is a rare location for these neoplasms and often gives rise to highly proliferative poorly differentiated tumors with aggressive features and dismal prognosis A 32yearold male presents with a large cell neuroendocrine carcinoma arising from an unusual location the descending colon The pa tient™s clinical and imaging characteristics resembles those seen in the much more common neoplasm colonic adenocarcinoma Computed tomography and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Pathologic correlation of a sur gical specimen is required to make the correct diagnosis The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 Introduction Neuroendocrine neoplasms NENs are uncommon slow growing neoplasms of the neuroendocrine system that most frequently occur in the ileum the rectum “ the appendix “ and the stomach “ [ ] The colon excluding the appendix and the cecum is a rare ori gin for NENs with a reported incidence of per per sons [] The overall median age at diagnosis is years [] Colonic NENs are not normally associated with hereditary tu mor syndromes such as multiple endocrine neoplasia type [] Colonic NENs are typically poorly differentiated on histol ogy and often appear as a large mass with aggressive growth rapid dissemination and distant metastases at the time of diagnosis [“] Once metastasized the prognosis is dismal with a median survival of months [] Patients with NENs typically present with nonspecific com plaints such as bleeding diarrhea abdominal pain gastroin testinal blood loss or weight loss [ ] Carcinoid syndrome is more often seen in patients with gastric or small intesti nal NENs with liver metastasis In contrast carcinoid syn drome is rare in patients with colonic NENs because these tumors rarely contain serotonin or secrete serotonin precur sors [ ] Urine levels of the serotonin metabolite 5HIAA are not significantly elevated in patients with colonic NENs [] Serum chromogranin A may be elevated in of all gas trointestinal NENs and correlate with tumor burden [] How ever its diagnostic accuracy can be lower for poorly differen tiated NENs [ ] Also it may be falsely elevated in proton œ Competing Interests The authors have declared that no competing interests exist ˆ—Corresponding author Email address alanmlarkinhospitalcom A Mo 101016jradcr202007045 The Authors Published by Elsevier Inc on behalf of University of Washington This is an access under the CC BYNCND license httpcreativecommonslicensesbyncnd40 0c R a d i o l o g y C a s e R e p o r t s “ Fig “ Axial a and coronal b images of CT with IV contrast with multiphasic acquisition Irregular circumferential wall thickening of the descending colon — cm heterogeneously with a contiguous enhancing mass — pump inhibitor use atrophic gastritis impaired renal func tion rheumatoid arthritis inflammatory bowel disease and nonneuroendocrine neoplasms such as prostate cancer ovar ian cancer breast cancer and colorectal cancer [] The crosssectional imaging features of colonic NENs include irregular circumferential wall thickening or large polypoidal mass with lymphadenopathy closely resembling those of colonic adenocarcinoma [ ] Metastasis to the liver is common and appear as hypervascular lesions that demonstrate moderatetointense homogenous or pe ripheral rim enhancement during hepatic arterial phase on multiphasic computed tomography or magnetic resonance imaging [ ] In111 octreotide scintigraphy utilizes a synthetic somatostatin analog to characterize NENs [] Neuroendocrine tumors containing somatostatin receptors demonstrate increased radiotracer uptake We present a rare case of large cell neuroendocrine carcinoma in the descend ing colon with metastasis in the liver which demonstrates clinical and imaging features closely resembling those of metastatic colonic adenocarcinoma Case presentation A 32yearold male with a past medical history of depression and schizophrenia presented with constant left abdominal pain radiating down to the hip and groin No pertinent surgi cal or family history was noted The patient admitted to daily use of alcohol and tobacco and denied recreational drug use The review of systems was positive for fatigue intermittent bloodstreak stools and unintentional weight loss of lbs The patient denied fever night sweats decreased appetite nausea vomiting diarrhea cutaneous flushing sweating or bronchospasm The physical exam was unremarkable except for tenderness over the left flank and mid abdomen Computed tomography of the abdomen and pelvis with IV contrast revealed irregular circumferential wall thickening of — cm het the descending colon with a contiguous erogeneously enhancing mass Fig Innumerable hypoat tenuating lesions with hypovascular peripheral enhancement — Fig “ Axial images of CT of the abdomen and pelvis at the level of the right portal vein Noncontrasted axial CT image a demonstrates innumerable illdefined hypoattenuating masses throughout the liver suspicious for metastatic lesions Contrasted axial CT image on arterial phase b demonstrates the same masses with peripheral rim enhancement and a hypoattenuating center The masses remain hypodense to the background hepatic parenchyma Each mass contains a faint hypoattenuating and nonenhancing halo Contrasted axial CT image on portal venous phase c and delayed phase d demonstrate persistent peripheral enhancement with no rapid washout were observed throughout the hepatic parenchyma Fig No skeletal metastasis was appreciated In111 octreotide scan demonstrated multiple phot ic lesions within the liver Fig After an unsuccessful attempt with colonoscopy left hemi colectomy and surgical pathology were pursued Surgical pathology of the colonic mass revealed poorly differentiated large cell neuroendocrine carcinoma with tumoral invasion into the visceral peritoneum and positive of lymph nodes Fig Additionally interventional radiology was consulted for CTguided biopsy of the liver lesions Tissue biopsy of the hepatic lesions confirmed metastasis from the colonic mass Evaluation of 5hydroxyindoleacetic acid 5HIAA in a hour urine specimen was within normal limits at 4mg24h cisplatin The patient was treated with intravenous mgm etoposide for first for weeks in combination with mgm days Discussion Colonic NENs demonstrate similar crosssectional imag ing features as colorectal adenocarcinomas [ ] Both 0cR a d i o l o g y C a s e R e p o r t s “ demonstrating moderatetointense homogenous or periph eral rim enhancement during arterial phase [ ] Hyper vascular hepatic metastasis are nonspecific and can be com monly seen in melanoma renal cell carcinoma choriocarci noma and thyroid carcinoma [] However in the setting of a patient with a colonic mass hypervascular hepatic metastatic lesions may be the differentiating imaging feature to sug gest colonic NENs rather than adenocarcinoma In our case the hepatic metastatic lesions demonstrated hypovascular peripheral enhancement that resemble those typically seen with colonic adenocarcinoma as opposed to those seen with colonic NENs We postulate that the appearance of these le sions may be attributed to the fibrogenic nature of NENs [ ] and central necrosis due to the poor cell differentiation of the mass In111 octreotide scintigraphy is commonly used in diag nosis of NENs with a sensitivity of for all NENs [] It utilizes a synthetic somatostatin analog that binds to somato statin transmembrane receptors which are expressed in of all NENs [] However poorly differentiated NENs may sometimes express fewer somatostatin receptors or may even completely lack them all together producing less reli able results [ ] In our case In111 octreotide scintigraphy demonstrates multiple phot ic lesions in the liver This may be secondary to the absence or fewer numbers of somato statin receptors in poorly differentiated NENs Our patient presents with abdominal pain fatigue weight loss and hematochezia without the characteristic symptoms of carcinoid syndrome despite having substantial hepatic Fig “ hours delayed anterior projection of the chest In111 octreotide scintigraphy demonstrates multiple phot ic lesions within the liver feature irregular circumferential wall thickening or polypoid intramural mass with areas of central necrosis and degener ation [ ] Also both malignancies often metastasize to the liver [] Colonic adenocarcinomas often produce hypo vascular hepatic metastatic lesions In contrast of gas trointestinal NENs metastases feature hypervascular lesions Fig “ HE stain scan power view shows sheetlike growth pattern of tumor cells involving whole layer of colon A On higher magnification B tumor cells show solid growth pattern Note the vesicular nuclei with saltandpepper chromatin Immunohistochemical staining for chromogranin A C and synaptophysin D reveals diffuse positive in tumor cells 0c R a d i o l o g y C a s e R e p o r t s “ metastasis Also laboratory analysis of urine 5HIAA is un remarkable CT and In111 octreotide scan are limited in diagnosing large cell neuroendocrine carcinoma Ultimately the correct diagnosis is made through immunohistochemical evaluation of the surgical pathologic specimen R E F E R E N C E S [] Turaga KK Kvols LK Recent progress in the understanding diagnosis and treatment of gastroenteropancreatic neuroendocrine tumors CA Cancer J Clinic “ [] Koenig A Krug S Mueller D Barth PJ Koenig U Scharf M et al Clinicopathological hallmarks and biomarkers of colorectal neuroendocrine neoplasms PloS One 20171212e0188876 [] Yao JC Hassan M Phan A Dagohoy C Leary C Mares JE et al One hundred years after ˜carcinoid™ epidemiology of and prognostic factors for neuroendocrine tumors in cases in the United States J Clin Orthodont “ [] Caplin M Sundin A Nillson O Richard PB Klaus JK Fahrettin K et al ENETS consensus guidelines for the management of patients with digestive neuroendocrine neoplasms colorectal neuroendocrine neoplasms Neuroendocrinology “ [] Grabowski P Sch¶nfelder J AhnertHilger G Foss HD Heine B Schindler I et al Expression of Neuroendocrine Markers A Signature of Human Undifferentiated Carcinoma of the Colon and Rectum Virchows Archiv Int J Pathol “ [] Chang S Choi D Lee SJ Lee WJ Park MH Kim SW et al Neuroendocrine neoplasms of the gastrointestinal tract classification pathologic basis and imaging features Radiographics “ [] Ganeshan Dhakshina Bhosale Priya Yang Thomas Kundra Vikas Imaging features of carcinoid tumors of the gastrointestinal tract AJR Am J Roentgenol “ [] Kaltsas GA Besser GM Grossman AB The diagnosis and medical management of advanced neuroendocrine tumors Endocr Rev “ [] Cimitan M Buonadonna A Cannizzaro R Canzonieri V Borsatti E Ruffo R De Apollonia L Somatostatin receptor scintigraphy versus chromogranin a assay in the management of patients with neuroendocrine tumors of different types clinical role Ann Oncol “ [] Gut P Czarnywojtek A Fischbach J B ˛aczyk M Ziemnicka K Wrotkowska E et al Chromogranin a unspecific neuroendocrine marker Clinical utility and potential diagnostic pitfalls Arch Med Sci AMS “ [] Levy AD Sobin LH From the archives of the AFIP gastrointestinal carcinoids imaging features with clinicopathologic comparison Radiographics “ [] Sahani DV Bonaffini PA Castillo CFD Blake MA Gastroenteropancreatic neuroendocrine tumors role of imaging in diagnosis and management Radiology “ [] Bader TR Semelka RC Chiu VC Armao DM Woosley JT MRI of carcinoid tumors spectrum of appearances in the gastrointestinal tract and liver J Magn Reson Imaging JMRI “ [] Intenzo CM Jabbour S Lin HC Miller JL Kim SM Capuzzi DM et al œScintigraphic imaging of body neuroendocrine tumors Radiographics “ [] Namasivayam S Martin DR Saini S Imaging of liver metastases MRI Cancer Imaging “ [] Laskaratos FM Rombouts K Caplin M Toumpanakis C Thirlwell C Mandair D Neuroendocrine tumors and fibrosis an unsolved mystery Cancer “ 0c'

" preproofsevere acute respiratory syndrome coronavirus sarscov2 is a highly contagious zoonotic pathogen that has exacted heavy public health social and economic tolls in february the world health anization acronymed the disease caused by sarscov2 as covid19 for coronavirus disease the number of confirmed covid19 infections which has been detected in at least countries has reached worldwide as of april with deaths according to the us centers for disease control and prevention cdc1 many cases of covid19 resolve quickly however the disease which like other respiratory pathogens that cause common cold symptoms is believed to be transmitted through respiratory droplets infection with covid19 can also lead to significant morbidity and death this is particularly the case for cancer patients moreover because the signs and symptoms of covid are easily misattributed to the sequelae of cancer itself such as pulmonary embolism or its treatment such as nausea and diarrhea diagnosis may be delayed or missed potential covid rule out criteria based on the wells˜ criteria for pulmonary embolism another protean disease entity are provided as a decisionmaking aid this review summarizes the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis rationale to treat the cancer or not treatment and prevention of covid19 with an emphasis on implications in cancer keywords covid19 sarscov2 cancer introduction sarscov2 the rna virus responsible for the illness which has been named covid19 for coronavirus disease2019 the year it was diagnosed has sent shockwaves and dominated the news cycle due to its pandemic spread from the point of origin in wuhan china to the rest of the world declared a public health emergency of international concern pheic by the world health anization who2 sarscov2 is the third highly pathogenic novel zoonotic bat coronavirus sonamed because of the crownlike spikes on its surface to have emerged the first was sars coronavirus now named sarscov1 in with a fatality rate of and 0c preproofthe second was middle east respiratory syndrome mers in with a fatality rate of where the camel was the intermediate host3 sarscov1 merscov and sarscov2 belong to the betacoronavirus genus which are enveloped positivestranded rna viruses whose approximately nucleotide genome serves as an mrna template for the translation of viral proteins4 the virion contains four proteins spike envelope membrane and nucleocapsid and the host receptor with which the spike surface glycoprotein of sarscov2 engages is angiotensin converting enzyme ace25 the biology of sarscov2 is described in more detail in figure a zinc metallopeptidase enzyme ace2 which is abundantly present in lung and gastrointestinal epithelial cells6 not only mediates viral entry through receptormediated endocytosis7 but also the efficiency of viral replication8 its expression is upregulated with older age smoking the antihypertensives angiotensin converting enzyme ace inhibitors and angiotensin receptor blockers arbs thiazolidinediones tzds a class of oral antidiabetic drugs and ibuprofen risk factors which may increase susceptibility to the covid19 virus infection and which are common in generally elderly multimorbid cancer patients9 preproofthe clinical spectrum of sarscov2 ranges from mild upper respiratory tract infection with fever sore throat headache cough and potentially nausea and diarrhea the majority of cases recover without serious complications to severe pneumonia with sequelae that include acute respiratory distress syndrome ards cytokine storm and death10 because sars is an acronym for severe acute respiratory syndrome digestive manifestations including inappetence nausea abdominal pain and diarrhea of covid19 resulting from binding of the virus to the ace2 receptor in the gi tract may precede respiratory symptoms11 gastrointestinal manifestations are potentially underappreciated and overlooked as sentinel symptoms that herald the onset or persistence of disease especially in cancer patients and may contribute to delayed or missed opportunities for testing diagnosis and containment unlike sarscov1 which seems to have disappeareddied out12 and mers which reappears only sporadically sarscov2 is less lethal with a fatality rate between although this number is highly uncertain and debated13 but much more infective consequently public panic and economic disruption have ensued resulting in wartimelike mobilization efforts to mitigate its spread old age smoking and comorbidities such as diabetes morbid obesity immunosuppression frailty and cardiovascular disease appear to predispose to worse outcomes possibly secondary to impaired t and b cell responses notably covid19 infection is associated with lymphopenia and delayed development of the adaptive immune response which appears to correlate with prolonged virus clearance and more severe disease progression15 the first pillar of defense against infection is hand washing avoidance of face touching and minimization of close contact ie social distancing with selfquarantine and selfisolation16 in case of exposure or evidence of covid19 symptoms respectively the second prophylactic pillar is vaccination with specific viral antigens or mrnas which are not yet publicly available although the company moderna has reportedly started testing an mrna vaccine in healthy volunteers and multiple other vaccination strategiesplatforms appear to be in progressunder development17 0c transmission and prevention according to the world health anization who21 sarscov2 is spread persontoperson mainly via aerosol inhalation from sneezing coughing or exhalation 22and via fomitetoface contact since depending on the surface material the virus may remain viable and infectious for hours to days figure fecaloral transmission has also been hypothesized because diarrhea was a common feature with sars and mers and diarrhea and other digestive issues have also been reported in patients with covid1925 26notably transmission of sarscov2 is not limited to symptomatic individuals ie those with fever cough sore throat myalgias or dyspnea but also to asymptomatic or subclinically infected carriers of the virus which is problematic from the perspective of disease control 27and highlights the importance of containment measures including isolation and quarantine the basic reproduction number r0 of sarscov2 is which means that on average for every patient an additional individuals are infected because coronaviruses may persist on inanimate surfaces like metal glass or plastic for up to days careful disinfection with or greater ethanol for small surfaces or sodium hypochlorite for larger surfaces is recommended preproofdiagnosis and clinical features preproofthe available evidence is limited but clinical courses and outcomes of covid19 are likely to be worse in patients with cancer especially given the clear association between severity of disease and older age and higher levels of comorbidity the overall case fatality rate cfr for the general covid19infected population is around but in cancer it rises to overall and to in italy19 this cfr in cancer patients compares to for no comorbid conditions for cardiovascular disease for diabetes for hypertension and for chronic respiratory disease the aim of this review is to summarize and condense the current understanding of the transmission clinical presentation diagnosis and differential diagnosis pathogenesis treatment and prevention of covid19 with a special focus on cancer in the united states the test of choice for sarscov2 is a nasopharyngeal swab specimen or sputum if a productive cough is present on which a reversetranscriptase pcr rtpcr assay or an enzymelinked immunoassay eia directed particularly at the envelope e rnadependent rna polymerase rdrp spike protein s and nucleocapsid n genes is performed the fda has also approved an antibody test29 a positive test for sarscov2 in a symptomatic patient generally confirms the diagnosis of covid19 with the caveat that false positive and false negative tests have been documented if initial testing is negative but a high index of suspicion and pretest probability for covid19 remains on the basis of patient signs and symptoms then retesting is indicated in patients with high indexes of clinical suspicion and equivocal or negative test results the who recommends that lower respiratory tract specimens which contain the highest viral loads should be obtained since nasopharyngeal swabs may miss some infections30 according to cdc guidelines disease is excluded on the basis of two 0c preproofconsecutive negative tests respiratory tests separated by ‰¥ hours however in the presence of suggestive symptoms rectal swabs may also be indicated since the ace2 enzyme to which the virus binds is abundantly present in rectal epithelia cells31 the differential diagnosis for sarscov2 in cancer is extremely broad and includes conditions such as foreign body aspiration toxicities from chemotherapy and radiation tumor progression postobstructive pneumonia malignant obstruction atelectasis pulmonary embolism pneumonitis pulmonary edemafluid overload immunotherapyrelated pneumonitis copd exacerbation q fever adenovirus bocavirus coronavirus 229e hcov 229e coronavirus hku1 hcov hku1 coronavirus nl63 hcov nl63 coronavirus oc43 hcov oc43 human metapneumovirus hmpv influenza a influenza a subtype h1n1pdm09 influenza a subtypes h1 and h3 influenza b parainfluenza virus ““ piv “ respiratory syncytial virus ab rsv ab rhinovirusenterovirus hrvev bordetella pertussis legionella pneumophila and mycoplasma pneumoniae32 the diagnosis of sarscov2 is complicated by the possibility of simultaneous coinfection with other respiratory viruses33 which is especially true for immunosuppressed cancer patients whose susceptibility to microanisms is increased the heightened infectious risk for cancer patients underscores the importance of screening them at presentation with extended viral respiratory panel testing given that coinfection may impact management decisions since conceptually at least the morbidity of covid19 and the risk of severe illness should increase in the presence of a second or third virus preproofunlike infection with influenza for example covid19 signs and symptoms may vary considerably depending on the dose of viral inoculum route of inoculation concomitant medications and underlying health status34 to include fever dry cough fatigue sputum production shortness of breath sore throat headache myalgia or arthralgia chills nausea or vomiting nasal congestion diarrhea hemoptysis and conjunctival congestion with an incubation period of to days after exposure36 presymptomatic or minimally symptomatic infection may majorly drive transmission especially since detected viral loads are similar in both symptomatic and asymptomatic patients3738 populations of concern include the elderly smokers vapers and dual users those of any age with preexisting chronic medical conditions those receiving particular medications or therapies which upregulate the ace2 receptor or suppress the immune system and those from lower socioeconomic classes a conglomeration of factors which are often present in cancer patients as depicted in figure while the surveillance focus for covid19 is on the respiratory tract enteric symptoms are a potentially underappreciated overlooked and misattributed manifestation of disease as stated earlier and this is especially the case for cancer patients where gastrointestinal toxicity occurs routinely from chemotherapy ie cisplatincarboplatinoxaliplatin irinotecan 5fluorouracil ifosfamide from targeted agents ie erlotinib imatinib bortezomib temsirolimus sunitinib regorafenibsorafenib and bevacizumab39 and from locally advanced or metastatic disease therefore abdominal complaints in cancer patients which are potentially but not automatically attributable to underlying disease justify further investigation especially if persistent worsening or new particularly because sarscov2 transmission may occur via the fecal“oral route40 0c preproofabnormal laboratory findings in covid19 include lymphopenia percent prolonged prothrombin time percent elevated lactate dehydrogenase percent elevated ast and alt “ percent elevated highly sensitive hs crp and elevated procalcitonin however because these parameters routinely fall well outside of the normal reference range in cancer patients it is difficult to confirm or refute the presence of disease on this basis alone chest radiographs and chest ct abnormalities are similarly nonspecific since the most common features multifocal groundglass opacities and consolidation mimic other pneumonias41 significant antibody production is observed after infection but it is unknown whether this helps or harms since antibodydependent enhancement ade may potentiate viral entry and the induction of a severe inflammatory response42 universal screening of cancer patients for covid19 is desirable but logistically impossible for the foreseeable future since diagnostic tests are in short supply and simply not always readily available43 hence covid19 rule out criteria are proposed in table as a potential decisionmaking aide mémoire which separates patients into low and highrisk groups by analogy to the wells˜ criteria for pulmonary embolism4445 preventive measures focus on selfisolation social distancing with a 6foot 2m separation46 frequent hand washing with soap and water andor use of hand sanitizers patient isolation during clinical care use of masks to help prevent aerosol transmission and flushing with the lid closed to control socalled •toilet plume– in an asco guidance immunocompromised cancer patients are advised to minimize exposure to sick contacts and large crowds48 for healthcare personnel the use of personal protective equipment such as n95 masks ffp3 masks gowns eye protection gloves and gowns is mandated49 preproof vaccination and immunity vaccination efforts and the related topic of whether those who have recovered from covid19 develop protective immunity have drawn great attention the latter has implications on whether people who test positive for sarscov2 antibodies can be safely assumed to be immune and at negligible risk of contracting or transmitting the disease there have been case reports of patients who have recovered from covid19 and had recurrence of rtpcr positivity approximately one month after initial diagnosis with only one patient exhibiting significant clinical symptoms and another having a mild intermittent cough50 but while not zero the risk of transmissibility or recurrence of symptomatic disease in recovered patients has yet to be quantified and the paucity of currently available reports of recurrence in the setting of a pandemic suggests that it is low a separate practical question will be whether antibodybased tests prove to have sufficient sensitivity and specificity to identify people who had asymptomatic infections developed immunity and can return to normal activities without jeopardizing disease containment efforts immunity may be due to antibodies cell mediated immunity or a combination of the two previous experience with using plasma from convalescent patients to treat severe cases of the first sars and mers as well as limited experience with covid19 suggests that antibody mediated immunity alone is clinically beneficial even during acute infection51 safety concerns about antibodies have been raised based on preclinical studies of sarscov vaccination in 0c preproofferrets showing hepatotoxicity52 and of vaccination against feline infectious peritonitis virus another coronavirus leading to more severe disease when kittens were subsequently challenged with the virus53 although animal models may not be representative of human hostpathogen interactions the nature of sarscov and sarscov2 antibodies are likely different as crossneutralization was not observed invitro54 and experience with convalescent plasma has not borne evidence of antibody mediated enhancement of infection in acutely infected patients the potential risk deserves attention if vaccination is proposed for the entire population t cell responses are also readily observable in patients who recover from coronavirus infections55 and memory t cell responses alone were protective in mice56 with the potential advantage of longer persistence of memory t cell responses compared to humoral immunity when clinical data on vaccine candidates becomes available cancer patients may face different considerations surrounding vaccination than the general population particularly patients with hematologic malignancies being treated with agents targeting b cells who would derive greater benefit from vaccines eliciting cell mediated than antibody responses preproofpathogenesis and pathology relating to ace2 and ras signaling the ace2 enzyme a key regulator of the reninangiotensin system ras57 to which the virus binds through its surface spike proteins is particularly abundant in the digestive tract lungs kidney heart and blood vessels where pathology from sarscov2 occurs58 a peptidase that catalyzes the conversion of angiotensin ii angii referred to as •the quintessential perpetrator of inflammation– to angiotensin ang ace2 mediates antiproliferative and vasodilatory functions that oppose the vasoconstrictive and inflammatory functions of angiotensin converting enzyme ace60 the binding of sarscov2 to ace2 leads to downregulation of ace2 expression potentially through increased internalization and shedding from the cell surface with decreased ang1“ generation and increased ang ii levels as a consequence61 this unfavorably shifts the balance of the renin angiotensin system ras from the vasoprotective ace2ang17 axis to the aceang iiangiotensin at1 receptor axis and drives a proinflammatory profibrotic and proliferative response62 as shown in figure fang et al63 contend that because thiazolidinediones ibuprofen and angiotensin converting enzyme ace inhibitors and angiotensin ii typei receptor blockers arbs substantially increase the expression of ace2 they facilitate sarscov2 infection and therefore the risk of severe and fatal covid19 in contrast alghatrif et al64 present a diametrically opposed hypothesis that downregulated ace2 signaling is responsible for sarscov2induced acute lung injury ali acute respiratory distress syndrome ards and cytokine storm and that aceis and arbs are beneficial precisely because they increase ace2 expression and activity furthermore according to alghatrif et al lower ace2 levels and hence higher baseline oxidative stress and inflammation6566 are present in older comorbid individuals such as cancer patients which renders them more susceptible to severe covid19 than younger noncomorbid individuals with increased ace2 levels and lower baseline inflammation as shown in figure furthermore low ace2 may promote tumor progression and conversely ace2 overexpression is associated with antiangiogenesis and tumor regression67 in summary then 0c preproofdespite the concerns and controversy68 surrounding the use and continuation of aceisarbs during the sarscov2 epidemic it is likely that the pros outweigh the cons especially in cancer patients due to their potential antitumor and anticovid19 effects69 in line with ras involvement emerging data suggest that sarscov2 infection may induce serious cardiovascular injury or exacerbate existing cardiovascular disease cardiovascular sequela includes heart failure arrythmias disseminated intravascular dissemination dic and troponin elevation which may closely correlate with disease severity and the likelihood of inhospital death70 liu et al71 propose a mechanism whereby the virus which lowers hemoglobin hb levels72 binds to the porphyrin of heme and displaces iron thereby compromising the oxygencarrying capacity of red blood cells and exacerbating the hypoxemia since chloroquine and the experimental anticancer agent rrx001 also bind to porphyrins they may competitively interfere with binding by the virus rationale for continuation or discontinuation of cancer therapy preproofthe benefitrisk calculus that informs the decision whether and how to treat with anticancer therapy falls into a •gray zone– about which no consensus exists leading to a therapeutic dilemma on the one hand zhang et al73 in annals of oncology reported a strong association in patients of them with lung cancer between antineoplastic therapy in the past days and severe effects of covid19 hr4079 ci p0037 on this basis the authors recommend treatment interruption dose reduction or substitution of cytotoxic chemotherapy with nonimmunosuppressive options eg checkpoint inhibitors if available especially in the case of lung cancer patients that are already prone to develop respiratory infections and complications74 similarly heavily immunosuppressed patients such as those who have undergone hematopoietic stem cell transplantation are also particularly susceptible to viral respiratory infections these findings are supported by a nationwide analysis of data75 in china from covid19 patients of which were diagnosed with cancer this patient cohort experienced a higher incidence of severe events vs p and the administration of chemotherapy or surgery was found to have increased the risk of death andor intensive care unit admission even after adjusting for age sex and comorbidities odds ratio or ci “ p while these studies are limited by small sample sizes the data suggests that cancer predisposes to more severe disease therefore since inperson contact increases the risk of transmission several institutions have mandated realtime video or telephone interactions alternatively referred to as telehealth77 postponed surgeries biopsies endoscopies scans and routine investigations when possible and in line with esmo guidelines78 encouraged conversion from the intravenous to the oral route eg 5fluorouracil to capecitabine etoposide and vinorelbine on the other hand the immediate existential threat of progressive disease for which death is an impending imminent certainty rather than a remote possibility in the absence of treatment likely outweighs the theoretical risk of sarscov2 infection even in lower risk disease for example in situ or localized prostate breast and head and neck cancer delayed treatment is 0c preproofpotentially conducive to tumor development and progression and thus may unfavorably impact prognosis79 hanna et al have proposed a triage strategy80 which prioritizes treatment for those patients with imminent risk of early mortality from acute leukemias aggressive lymphomas metastatic germ cell tumors oncologic emergencies such as spinal cord compression chemoradiotherapeuticresponsive cancers such as head and neck cervical and anal cancers and neoadjuvant or adjuvant therapyresponsive tumor types such as stage iii colon cancer and deprioritizes visits for surveillance and survivorship however in the absence of a •one size fits all– consensus recommendation which is unlikely since cancer is so genetically diverse and heterogeneous the decisionmaking process and the subsequent treatment plan are individualized and to be determined tbd on casebycase basis taking into account multiple factors including the risk of cancer recurrence if therapy is delayed modified or interrupted the type of therapy eg surgery radiation chemotherapy checkpoint inhibitors and stem cell transplantation extent of comorbidities concomitant medications patient preferences physicianpatient relationship race age the number of cycles of therapy completed and treatment tolerance in terms of specific cancerrelated conditions asco makes the following heavily qualified recommendations81 \uf0b7 growth factor prophylaxis for neutropenia and neutropenic fever even at lower levels of risk as well as empiric antibiotics for acute care \uf0b7 erythropoietinstimulating agents for anemia prophylaxis and transfusion when necessary depending on the patient context and underlying comorbidities preproof treatment based on the high transmissibility of the virus82 the main nonpharmacologic countermeasures to mitigate or delay the impact of covid19 include rigorous hand hygiene use of facemasks respiratory etiquette ie coughing or sneeze into the upper sleeve or elbow not the hands flushing with the lid down to prevent bioaerosolization as well as quarantine stay at home policies and workplace and school closures which have upended the social cultural political and economic status quo no specific treatment or vaccine is currently available although promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir the mainstay of medical therapy includes symptomatic care such as supplemental oxygen antibiotics and hemodynamic and mechanical ventilatory support if indicated for septic shockmultiple an failure and respiratory failure respectively83 over active clinical treatment trials are underway84 these include vaccines as well as a number of different agents some with promising preliminary data as mentioned above and also those with potential anticancer activity which will hopefully serve a double purpose first to treat covid19 and second as an adjunct to bridge the time gap until the patient is recovered and the primary antineoplastic is startedrestarted as shown in table 0c preproof conclusions the alarming spread of the covid19 pandemic has disproportionately affected cancer patients an atrisk population both from the standpoint of increased disease severity and disruption to care which includes widespread suspension of clinical trials in the united states that are already fraught with barriers to enrollment and participation85 86because the symptoms of covid19 are nonspecific underlying symptoms from the cancer eg dyspnea cough fever fatigue diarrhea etc which overlap with those from the viral infection may obscure and delay the diagnosis hence if the covid19specific rapid reverse transcriptase polymerase chain reaction rtpcr test is not readily available andor in short supply which is currently the case diagnosis will depend on the maintenance of a high index of clinical suspicion especially in advanced cancer patients who check all the boxes for risk factors such as older age frailty disability immunosuppression generalized systemic inflammation and multiple comorbidities eg hypertension diabetes and cardiorenovascular diseases that predispose to severe disease and death preproofthese comorbidities are commonly treated with renin angiotensin system blockers such as angiotensinconverting enzyme inhibitors aceis or angiotensinreceptor blockers arbs which increase levels of ace2 the continued use of aceisarbs is the centerpiece of an intense debate because on the one hand sarscov2 coopts ace2 for target cell entry but on the other ace2 overexpression may counterbalance vasoconstriction and profibrotic processes and thereby reduce the incidence or mortality associated with covid19“associated ali or acute respiratory distress syndrome another controversy involves whether or not to continue cancer treatment given the high transmissibility potential of the virus however since no expert consensus recommendations have been issued to date and prognosis stage and responses to therapy are highly heterogeneous the riskbenefit tradeoff and subsequent treatment plan are highly individualized and context dependent currently the focus of treatment is infection control appropriate symptomatic care and oxygen therapy no approved medication or vaccine has been developed but promising activity has been reported for hydroxychloroquine chloroquine arbidol remdesivir convalescent sera and favipiravir and several repurposed agents with antitumor properties are under investigation including thalidomide and rrx001 which may hopefully bridge the gap from the time covid is first diagnosed until the primary anticancer therapy is restarted finally multiple comparisons have been made between the allout mobilization efforts to combat covid19 with the massive scaleup of human and material resources that occurred during world war ii8788 in the words of winston churchill prime minister of great britain from whose intrepid fighting spirit iron will and intransigent defiance of tyranny galvanized the resolve of an entire nation to fight on in the face of seemingly impossible odds oncologists on the frontlines that have answered the call should •never worry about action only inaction– 0c preproof declaration of interests the authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper references sitammagari k skandhan a dahlin awhat hospitalists need to know about covid19 medscape mar httpswwwmedscapecomviewarticle924596 meo sa alhowikan am alkhlaiwi t meo im halepoto dm iqbal m usmani am hajjar w ahmed n novel coronavirus 2019ncov prevalence biological and clinical characteristics comparison with sarscov and merscov eur rev med pharmacol sci feb24420122019 chan jf kok kh zhu z et al genomic characterization of the novel humanpathogenic coronavirus isolated from a patient with atypical pneumonia after visiting wuhan emerg microbes infect kuba k imai y rao s et al a crucial role of angiotensin converting enzyme ace2 in sars coronavirusinduced lung injury nat med zhang h kang z gong h et al the digestive system is a potential route of 2019ncov infection a bioinformatics analysis based on singlecell transcriptomes [epub ahead of print] biorxiv de wit e van doremalen n falzarano d munster vj sars and mers recent insights into emerging coronaviruses nat rev microbiol “ w li et al angiotensinconverting enzyme is a functional receptor for the sars coronavirus nature hughes s covid19 and angiotensin drugs help or harm medscape march httpswwwmedscapecomviewarticle927542 yang x yu y xu j et al clinical course and outcomes of critically ill patients with sarscov2 pneumonia in wuhan china a singlecentered retrospective observational study [published online ahead of print feb ] [published correction appears in lancet respir med feb ] lancet respir med 2020s2213 gu j han b wang j covid19 gastrointestinal manifestations and potential fecaloral transmission [published online march ] gastroenterology ruiheng x chance missed but still there memoirs at the 10th anniversary of sars outbreak j thorac dis aug 5suppl s90“s93 jiang s don't rush to deploy covid19 vaccines and drugs without sufficient safety guarantees nature mar5797799321 doi 101038d41586020007519 peiris js guan y yuen ky severe acute respiratory syndrome nat med dec suppls8897 zheng h zhang m yang c et al elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid19 patients cell mol immunol2020 tognotti e lessons from the history of quarantine from plague to influenza a emerg infect dis “ httpswwwnihgovnewseventsnewsreleasesnihclinicaltrialinvestigationalvaccinecovid19begins httpswwwascoascocoronavirusinformationcareindividualscancerduringcovid19 onder g rezza g brusaferro s casefatality rate and characteristics of patients dying in relation to covid in italy published online march doi101001jama20204683 hanna tp evans ga booth cm cancer covid19 and the precautionary principle prioritizing treatment during a global pandemic nat rev clin oncol who advice on the use of masks in the community during home care and in healthcare settings in the context of the novel coronavirus 2019ncov outbreak jan rodriguezmorales aj macgregor k kanagarajah s patel d schlagenhauf p going global travel and the novel coronavirus travel med infect dis httpsdoi101016jtmaid2020101578 httpswwwnejmdoifull101056nejmc2004973 lu cw liu xf jia zf 2019ncov transmission through the ocular surface must not be ignored the lancet preproof 0c preproof holshue ml et al first case of novel coronavirus in the united states n engl j med yeo c kaushal s yeo d enteric involvement o

"Lactation has a negative effect on female sexual function Hormonal changes during lactation causechanges which might lead to dyspareunia lack of libido and anasmia There are various pharmacological andnonpharmacological approaches to treat sexual dysfunction While pharmacological treatment has multipleunwanted side effects nonpharmacological therapies such as complementary medicine are a potential saferalternative The aim of this study is to evaluate the effect of ear acupressure on sexual function of lactating womenMethodsdesign This is a randomized clinical trial with a parallel sham control group In this study lactatingwomen between months and year after childbirth were referred to health care centers in Qazvin City andwould be invited to participate Participants will be divided into intervention n and control n groupsusing simple block randomization Both intervention and sham control groups will be visited over sessionswithin a 4day interval At each visit the adhesives containing Vaccaria seed will be adhered for the interventiongroup while nonlatexbased adhesives with no Vaccaria seeds will be placed on the same ear acupoints for thesham control group Selected ear acupoints include genitalia two ear points pelvic point master shoulder andposterior pituitary gland The women will be asked to hold the seeds on their ears for days and press each earpoint three times a day for s After days they will be asked to remove the seeds from their ears and rest for day Sexual function as primary outcome in both groups will be assessed using the Female Sexual Function Indexbefore and immediately after and months after the intervention Also Sexual Quality of Life as secondaryoutcome will be assessed using Sexual Quality of LifeFemale SQOLF before and months after intervention Datawill be analyzed using repeated measure ANOVA at the significant level of Discussion This study is expected to support the impact of ear channel ear acupressure on sexual function inlactating womenTrial registration Iranian Clinical Trial Registration Center IRCT20190626044028N1 Registered on August Keywords Ear acupressure Sexual function Lactation Correspondence nbahramiqumsacir2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin IranFull list of author information is available at the end of the The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cBarghamadi Trials Page of BackgroundThe postchildbearing period is a crucial stage in thewoman™s life that involves physical hormonal mentalsocial and cultural changes [] Fatigue insomnia somestressors such as child care and changes in the bodyimage along with hormonal changes can reduce interestin sexual life and decrease the number of sexual intercourses during this period [] Endocrine function of lactation has a negative effect on sexual function becauseprolactin decreases sexual hormones such as androgenand estrogen This mechanism reduces sexual functionbecause of vaginal dryness vaginal epithelium atrophyand dyspareunia [] About two thirds of women duringlactating period experience at least one sexual problemsuch as decreased libido lack of sexual pleasure dyspareunia and vaginal dryness which usually are resolvedwithin year after childbirth [ ] The prevalence ofsexual dysfunction increases from to in the prepartum period [] to to in the postpartum period[] The results of various Iranian studies confirmed thehigh prevalence to of sexual dysfunctionduring lactation [ ] Therefore sexual dysfunction is acommon problem during lactation that needs moreattention from health care providersIn the postchildbearing period most women try to return to their sexual life within to weeks after childbirth Howeverit may take up to year to resumesexual relationships with the same quality of beforepregnancy [] Psychotherapy and pharmacotherapy aretwo main methods for treating female sexual dysfunctionFSD [] However the US Food and Drug Administration FDA does not recommend any foods or medicines for the treatment of FSD [] Therefore nonpharmacological therapies such as complementary medicine are a potentially safer alternative to pharmacologicaltherapy Acupressure is one of the noninvasive complementary methods oftraditional Chinese medicineTCM based on the principles of acupuncture []Acupuncture stimulates medical points and meridiansthat are distributed throughout the body to regulatephysiologic reactions [] Acupressurelike acupuncture modulates a person™s vital energy by stimulatingacupoints and meridians that are distributed throughoutthe body []The external ear is one of the several somatotopicmicrosystems that can be used in acupressure Ear acupressure also known as auriculotherapy or auricularacupressure [ ] was first presented in ancient Chinese medicine to years BC but Dr Paul Nogierwas the first Western physician who put forward auriculotherapy in a scientific way [ ] Accordingto this theory each part of the body is associated with aspecific part of the ear that reflects the physiological orpathological state of the body [] In this method theouter surface of the ear auricle can be stimulated to reduce pathological conditions in other parts of the body[] Ear stimulation can be performed in various wayssuch as manual finger pressure electrical stimulation lasers differenttypes of needles magnetic seeds andseeds to strengthen neural connections [] The WorldHealth anization WHO has recognized ear acupuncture as a promising therapeutic approach becauseof its efficacy in managing health disorders [] Ear acupuncture has been recognized as a form of acupuncturethat can affect all body ans [] Ear acupressure isan easy noninvasive and safe therapy [ ] It is easyto use for mothers who need to take care of their childin the postchildbearing period []There are several studies supporting the effects of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] However the effect of ear acupressure on sexualfunction has not been studiedResearch aimThis study is designed to investigate the effect of earacupressure on the sexual function of lactating womenMethodsDesign and settingThis is a randomized clinical trial with a parallel shamcontrolled group The study is designed in accordancewith the CONSORT standards Lactating women referred to health care centers in Qazvin City Iran will beinvited to participate Figure shows the CONSORTflow diagram The current protocol is anized basedon the SPIRITTCM extension []ParticipantsIn this study breastfeeding women between monthsand year after childbirth will be referred to health carecenters in Qazvin City Iran and they will be invited toparticipate in the study Inclusion criteria will be willingness to participate in the study being literate beingprimiparous breastfeeding fullterm singleton deliverylesionfree auricle no ulcer and pain in the ear and ability to present for all intervention sessions Exclusion criteria will be being away from their spouse for more than month having complications during pregnancy orhaving postpartum depression in the postchildbearingperiod These criteria were diagnosed using the Edinburgh Postpartum Depression Scale substance abuse inthe individual or the spouse and history of illnessesaffecting sexual function in a woman or her spouse egpremature ejaculation cardiovascular mental health disorders thyroid diseases any form of cancer or injuries 0cBarghamadi Trials Page of Fig The CONSORT flow diagramof the genital area The use of drugs affecting sexualfunction such as psychotropic cardiovascular neurological and hormonal drugs will also be excluded Afterthe initial screening the Demographic and ObstetricQuestionnaire and Female Sexual Function Index FSFIwill be completed by the participants before anyinterventionRole and qualification of practitionersThe research team consists of one specialist in auriculotherapy TO two specialists in reproductive health ZAand NB and one midwifery postgraduate student Theprotocol of intervention was designed based on the literature review and expert opinion of TO who is an expert inauriculotherapy and is a journal editor for several international journals Other team members have been qualified in this technique before designing present researchParticipant screening and care providing will be done bySB and ZA and NB will monitor her during the first sessions to ensure the fidelity of providing interventionSample size calculationSample size is estimated according to the study ofBokaie with the mean and standard deviation of 0cBarghamadi Trials Page of total sexual function score has been reported as ± and ± in the intervention telephonecounseling group and control groups respectively []type error α Therefore considering the first confidence the second type error β power and error d and the possibility of loss tofollowup individuals for each group are requiredSo the total sample size will be peopleInterventionEar acupressure groupThe researcher will clean the auricle using alcoholand then place Vaccaria seeds using nonlatexbased adhesives on ear acupoints for genitalia pelvis shoulderand the posterior pituitary gland Figure illustrates theintended ear acupoints Intervention is designed for sessions within a 4day interval During these sessionsthe seeds are placed on the designated ear acupressurepoints The women will be instructed to keep the seedson their ears for days during which each point shouldbe compressed three times a day for s The compression should be performed with moderate stimulationthrough pressing steadily and slightly tighter until feelinga slight tingling and discomfort After days they willbe asked to remove the seeds from their ears and restfor a day [] The women will be reminded daily bysending the text and will be reminded by phone for thenext interventional session This procedure will be performed for ten sessions for each participant It should benoted that if a participant has problems during the useof the method for any reason displacement of seedsdiscomfort etc she can remove the previous seed andask the researcher to reattach new seedsSham control groupThe control group will have sessions that will be thesame as the intervention group except that no Vaccariaseeds will be placed on the ear acupointsMeasuresIn this studycollectionfour measures will be used for dataDemographic and Obstetric QuestionnaireThe demographic section includes questions such asage education level occupation place of residence economic status age at marriage and length of marriageThe obstetric section includes questions regarding thenumber of pregnancies type of contraceptive methoddelivery date type of delivery perinealinstrumental delivery history of painful intercourse in prenatalperiod infant gender birth weight first lactation number of intercourse per month before pregnancy onset offirst intercourse after delivery and average number ofintercourses per week during lactation Validity of thisquestionnaire will be confirmed by some of the facultymembers ofthe midwifery department of QazvinUniversity of Medical Sciences IraninjuryFig The selected ear acupoints 0cBarghamadi Trials Page of Female Sexual Function Index FSFIIt has been designed by Rosen to evaluate sexualfunction in women over the past weeks [] It consistsof items subdivided into six subcategories of sex desire items arousal items lubrication items asm items satisfaction items and pain itemsThese subcategories have response ranges from or to with higher scores indicating better sexual performance [] The questionnaire has been used in manystudies abroad and has shown to have a high degree ofinternal consistency reliability and validity [] Thefindings of Fakhri and Mohammadi™s study showed thatthe Farsi version of FSFI FSFIIV is a reliable and validinstrument with appropriate psychometric properties toevaluate women™s sexual function [ ] Reliability ofthe scale has been calculated through stability analysisor calculation of the internal consistency coefficient TheCronbach™s alpha coefficient was and was higher forall domains and for the whole scale [] In addition reliability was confirmed by the testretest method and thecorrelation coefficient was reported high indicating thatFSFIIV is reusable within a 4week interval [ “]The maximum score for each domain is and for thewhole scale is Also any score less than will beused to indicate sexual dysfunction []The Edinburgh Postpartum Depression Rating ScaleThis 10item scale has been designed to detect depression from weeks after delivery The Edinburgh Scalescores vary between and with cutoff point andabove to detect postpartum depression [] Psychometric evaluation of the Farsi version of this scale is carriedout in [] The Cronbach™s alpha for the Edinburgh Scale has been reported as Validity of the Edinburgh Beck Scale has been reported as Thefindings of the study confirmed the high validity of theFarsi version of the Edinburgh Scale for the diagnosis ofpostpartum depression []The Sexual Quality of LifeFemale SQOLFIt is a short tool that specifically assesses the relationshipbetween sexual function and women™s quality of life Ithas been developed by Symonds [] This 18itemquestionnaire focuses on sexual selfesteem emotionalaffairs and relationships Each item is responded with aLikert scale of strongly agree score to strongly disagree score Higher scores indicate better quality ofsexual life for women [] Validity and reliability of theFarsi version ofthis scale have been confirmed byMaasoumi []Primary outcomePrimary outcome of this study is to investigate changesof sexual function using the FSFISecondary outcomeThe secondary endpoint is investigating the quality ofwomen™s sexuallife which will be assessed using theSexual Quality of Life Questionnaire Any harm or sideeffects will be also reportedStudy procedureAfter obtaining the necessary permissions from the Ethics Committee of Qazvin University of Medical Sciencesregistration at the Iranian Clinical Trial RegistrationCenter IRCT and obtaining the permission from Qazvin Nursing and Midwifery School the researcher willattend for recruitment in Qazvin Health Centers Thelactating women of each center will be invited for participation The process and purpose of the research willbe explained to those who meet the inclusion criteriaAlso they will be ensured that their information will bekept confidential and that the questionnaires will be anonymous and coded in accordance with the researchethics They will enter into the study after signing thewritten consent form The researcher will ensure thatthey can leave the study at any time After enrolling eligible individuals a simple block randomization will beused Participants will be randomly assigned to the intervention and control groupsThe questionnaires will be filled out by the participants before the intervention Then the interventionacupressure will be performed as mentioned aboveEvaluation of sexual function in both the interventionand control groups will be performed using the FSFIquestionnaire immediately and months after theintervention Figure provides the timeline of recruitment allocation and assessmentStatistical analysisData will be analyzed using the chisquare test Fisherexact test and independent t test via the SPSS v24 software The KolmogorovSmirnov and Shapiro tests willalso be used to check for the normal distribution of sexual function scores Descriptive statistics number meanand standard deviation will be used to describe thedemographic characteristics of the participants The repeated measure ANOVA will be used to compare themean sexual function of women before and after theintervention The significance level of all tests is set asp Methods to protect against biasRandomization and allocationSampling will be made using a twostep samplingmethod In the first step Qazvin City will be consideredas five geographical districts Next two health centerswill be randomly selected in each of these five districtsof Qazvin City Secondly women referred to selected 0cBarghamadi Trials Page of STUDY PERIODEnrolment AllocationPostallocationCloseoutTIMEPOINT1t1t2t3t4t5t6t7t8t9t10t11t12t13tXENROLLMENTEligibility screenInformed consentRandomized by independent personAllocationINTERVENTIONSGroup A Auricular acupressureGroup B ControlASSESSMENTSFSFISQOLXXXXXXX X XXFig Schedule of enrolment interventions and assessmentshealth centers will be assessed for eligibility and in caseof willingness to participate in the study will be recruited They will be randomly assigned to two groupsas follows One letter is assigned to each group A intervention group B control group and all possible conditions for the 4block will be written and numbered asfollows 1AABB 2ABAB 3BBAA 4BABA 5ABBA6BAAB In a simple block randomization method using thetable of random numbers numbers of blocks will be selected until the specified sample size is achieved Therandom allocation sequence is specified For example ifthe numbers given are and respectively theallocation sequence will be as follows AABB ABABABAB BBAA As a result each participant will have aunique code n Allocation concealmentFor this purpose after preparing the allocation sequencethe sequence is annotated on paper and placed inopaque sealed envelopes which will be numbered consecutively The questionnaires will be coded in the samemanner A questionnaire with the same code will becompleted by the person who receives code intervention The assignment sequence and its concealment willbe performed by someone outside the research teamBlindingParticipants outcome assessor and statistical analyzerwill be blind to which group a participant was placedThe individual entering the data from the participantquestionnaires on demographic information postpartumdepression female sexual functioning and female qualityof life will be blind to which participant was assigned tothe auricular acupressure seed group or the sham control groupTreatment fidelityOne of the researchers SB is responsible to performintervention She has participated specific course on auriculotherapy and is supervised by acupuncturist MHAabout how to perform the intervention Approximately of intervention sessions will be run under supervision of the acupuncturist to ensure that all acupoints arechosen correctlyData managementOne of the researchers SB will be responsible for collecting data at each study stage and will assign anotherindividual for the task of data entry into the SPSS software This process will be performed under supervisionof NB and ZA 0cBarghamadi Trials Page of Ethical and safety issuesThe research protocol will be reviewed and approved bythe Ethics and Human Research Committee of QazvinUniversity of Medical Sciences decree code IRQUMSREC1398056 It is registered on the Iranian ClinicalTrial Registration Center underofIRCT20190626044028N1 In addition the following ethical considerations will be considered throughout this research obtaining written informed consent the volunteernature of participation in the study ability to withdrawfrom the study at any time and confidentiality of information even during the publication of findings All ethical issues related to clinical trials will be considered Informedconsent during recruitment phase will be obtained by SBdecreecodeDiscussionTo the best of our knowledge this study is the first randomized clinical trial investigating the effect of ear acupressure on the sexual function of lactating women Thestrength of this study is the randomized design with parallel treatment procedures and a large sample size butdue to the importance of the treatment practitionerknowing whether they are placing an adhesive patchwith a Vaccaria seed or without one a true double blindstudy is not possible Acupressure has no known side effects is noninvasive and is easy to use [ ] Therefore the therapist and clients can develop a sense ofclosenesstrust and confidence [] Several studieshave shown promising results about the effect of earacupressure on insomnia [ ] menstrual cramps anddysmenorrhea [ ] premenstrual pain []chemotherapyinduced nausea and vomiting [] smoking cessation [] obesity [] and shortness of breath[] In the study of Oakley acupuncture wasaffective in premenopausal women with sexual function[] In line with previous literature this study wouldprovide insights about the impact of ear acupressure onsexual function in lactating womenAbbreviationsCONSORT Consolidated Standards of Reporting Trials SQOLF Sexual Qualityof LifeFemaleAcknowledgementsNot any in this stageTrial statusThe recruitment has not yet begun and necessary permissions are acquiredthe estimated date of recruitment is November The expected timefor completing the recruitment is March Protocol version registered on August Authors™ contributionsAll authors SB ZA NB and TO contributed to the design of the study SBNB and ZA drafted the preliminary manuscript TO revised the manuscriptand prepared the final version of the manuscript All authors revised themanuscript agreed to be fully accountable for ensuring the integrity andaccuracy of the study and read and approved the final version of themanuscript to be published All the authors met the criteria for authorshipand listed as coauthors on the title pageFundingThe research deputy of Qazvin University of Medical Sciences will providefinancial support Study funders have no role in the study design thecollection management analysis and interpretation of data the writing ofthe report and the decision to submit the report for publicationAvailability of data and materialsAnalyzed data and materials will be deidentified and publishedEthics approval and consent to participateResearch protocol is approved by the Ethics and Human ResearchCommittee of Qazvin University of Medical Sciences decree codeIRQUMSREC1398056 Permissions from responsible authority will beobtained before recruitment Informed consent will be acquired beforeparticipationConsent for publicationNot applicableCompeting interestsNone to declareAuthor details1Student Research Committee Qazvin University of Medical Sciences QazvinIran 2Social Determinants of Health Research Center Research Institute forPrevention of NonCommunicable Diseases Qazvin University of MedicalSciences Shahid Bahonar Blvd Qazvin Iran 3Emperor™s College of TraditionalOriental Medicine Santa Monica CA USAReceived November Accepted August ReferencesAbdelhakm EM Said AR Elsayed DMS Effect of PLISSIT model sexualcounseling program on sexual quality of life for postpartum women Am JNurs Sci Avery MD Duckett L Frantzich CR The experience of sexuality duringbreastfeeding among primiparous women J Midwifery Womens Health“Acele EÖ Karaçam Z Sexual problems in women during the firstpostpartum year and related conditions J Clin Nurs ““ Mohammed YF Hassan HM AlDinary AM Rashed NS Sexual function afterchild 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surgically menopausal women a randomizedplacebocontrolled trial Menopause “ Belkin ZR Krapf JM Goldstein AT Drugs in early clinical development forthe treatment of female sexual dysfunction Expert Opin Investig Drugs“ Ozgoli G Mobarakabadi SS Heshmat R Majd HA Sheikhan Z Effect of LI4and BL32 acupressure on labor pain and delivery outcome in the first stage 0cBarghamadi Trials Page of of labor in primiparous women a randomized controlled trial ComplementTher Med “ Oleson T Auriculotherapy manual Chinese and Western systems of ear Mohammadi KH Heydari M Faghihzadeh S The Female Sexual FunctionIndex FSFI validation of the Iranian version Health Monit J Iran Inst HealthSci Res Burri A Cherkas L Spector T Replication of psychometric properties of theFSFI and validation of a modified version FSFILL assessing lifelong sexualfunction in an unselected sample of females J Sex Med “Sidi H Abdullah N Puteh SEW Midin M The female sexual function indexFSFI validation of the Malay version J Sex Med “Sun X Li C Jin L Fan Y Wang D Development and validation of Chineseversion of female sexual function index in a Chinese population”a pilotstudy J Sex Med “Ter Kuile MM Brauer M Laan E The female sexual function index FSFI andthe female sexual distress scale FSDS psychometric properties within aDutch population J Sex Marital Ther “ Cox JL Holden JM Sagovsky R Detection of postnatal depressiondevelopment of the 10item Edinburgh Postnatal Depression Scale Br JPsychiatry “ Ahmadi kani Golzar A GoliZadeh Z Validation of Edinburgh PostpartumDepression Scale EPDS for screening postpartum depression in Iran J NursEduc “Symonds T Boolell M Quirk F Development of a questionnaire on sexualquality of life in women J Sex Marital Ther “ Maasoumi R Lamyian M Montazeri A Azin SA AguilarVafaie ME HajizadehE The sexual quality of lifefemale SQOLF questionnaire translation andpsychometric properties of the Iranian version Reprod Health Oakley SH WaltherLiu J Crisp C Pauls R Acupuncture in premenopausalwomen with hypoactive sexual desire disorder a prospective cohort pilotstudy Sexual medicine 201643e176“e81Publisher™s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsacupuncture Yeh ML Chang YC Huang YY Lee TY A randomized controlled trial ofauricular acupressure in heart rate variability and quality of life forhypertension Complement Ther Med “Tan JY Molassiotis A Wang T Suen LK Current evidence on auriculartherapy for chemotherapyinduced nausea and vomiting in cancer patientsa systematic review of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chien LC Chiang YC Lin SW Huang CK Ren D Reduction innausea and vomiting in children undergoing cancer chemotherapy byeither appropriate or sham auricular acupuncture points with standard careJ Altern Complement Med “ Abbate S Chinese auricular acupuncture Florida Routledge Yeh TL Chen HH Pai TP Liu SJ Wu SL Sun FJ The effect ofauricular acupoint stimulation in overweight and obese adults a systematicreview and metaanalysis of randomized controlled trials Evid BasedComplement Alternat Med Yeh CH Chiang YC Hoffman SL Liang Z Klem ML Tam WW Efficacyof auricular therapy for pain management a systematic review and metaanalysis Evid Based Complement Alternat Med Kim JY Ryu HS Nam SH Park KS Effects of auricular acupressure therapy onnocturia and insomnia in the elderly Korean J Rehabil Nurs “ Wang YJ Hsu CC Yeh ML Lin JG Auricular acupressure to improvemenstrual pain and menstrual distress and heart rate variability for primarydysmenorrhea in youth with stress Evid Based Complement Alternat MedShergis JL Ni X Jackson ML Zhang AL Guo X Li Y A systematicreview of acupuncture for sleep quality in people with insomniaComplement Ther Med “ Dantas KKdL Auriculoterapia chinesa com o uso de sementes de colza nadismenorreia primaria relato de caso Universidade Federal do Rio Grandedo Norte Portman DJ Bachmann GA Simon JA Group OS Ospemifene a novelselective estrogen receptor modulator for treating dyspareunia associatedwith postmenopausal vulvar and vaginal atrophy Menopause “ Oleson T Flocco W Randomized controlled study of premenstrualsymptoms treated with ear hand and foot reflexology Obstet Gynecol“Kwon SJ Park JS Effects of auricular acupressure on chemotherapyinducednausea vomiting and serum serotonin level Korean J Adult Nurs “ Di YM May BH Zhang AL Zhou IW Worsnop C Xue CC A metaanalysis ofearacupuncture earacupressure and auriculotherapy for cigarette smokingcessation Drug Alcohol Depend “ Huang ETY Di PhD YM Acupuncture therapies for chronic obstructivepulmonary disease a systematic review of randomized controlled trialsAltern Ther Health Med Dai L Cheng CW Tian R Zhong LL Li YP Lyu AP Standard ProtocolItems for Clinical Trials with Traditional Chinese Medicine recommendations explanation and elaboration SPIRITTCM extension Chin J Integr Med “ Bokaie M Hajimaghsoudi S Dehghani A Hosseini F The effect of sexualhealth counselling on the sexual function and satisfaction of breastfeedingwomen in the form of group consultation and telephone consultancyJ Adv Pharm Educ Res 20199S2191“ Rosen CB Heiman J Leiblum S Meston C Shabsigh R Ferguson DD'Agostino R The Female Sexual Function Index FSFI a multidimensionalselfreport instrument for the assessment of female sexual function J SexMarital Ther “Fakhri A Pakpour AH Burri A Morshedi H Zeidi IM The Female SexualFunction Index translation and validation of an Iranian version J Sex Med“ Wiegel M Meston C Rosen R The female sexual function index FSFI crossvalidation and development of clinical cutoff scores J Sex Marital Ther“ 0c"

"an-specific analysis demonstrated that pre-treatment diameter of lung metastatic lesions had a moderately positive association with percent change in post-treatment tumor diameter (R?=?0.341 ). There were fewer target lesions in the other three ans than in the lung and there was no association between liver lymph node or kidney lesion size and percent change in post-treatment tumor diameter (). The percent changes in target lesion size differed significantly between the four individual ans (P?=?0.0007 by the Kruskal Wallis test). The mean (± SD) percent changes in target lesion size in the lung liver lymph nodes and kidney were ?27.1?±?33.5 0.5?±?29.4 ?16.7?±?19.6 and ?2.7?±?21.7 respectively. Target lesions in the lung showed the greatest change in size which was significantly greater than in the liver and kidney (P?=?0.007 and 0.002 respectively). There was no difference in the percent change in target lesion size between the lung and lymph nodes (P?=?0.114) (). Association between pre-treatment tumor size and percent change in lesion size after sunitinib treatment. Association between pre-treatment tumor size and percent change in size was analyzed for lesions in the lung (a) liver (b) lymph nodes (c) and kidney (d). The pre-treatment size of lung lesions showed a moderately positive association with percent size change after treatment with sunitinib while no association was observed in the liver lymph nodes and kidney. Percent change in target lesion size in different ans. The reduction in lesion size was significantly greater in lung lesions compared with liver and kidney lesions. Lung lesions with an initial diameter?<?17.3 mm (median) showed a significant percent reduction in size compared with lesions???17.3 mm. No significant differences in relation to initial lesion size were observed in the liver lymph nodes and kidney. Lesions in each an were divided into two groups according to the median pre-treatment diameter and percent changes in tumor diameter were compared between the two groups. Lung lesions with a pre-treatment diameter less than the median value of 17.3 mm showed a significant percent reduction in diameter compared with tumors???17.3 mm (P?=?0.002). There were no differences in the percent change in relation to size above or below the median value in other ans (). Cut-off value for pre-treatment tumor diameter predicting response to sunitinib in lung metastasis ROC curves were drawn to determine cut-off values predicting 30% and 50% reductions in the diameter of lung metastatic lesions (). The cut-off predicting a 30% reduction in diameter was 16.5 mm with a sensitivity of 69.6% specificity of 58.2% and an area under the curve (AUC) of 0.662. The cut-off predicting a 50% reduction in diameter was also 16.5 mm with a sensitivity of 67.0% specificity of 77.8% and an AUC of 0.752. Using this cut-off value the percent change in lesion size for lesions?<?16.5 mm was ?41.7?±?35.5% while that for lesions???16.5 mm was ?16.2?±?26.9% (P?=?0.0005). Cut-off values of initial lung tumor size for predicting 30% and 50% reductions in diameter after sunitinib treatment. Based on ROC analysis the cut-off values of initial lung lesion size for predicting reductions in diameter of both 30% and 50% were 16.5 mm. Influence of pre-treatment CRP value cytoreductive nephrectomy and treatment line on percent change in target lesion size in the lung Metastatic lung lesions were categorized into two groups based on pre-treatment C-reactive protein (CRP) levels. The mean diameter and percent change in lesion size in patients with CRP?<?2.0 mg/ml were 19.0?±?9.3 mm and ?38.3?±?30.5% respectively while those in patients with CRP???2.0 mg/ml were 28.3?±?20.6 mm and ?9.6?±?33.9% respectively. The lesions were further divided into three subgroups according to pre-treatment diameter?<?20 mm ? 20 to?<?40 mm and???40 mm. Percent changes were compared between lesions in patients with CRP?<?2.0 mg/dl (low CRP) and those with CRP???2.0 mg/dl (high CRP) in each subgroup. For lesions?<?20 mm patients with low CRP had significantly greater reductions in tumor diameter than patients with high CRP (?43.8?±?33.4% vs. ?15.3?±?37.7% P?=?0.0019). In patients with lesions ?20 to?<?40 mm there was a tendency towards a greater reduction in patients with low CRP compared with high CRP though the difference was not significant (?29.0?±?17.3% vs. ?12.4?±?30.2% P?=?0.054) and similarly there was no significant association between tumor reduction and CRP level in patients with lesions???40 mm (?13.3?±?20.6 vs. 6.8?±?17.4 P?=?0.151) (). Influence of CRP on percent change in lung lesion size. In patients with lung lesions?<?20 mm the percent reduction in size was significantly greater in patients with low compared with high CRP levels. Meanwhile CRP level had a marginal effect on size reduction in lesions???20 to?<?40 mm and no effect in lesions???40 mm. Percent changes in size were compared between lung lesions in patients in each diameter subgroup who did and did not undergo cytoreductive nephrectomy. There were no lung lesions???40 mm in patients who did not undergo cytoreductive nephrectomy. There was no significant difference between mean percent change in lesion size in patients with and without cytoreductive nephrectomy (?36.1?±?32.6 vs. ?28.2?±?41.9 P?=?0.421 and ?20.4?±?25.1 vs. ?32.0?±?21.9 P?=?0.307 in lesions?<?20 mm and???20 mm respectively). Similarly there was no significant difference in percent change in lesion size between lung lesions in patients treated as first-line therapy and those treated as second-line or later therapy in any diameter subgroup (?38.1?±?37.7 vs. ?32.1?±?33.9 P?=?0.280; ?23.5?±?21.6 vs. ?17.5?±?23.3 P?=?0.803; and 3.5?±?2.6 vs. 1.9?±?22.9 P?>?0.9 in lesions?<?20 mm ? 20 to?<?40 mm and???40 mm respectively). Discussion A previous study on metastatic RCC by Yuasa et al. demonstrated that: 1) smaller initial tumor size predicted a good response to TKIs; 2) the greatest response was achieved in patients with lung lesions; and 3) there was no difference in tumor response between patients treated with sorafenib and sunitinib [6]."

99mtc‘labeled nanocolloid drugs development methodsVladimir Sadkin1 Viktor Sкuridin1 Evgeny Nesterov1 Elena Stasyuk1 Alexander Rogov1 Natalya Varlamova1 Roman Zelchan2The work considers the problem of obtaining nanocolloid radiopharmaceuticals RPs and studying their functional suitability for diagnosing sentinel lymph nodes SLN in cancer patients Two principal approaches to the formation of technetium99mlabeled ps based on inanic and anic matrices were considered when carrying out research to develop methods for the production of nanocolloid RPs The composition of the reagents and the conditions for obtaining nanocolloid radiopharmaceuticals were determined The functional suitability of new RPs for scintigraphic diagnostics of sentinel lymph nodes has been studiedThe identification of sentinel lymph nodes”the first nodes that stand in the way of metastasizing of malignant neoplasms attracts increasing interest in modern oncological practice1“ It is believed that if the SLN is not affected by the metastatic process then all other regional lymph nodes are intact so the results of biopsy of these nodes are an objective diagnostic criterion for the spread of malignant process Fig a0 The optimal method of detecting SLN is the use of colloid nanomaterials labeled with technetium99m for scintigraphic or radiometric determination of node localization6“ Not so much the chemical nature of such ps but their size is the determining factor in the choice of the indicator in this case Thus according to Schauer14 a colloid with a p size of less than a0nm can accumulate not only in the SLN but also at nodes of and orders of magnitude Ps with the sizes of more than a0nm slowly migrate from the injection site The colloid with the p size from to a0nm was recognized as the optimal one for detecting SLNThe simplest method of obtaining colloids with given sizes and properties is immobilization of 99mTc on the surface of nanosized materialsTechnetium99m is by far the most popular radionuclide for conducting diagnostic studies practically in all fields of medicine15“ This is primarily due to its nuclearphysical characteristics a relatively short halflife a0h and Îradiation energy of a0meV providing a low exposure dose and at the same time sufficient penetrating power for radiometric measurementsToday the Tc99m Tilmanocept radiopharmaceutical is widely used which has proven itself well and gives good results But its production is quite timeconsuming and requires expensive components We offer a less laborious method from the simple components1920Materials and methodsMaterials All the reagents were purchased from SigmaAldrich ACS grade and used without further purification Technetium99m was obtained from chromatographic 99Mo99mTc generator œ99mTcGTTOM produced by Tomsk Polytechnic University TPU”Tomsk RussiaThree types of nanops were selected to obtain nanocolloids labeled with 99mTc The first type of colloids was created on the basis of metal chelates with chemically modified complexons of diethylenetriaminepentaacetic acid DTPA It should be noted that the DTPA molecule itself like its complexes with metals is hydrophilic and does not tend to form colloidal ps The introduction of hydrophobic fragments into its structure allowed the preparation of waterinsoluble modified DTPA complexes21 The original substance of the modified DTPA DTPAmod was synthesized in Tomsk Polytechnic University Preparation of colloid solution DTPAmod was produced using the following method A sample of modified DTPA with the mass of a0mg was quantitatively transferred to a volumetric flask of a0ml and dissolved in a0ml of NaHCO3 solution by heating to a0°C After that the volume was adjusted with the same solvent up to the mark In order to reduce the p size the container with suspension was heated in water to a0°C and treated with ultrasound for a0min 1Tomsk Polytechnic University Lenina Avenue Tomsk Russia 2Tomsk National Research Medical Center Russian Academy of Sciences Kooperativny street Tomsk Russia email sadkintpuruScientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Scheme for determining the sentinel lymph node using nanocolloid radiopharmaceuticals radiopharmaceutical sentinel lymph node detectorFigure a0 The general scheme for the synthesis of 99mTcDTPAmodwhich reduced the average p radius up to a0nm The general scheme for the synthesis of 99mTcDTPAmod is shown in Fig a0The second type of colloids is iron nanops coated with a carbon shell of FeC Fig a03a These ps were obtained from the Institute of Metal Physics UrB RAS Ekaterinburg Russia In order to impart lipophilic properties to ironcarbon ps and to increase their stability in solution in the form of a colloid a technique for preliminary deposition of anic radicals aryldiazonium tosylates ADT onto the surface of these ps has been developed An effective method for the synthesis of ADT followed by their application onto the carbon surface of ps was developed at the Tomsk Polytechnic University22 The general scheme for the synthesis of FeC ps and their subsequent interaction with 99mTc is shown in Fig a03bIn the third type of colloids technetium99m was adsorbed on aluminum oxide powder A powder of lowtemperature cubic modification of gammaoxide Al2O3 prepared from aluminum hydroxide powder AlOH3 by its calcination in a muffle furnace was used The substance was synthesized in Tomsk Polytechnic UniversityA reducing agent”tin II chloride dihydrate was used in order to obtain complexes of 99mTc with colloidsGelatin powdered Ph Eur USPNF pure pharma grade CAS Number was purchased from AppliChem GmbH Darmstadt GermanyMethods Method for preparation of 99mTc labeled nanocells The introduction of the radioactive label 99mTc into a colloidal substance was carried out by mixing of the selected substance with the reducing agent SnCl2ˆ™2H2O “ a0mgml in different ratios and then adding a a0ml of eluate of 99mTc “ a0MBqml to the mixtures The mixtures were incubated for a0min at a temperature of “ a0°C The preparation is ready for use after cooling at room temperature The reducing agent SnCl2ˆ™2H2O was used as a hydrochloric acid solution The amount of a0g of tin chloride II is added to the vial and a0ml of a0M hydrochloric acid HCl Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cFigure a0 A Carbon encapsulated iron nanop B the general scheme for the synthesis of FeC psis then added for its preparation After dissolution the volume is adjusted with distilled water to a0ml Dissolution was carried out in an inert gas argon mediumDetermination of the size of 99mTc labeled colloidal ps The determination of the size of the labeled nanocolloids was carried out by spectroscopy on a Nanophox p size analyzer œSympatec GmbH Germany and also by a technique based on measuring the activity of the suspension before and after filtering it successively through filters with predetermined pore sizes and a0nm Three samples were taken with a volume of a0μl from each initial solution and filtrates for the subsequent measurement of their activity Calculations of the yield of products with different p sizes were determined according to the formulas given belowC220 Avc ˆ’ A1Avc C100 A1 ˆ’ A2A1 C50 A2 ˆ’ A3A2where Avc is the activity of the initial suspension prior to filtration A1 is the activity measured after filtration through a a0nm filter A2 is the activity after filtration through a0nm filter A3 is the activity measured after filtration through a0nm filterIn parallel determination of the radiochemical purity RCP of preparations by thin layer chromatography method was carried outThin‘layer chromatography TLC procedure To determine radiochemical purity of 99mTc“nanocolloid a0 µl of prepared sample was spotted on silicagel impregnated strip Sorbfil Russia — a0 cm To determine Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cAmount of SnCl2ˆ™2H2O mgA[Sn99mTc]A ATcVIIA Table Change in relative activities of the complex [Sn99mTc] and 99mTc VIIpertechnetate content of the radiopharmaceutical sample first strip was developed using acetone as the mobile phase time of chromatography a0min In this system pertechnetate migrated with the front of the mobile phase Rf To determine the colloid content of the preparations the second strip was developed using ethanolwaterammonium hydroxide as the mobile phase time of chromatography a0min In this system the 99mTc“nanocolloid migrated with the front of the mobile phase Rf Stability The stability of 99mTc“nanocolloid was studied in a0vitro by mixing of a0ml of normal serum and a0ml of 99mTc“nanocolloid following by incubation at a0°C for a0h At different time points a0h a0h and a0h a0ml aliquots of complex were removed and evaluated for radiochemical purity using TLC24Determination of the functional suitability of preparations for scintigraphic detection of SLN A study to assess the functional suitability of new nanocolloid RPs was performed in series of experiments involving white Wistar male rats weighing “ a0g Injection of RP in a dose of “ a0MBq was performed between the first and second fingers of the rat™s hind paw The animals were anesthetized with ether before the subcutaneous injection and during the scintigraphic study Since the introduction the kinetics of radiopharmaceutical distribution throughout ans and tissues was recorded by a framebyframe recording for a0min one frame” a0s in a — pixel matrix Static scintigraphy was performed after and a0h in the anterior and posterior projections in a matrix of — with a set of pulses scintigraphy of animals was performed on an ECAM Signature gamma camera Siemens Germany The results of scintigraphic studies determined the percentage of accumulation of RP in the lymph node and the injection site The maintenance and participation of the animals in the experiment was carried out in accordance with the rules adopted by the œEuropean Convention for the Protection of Vertebrates Used for Experiments or Other Scientific Purposes Strasbourg The experimental protocols were approved by Cancer Research Institute Biomedical Ethics Committee Protocol number All invasive manipulations with animals were performed using inhalation or drug anesthesiaStatistical analysis All mean values are expressed as IDg ± SD Data were analyzed statistically using methods of general statistics with a commercially available software package œStatistics for Windows StatSoft Inc Version Results and discussionTo carry out the labeling of colloids 99mTc extracted from a standard generator in the form of pertechnetate ions contained in a NaCl solution was used It has a higher degree of oxidation VII in this chemical form and is not prone to complex formation A reducing agent”tin II chloride dihydrate widely used for the preparation of various compounds labeled with 99mTc to was used to reduce its valence state in order to obtain complexes with nanoscale ps25 As a result of the reaction of these components the appearance of an untargeted colloid is also possible due to the hydrolysis of excess SnCl2·2H2O or the additional formation of a complex of reduced 99mTc with tin26 All this required preliminary experimental studies to establish the necessary and sufficient amount of SnCl2·2H2O in the reaction mixtureDuring the experiments it was found that the optimal concentration of Sn II in the composition of the reaction mixture when it interacts with 99mTc should be in the range of “ a0mgml Table a0It was found that when the eluate with the preliminarily reduced 99mTc VII was added to the nanops the Sn II concentration introduced in the RP was CSn a0mgml almost the entire amount of 99mTc has time to enter the composition of the largesize complex with tin even before its mixing with nanops This means that the sequence of the introduction and mixing of the reagents has a great influence on the labeling process especially it concerns the introduction of the Sn II solution into the reaction mixture In this connection the reduction of 99mTc with tin II was carried out in the presence of the selected substance In this case we can observe a competitive redistribution of the radionuclide between the substance and the tin complex The technique of applying of the 99mTc label to the surface of nanosized ps is given in the previous sectionAs a result of the studies reagent compositions and conditions for obtaining three nanocolloid RPs were determined Table a0 shows their components as well as the radiochemical purity and yield of the target colloid with p sizes of “ a0nmProceeding from the chromatograms it follows that the content of radiochemical impurity of unreduced 99mTc VII in the obtained preparations is “ Preliminary tests of these preparations on experimental animals showed that accumulation in lymph nodes is practically not observed although colloids have p sizes in the required range from to a0nm Scintigrams of rats obtained after subcutaneous administration of a technetium99m labeled nanocolloid based on aluminum oxide are shown in Fig a0Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cComposition of the preparation per a0mlDTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Al2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg n Colloid yield “ a0nm RCP ± ± ± Table Composition of reagents for production of technocium99 a0m nanocolloidsFigure a0 Distribution of the preparation in the rat when the preparation is administered [Al2O3 99mTc Sn II] A immediately after the administration of the drug B a0min after the administration C a0min after the administrationComposition of preparations per a0mlAl2O3 a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n DTPAmod a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n FeC a0mg 99mTc “ a0MBq SnCl2ˆ™2H2O a0mg G a0mg n Yield of colloid “ a0nm RCP ± ± ± Table Indicators of RCP and the yield of a colloid with a fraction of “ a0nm after the introduction of gelatin in the reagentsScintigrams showed that the drug remains at the injection point for a0h without significant accumulation of 99mTc in the blood of animals which indicates a strong fixation of the radionuclide on the surface of the nanocolloid Along with this positive point it should be noted that accumulation of the preparation in the lymph nodes is not observed Gelatin G was introduced into the reaction mixture in this connection to increase the œmobility of the labeled ps and increase the speed of their movement through the lymph system Matrix systems based on gelatin provide a fairly uniform distribution of the immobilized substance and in this case prevents the formation of a large tin colloid with 99mTc The results of the experiments showed that the addition of gelatin “ a0mgml to the reagent additionally provides an increase in the yield of the target colloid with p sizes of “ a0nm Table a0In addition the size of these ps was determined on a Nanophox p analyzer The obtained dependence of the change in the density of the distribution of the number of ps on their size constructed from the results of a threedimensional measurement of the preparations is shown in Fig a0 A B C The average p size diameter is and a0nm respectivelyStability test showed that complex 99mTc“nanocolloid was stable in normal serum at least for a0h Radiochemical purity of the tracer at the end of the experiment was ± A study of the functional suitability of the obtained radioactive colloids for the scintigraphic imaging of the sentinel lymph nodes showed that these preparations provide a good level of accumulation in the sentinel lymph nodes Fig a0 Table a0 displays the Al2O3 99mTc DTPAmod 99mTc FeC 99mTc biodistribution data at different time points postinjectionThe level of accumulation of preparations in the lymph nodes is “ of the total injected activityconclusionAs a result of the studies the composition of the reagents and the conditions for the synthesis of three nanocolloid RPs were determined An experimental dependence of the change in the content of 99mTc VII impurities on the concentration of tin II was established and its minimum amount a0mgml was determined to reach a RHP greater than In this case the yield of the target colloid with p sizes of ± a0nm is “ Preliminary tests of the developed preparations on experimental animals showed that accumulation of RP in lymph nodes is practically not observed although the sizes of colloidal ps are in the required range Increase in the speed of transportation of colloids through the lymphatic system was achieved by the introduction of gelatin in the composition “ a0mgml In addition there was an increase in the yield of the colloid Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0cFigure a0 Change in the density of the distribution of the number of ps from their size in radiopharmaceuticals A œ99mTcAl2O3 B œ99mTcFeC C œ99mTcDTPAmodwith p sizes of “ a0nm to “ with radiochemical purity of the preparations of “ Repeated studies in experimental animals have shown that all synthesized nanocolloid preparations provide a good level of Scientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0cStomachTime h99mTcAl2O399mTcDTPAmod99mTcFeC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Figure a0 Distribution of the preparation in the rat with injection of suspension [Al2O3 99mTc Sn II Gelatin] A immediately after the administration of the preparation B a0min after the administration C a0min after the administration D a0min after the administration The numbers indicate ”lymph node ”site of preparation administrationg ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Liver ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Spleen ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± BloodmlHeart ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Lungs ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± g ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Table Biodistribution data up to a0h after injection of “ a0MBq of 99mTc in healthy male rats Data represent the average value n accumulation in the SLN Thus the level of accumulation of RP œ99mTcDTPAmod and RP œ99mTcFeCADT in the SLN is and respectively At the same time the accumulation level of the preparation based on aluminum oxide is of the total input activityReceived March Accepted July References Jakobsen J K Sentinel node biopsy in urooncology A history of the development of a promising concept Urol Oncol “ Weixler B et al Sentinel lymph node mapping with isosulfan blue or indocyanine green in colon cancer shows comparable results and identifies patients with decreased survival A prospective singlecenter trial World J Surg 101007s0026 Beasley G M et al Sentinel Lymph node biopsy for recurrent elanoma A multicenter study Ann Surg Oncol Moser J et al Sentinel node biopsy in melanoma A singlecentre experience with consecutive patients Br J Dermatol 101245s1043 “ Buda A et al Optimizing strategies for sentinel lymph node mapping in earlystage cervical and endometrial cancer Comparison of realtime fluorescence with indocyanine green and methylene blue Int J Gynecol Cancer “ Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c“ Sahbai S et al Pericervical injection of 99mTcnanocolloid is superior to peritumoral injection for sentinel lymph node detection of endometrial cancer in SPECTCT Clin Nucl Med “ Hoogendam J P et al 99mTcnanocolloid SPECTMRI fusion for the selective assessment of nonenlarged sentinel lymph nodes in patients with earlystage cervical cancer J Nucl Med “ Stoffels I Leyh J P¶ppel T Schadendorf D Klode J Evaluation of a radioactive and fluorescent hybrid tracer for sentinel lymph node biopsy in head and neck malignancies Prospective randomized clinical trial to compare ICG99mTcnanocolloid hybrid tracer versus 99mTcnanocolloid Eur J Nucl Med Mol Imaging “ Beisani M et al Initial experience in sentinel lymph node detection in pancreatic cancer Rev Esp Med Nucl Imagen Mol Schubert T Uphoff J Henke R P Wawroschek F Winter A Reliability of radioisotopeguided sentinel lymph node biopsy in penile cancer Verification in consideration of the European guidelines BMC Urol “ Jaukovic L et al Lymphoscintigraphy and sentinel lymph node biopsy in cutaneous melanoma staging and treatment decisions Hell J Nucl Med “ Subramanian S Pandey U Shah S Rangarajan V Samuel G An indigenous singlevial kit formulation of human serum albumin nanocolloid for use in sentinel lymph node detection Nucl Med Commun “ RuizDom­nguez J M IbarzServio L Garc­ade Manuel G Calaf Peris© O Intraoperative injection of 99mTcnanocolloid for localization of nonpalpable intratesticular tumours in ansparing surgery Actas Urol “ Schauer A J Specific developments in sentinel node labling using 99mTccolloids In The Sentinel Lymph Node Concept Springer Berlin Wang Y et al Gasphase chemistry of technetium carbonyl complexes Chem Phys “ O™Connor M K et al Comparison of Tc99m maraciclatide and Tc99m sestamibi molecular breast imaging in patients with Wang J Zhang R Evaluation of 99mTcMIBI in thyroid gland imaging for the diagnosis of amiodaroneinduced thyrotoxicosis suspected breast cancer EJNMMI Res Br J Radiol Costa P et al Scintigraphic imaging with technetium99Mlabelled ceftizoxime is a reliable technique for the diagnosis of deep sternal wound infection in rats Acta Cir Bras “ Vera D R Wallace A M Hoh C K Mattrey R F A synthetic macromolecule for sentinel node detection 99mTcDTPAmannosyldextran J Nucl Med “ Hoh C K Wallace A M Vera D R Preclinical studies of [99mTc]DTPAmannosyldextran Nucl Med Biol “ Skuridin V et al Modified DTPA moleculebased nanocolloid radiopharmaceuticals J Radioanal Nucl Chem “ Filimonov V D et al Unusually stable versatile and pure arenediazonium tosylates Their preparation structures and synthetic applicability Lett “ Lukasz K Thin Layer Chromatography in Drug Analysis “ CRC Press London Skuridin V et al Radiopharmaceutical drug based on aluminum oxide Indian J Sci Technol 1017485 ijst2015v8i36 Sazonova S I et al Synthesis and experimental study of norfloxacin labeled with technecium99m as a potential agent for infection imaging Iran J Nucl Med “ Skuridin V S et al Synthesis and biological characterization of 99mTclabeled ciprofloxacin Pharm Chem J “ AcknowledgementsThis work was financially supported by Ministry of Education and Science of the Russian Federation RFMEFI57514X0034Author contributionsVS Conducting experimental research analysis and interpretation of the data Final approval for manuscript publication VS development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication EN development of the concept and direction of research analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication ES development of the concept and direction of research experimental research development of analytical control methods for the developed kits and radiopharmaceuticals analysis and interpretation of the data Validation of critical intellectual content final approval for manuscript publication AR conducting experimental research analysis and interpretation of the data Final approval for manuscript publication NV conducting experimental research analysis and interpretation of the data Final approval for manuscript publication RZ conducting tests of the functional suitability of drugs Preparation of the section Evaluation of the functional suitability of the preparation by determining its pharmacokinetic characteristics and Figures „– Final approval of the manuscript for publication of the manuscriptCompeting interests The authors declare no competing interestsAdditional informationCorrespondence and requests for materials should be addressed to VSReprints and permissions information is available at wwwnaturecomreprintsPublisher™s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsScientific RepoRtS 101038s41598020709912Vol1234567890wwwnaturecomscientificreports 0c Access This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons license and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons license unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this license visit httpcreat iveco mmons licen sesby40 The Authors Scientific RepoRtS 101038s41598020709912Vol0123456789wwwnaturecomscientificreports 0c'

"The principal function of iodine acts on thyroid function but in recent years the role of iodinedeficiency in metabolism has also been gradually revealed We aimed to investigate the current status of iodizedsalt consumption and urinary iodine concentration UIC in an urban Chinese population with type diabetes andto further explore whether UIC was associated with diabetic microvascular complicationsMethods Four thousand five hundred fiftynine subjects with diabetes from communities in downtownShanghai were enrolled in the crosssectional Metal Study in UIC was detected using an inductively coupledplasmamass spectrometer Diabetic kidney disease DKD was defined as urinary albumintocreatinine ratioUACR mgg or estimated glomerular filtration rate mLmin173 m2 Diabetic retinopathy DR wasevaluated by highquality fundus photographs and was remotely read by ophthalmologistResults The median UIC of subjects with diabetes was μgL “ in downtown Shanghai Among allthe subjects consumed noniodized salt and were iodine deficient Iodine deficiency UIC μgLwas associated with an increased odds of DKD OR 95CI “ after adjustment for age sex educationcurrent smokers BMI HbA1c duration of diabetes dyslipidemia thyroidstimulating hormone and free thyroxineNo association was observed between UIC and DR after multivariable adjustmentConclusions A concerning number of subjects with diabetes consumed noniodized salt and suffered from iodinedeficiency in coastal regions of China Low UIC might be a risk factor for DKD which should be further confirmedby longitudinal prospective studiesKeywords Iodized salt Type diabetes Diabetic kidney disease Urinary iodine concentration Epidemiology Correspondence wnj486126com luyingli2008126com Chi Chen and Yi Chen contributed equally to this workInstitute and Department of Endocrinology and Metabolism Shanghai NinthPeople™s Hospital Shanghai JiaoTong University School of MedicineShanghai China The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cChen Nutrition Metabolism Page of IntroductionType Diabetes Mellitus T2DM has become a seriousglobal health care burden causing microvascular complications which are associated with increased disabilityreduced quality of life and life expectancy [ ] It wasestimated that there were million cases of adult diabetes worldwide in and the number was projectedto increase to million by [] leading to highincidence and prevalence of microvascular complications Approximately one third with T2DM will developdiabetic retinopathy DR and one quarter will developdiabetic kidney disease DKD [] Thus it is critical toidentify and control some novel modifiable risk factorscontributing to microvascular complications in patientswith T2DMIodine is an indispensible micronutrient for the synthesis of thyroid hormones Iodine deficiency ID in early lifeimpairs neurodevelopment and also has many adverse effects throughout various life stages [] Over the past years substantial progress has been achieved in the worldwide effort to eliminate iodine deficiency disorders IDDby salt iodization program However this program is vulnerable and requires a longterm commitment from governments In several countries where ID had been onceeliminated salt iodization programs were discontinuedand ID has now reappeared []The principal function of iodine acts on thyroid function but in recent years the role of ID in metabolismhas also been gradually revealed Analysis of data fromthe National Health and Nutrition Survey NHANES“ found that in US adults low urinary iodineconcentration UIC was associated with dyslipidemia[] and coronary artery disease [] Moreover O S AlAttas [] reported that UIC is markedly decreasedin T2DM and urinary iodine was negatively associatedwith insulin resistance in patients with T2DM Most recently Mingyue Jin [] also found that at a lowerUIC μgL the prevalence of diabetes significantlyincreased relative to an UIC of “ μgL Animalstudies also showed that iodine supplementation couldreduce the blood glucose levels and improve the insulinsensitivity in goats [] However the role of iodine nutrition on diabetic microvascular complications has notbeen studiedThe aim of this study was to investigate the currentstatus of iodized salt consumption and UICs in an urbanChinese population with T2DMand additionallywhether UIC is associated with diabetic microvascularcomplications including DKD and DRMethodsStudy populationThe crosssectional METAL study Environmental Pollutant Exposure and Metabolic Diseases in Shanghaiwwwchictrcn ChiCTR1800017573 was launchedto investigate the association between iodine nutritionand microvascular complications in Chinese adults withdiabetes We recruited study participants from sevencommunities in Huangpu and Pudong new districtShanghai China Huangpu district located in downtownShanghai is the administrative economic and culturalcenter of the metropolitan coastal city [] Pudong newdistrict is the symbol of China™s reform and ingup[] We randomly selected half of patients with diabetesfrom the registration platform in each communityhealthcare center Chinese citizens ‰¥ years old whohad lived in their current area for ‰¥ months were included In August a total of subjects withT2DM who were “ years of age received an examination Participants with missing UIC values n were excluded Finally participants were involvedin the present analysisThe study received ethical approval from the EthicsCommittee of Shanghai Ninth People™s Hospital Shanghai Jiao Tong University School of Medicine All procedures followed were abided by the ethical guidelines ofthe Declaration of Helsinki as reflected in a prioriapproval by the appropriate institutional review committee Informed consent was received from all participantsincluded in the study prior to the data collectionMeasurementsinThe same welltrained and experienced personnelSPECTChina study [“] used a questionnaire to collect information on sociodemographic characteristicseducation medical history family history and lifestylerisk factors Weight and height were measured using abalance beam and a vertical ruler in light clothing andwithout shoes Body mass index BMI was calculated asthe ratio of weight in kilograms divided by height in meters squared Current smoking was defined as havingsmoked at least cigarettes in one™s lifetime and currently smoking cigarettes [] Especially œFor the pastthree years which type of salt was used in your familywas applied to collect information about type of saltThree options for this item were provided only iodized salt only noniodized salt bothBlood samples were drawn between am and am after an overnight fast Blood was refrigerated immediately after phlebotomy and in hours it was centrifugated and the serum was aliquoted and frozen in acentrallaboratory Glycated hemoglobin HbA1c wasmeasured by highperformance liquid chromatographyMQ2000PT Medconn Shanghai China Fastingplasma glucose serum creatininetotalcholesterol high HDL and lowdensity lipoproteinLDL were performed with a Beckman Coulter AU Brea USA Serum thyroidstimulating hormone TSHtriglycerides 0cChen Nutrition Metabolism Page of and free thyroxine FT4 were measured by electrochemiluminescence Roche E601 GermanyMorning fasting spot urine samples collected were refrigerated immediately and frozen at ˆ’ °C in hoursUIC was detected using an inductively coupled plasmamass spectrometer ICPMS No 7700x Agilent Technologies Inc USA The concentrations of urine albuminand creatinine were determined with a Beckman CoulterAU Brea USA using a turbidimetric immunoassayand an enzymatic method respectively Urinary albumintocreatinine ratio UACR was calculated as the urinaryalbumin concentrations divided by the urinary creatinineconcentrations and expressed in mggDR screening was evaluated by mydriatic binocular indirect ophthalmoscopy Topcon TRCNW400 NonMydriatic Retinal Camera Oakland USA Fundus photographs were read by an experienced ophthalmologistspecialized in retinaOutcome definitionDyslipidemia was defined as total cholesterol ‰¥mmolL mgdL triglycerides ‰¥ mmolL mgdL LDL ‰¥ mmolL mgdL HDL mmolL mgdL or selfreported previous diagnosisof hyperlipidemia by physicians according to the modified National Cholesterol Education ProgramAdultTreatment Panel IIIThe estimated glomerular filtration rate eGFR was calculated using the Chronic Kidney Disease EpidemiologyCollaboration CKDEPI equation for œAsian origin []As suggested by American Diabetes Association ADAhigh UACR was defined as UACR ‰¥ mgg reducedeGFR as eGFR mlmin173m2 and DKD as UACR mgg or eGFR mLmin173 m2 []The internationally accepted DR classification by theœGlobal Diabetic Retinopathy Project Group in was applied [] The classification was no retinopathynonproliferative DR intraretinal microaneurysmshemorrhages venous beading prominent microvascularabnormalities and proliferative DR neovascularizationor vitreouspreretinal hemorrhagesStatistical analysisStatistical analysis was run with SPSS IBM Corporation Armonk NY USA General characteristics arepresented as median with the interquartile range IQRfor continuous variables or as proportion for categoricalvariables MannWhitney U test and the KruskalWallistest were used for comparison of two or more groups ofnonnormally distributed data Pearson™s χ2 tests wereperformed to compare categorical variablesThe associations of UIC with elevation of UACR reduction of eGFR DKD and DR were analyzed with logistic regression analyses The results are presented as oddsratio OR and confidence intervals CIs Model was unadjusted Model was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Table General characteristics of study participants by UIC categoriesUrinary iodineAge yrAdequate “ “ “ “ “ “Low “ “ “ “ “ “Women UIC μgLFPG mmolLHbA1c BMI kgm2Duration of diabetes yrCurrent smokers Beyond high school education TSH mIULFT4 pmolLBlood lipidsTotal cholesterol mmolLLDLC mmolLHDLC mmolLTriglycerides mmolLMore than adequate “ “ “ “ “ “ “ “Excessive “ “ “ “ “ “ “ “ “ “ “ “P “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “Data are summarized as median interquartile range for continuous variables or as number with proportion for categorical variablesUIC Urinary iodine concentration FPG Fasting plasma glucose HbA1c Glycated hemoglobin BMI Body mass index LDLC Low density lipoprotein cholesterol HDLC High density lipoprotein cholesterol TSH Thyroidstimulating hormone FT4 Free thyroxineUrinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL 0cChen Nutrition Metabolism Page of ResultsGeneral characteristics of the study populationThe general characteristics are presented in Table The mean age of the study population was nearlyone half were women About of the studypopulation were overweight or obese BMI ‰¥ kgm2and were current smokers The percentage of aneducational level beyond high school was and theaverage duration of diabetes was yearsCompared to those with adequate iodine nutritionsubjects with ID were slightly older and were more likelyto be women These subjects also had higher TSH lowerBMI and a lower percentage of current smokers Inaddition subjects with more than adequate and excessiodine nutrition were slightly younger and had higherBMI but comparable TSHUrinary iodine concentration and type of salt intake inthe populationThe distribution of UICs in the study population is presented in Fig The median 25th“75th percentile UICof subjects with diabetes was μgL “ indowntown Shanghai which falls within the range of μgL that WHOUNICEFICCIDD categorize asadequate Urinary iodine measurements indicative of IDUIC μgL were present in of the studypopulation Meanwhile and of the populationshowed more than adequate UIC “ μgL andexcess iodine intake UIC ‰¥ μgL respectivelyThe distribution of type of salt intake is presented inFig As high as of the study population consumed noniodized salt consumed iodized saltand consumed both Logistic regression analysisshowed that compared to those who consumed iodizedsalt subjects consumed noniodized salt were morelikely to be women OR 95CI “ and havea higher educational attainment OR 95CI “ but a comparable age OR 95CI “Association of urinary iodine concentration with elevationof UACR reduction of eGFR and DKDThe association of UIC with elevation of UACR reduction of eGFR and DKD is shown in Table Comparedwith those with adequate iodine nutrition subjects withID had an increased risk of elevation of UACR OR 95CI “ reduction of eGFR OR 95CI“ and DKD OR 95CI “ Adjustment for age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 did not attenuate the association of ID with UACRand DKD However multivariable adjustment weakenedthe association between ID and reduction of eGFR further such that it was no longer significant Meanwhilesubjects with more than adequate and excess iodine nutrition did not have an increased risk of elevation ofUACR reduction of eGFR and DKD after multivariableadjustmentAssociation of urinary iodine concentration with DRTable presents the association of UIC with nonproliferative and proliferative DR Compared with thosewith adequate iodine nutritionthe ORs of nonproliferative and proliferative DR in subjects with morethan adequate iodine nutrition were 95CI “Fig Distribution of UICs in the study population 0cChen Nutrition Metabolism Page of Fig Distribution of type of salt consumed in the study populationthan adequate and 95CI “ respectively Multivariable adjustment weakened the association betweenmoreiodine nutrition and nonproliferative DR further such that the association was nolonger significant There was no significant associationobserved between DR and ID and excessiodinenutritionDiscussionIn this study among over communitydwellingChinese adults with diabetes we found that of thesubjects consumed noniodized salt and had IDIodine deficiency was significantly associated with ahigher prevalence of elevated UACR and DKDindependently of age sex education current smokers BMIHbA1C duration of diabetes dyslipidemia TSH andFT4 To the best of our knowledge this is the first studyto investigate the current status of iodized salt consumption and iodine nutrition status in a relatively largepopulation with diabetes and further investigate the association between UIC and diabetic microvascularcomplicationsChina was once severely affected by IDD and hence amandatory Universal Salt Iodization USI program wasTable Association of urinary iodine with elevation of UACR and reduction of eGFRUrinary iodineHigh UACRAdequateLowMore than adequateExcessivePrevalenceOdds RatioModel Model Reduced eGFRPrevalence Odds RatioModel Model DKDPrevalence Odds Ratio Ref Ref Ref Ref “ “ “ “ “ “ “ “ “ “ “ “Model Model Ref Ref “ “Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with elevation of UACR reduction of eGFRand DKD P Model was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4High UACR was defined as UACR ‰¥ mgg reduced eGFR as eGFR mlmin173 m2 and DKD as UACR mgg or eGFR mLmin173 m2Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgL “ “ “ “ 0cChen Nutrition Metabolism Page of Table Association of urinary iodine with DRUrinary iodineNonproliferative DRAdequatePrevalence Odds RatioModel Model Proliferative DRPrevalence Odds RatioModel Model Ref Ref Ref RefLowMore than adequateExcessive “ “ “ “ “ “ “ “ “ “ “ “P Data are expressed as odds ratios 95CI Logistic regression analyses were used for the association of urinary iodine with DRModel was unadjustedModel was adjusted for age sex education current smokers BMI HbA1C duration of diabetes dyslipidemia TSH and FT4Urinary iodine concentrations low μgL adequate to μgL more than adequate to μgL excessive ‰¥ μgLintroduced in and was successful in eliminatingIDD However since the prevalence of thyroid diseaseshas markedly increased in recent years some concernsabout the USI have circulated especially among coastalresidents in urban areas [] In the present analysis themedian UIC of residents with diabetes in downtownShanghai has fallen to marginal levels of iodine sufficiency μgL and more surprisingly more thanhalf of the subjects consumed noniodized saltand were iodine deficient Compared with a studyconducted by the Shanghai Municipal Center for DiseaseControl and Prevention CDC in of participants used iodized salt and were iodine deficientat that time [] During “ Zhongyan Shanand her colleagues performed a crosssectional study ineastern and central China and reported that the medianUIC was μgL in schoolaged children and μgLin the total cohort population [] Our previous studyconducted on the general population found that consumed noniodized salt in the urban area of Shanghai in []The present study indicates that an increasing numberof urban residents in downtown Shanghai prefer to usenoniodized salt in recent years and suffer from IDWomen and those with a higher educational level weremore tended to consume noniodized salt WhyChanges in the reported spectrum and growing incidence of thyroid disorders have been linked to the increased iodine intake resulting from USI in the localmedia and international medical literature [] Thosewith high educational attainment were more likely to beconfused by these information and worry about theirthyroid health and call for liberalizing provincial controlof sales of noniodized salt Formerly needed a prescriptionthe sale of noniodized salt has now beenunofficially allowed by some coastal city authorities []In addition T2DM is associated with an increased riskof multiple thyroid disorders such as thyroid nodule[] thyroid cancer [] and autoimmune thyroid diseases [] Since the prevalence of thyroid abnormalitieswere found to be much higher in females than males[] it is reasonable to deduce that these women wouldhave a higher tendency towards consuming noniodizedsalt after diagnosis of thyroid abnormalitiesseafood alone to provide sufficientConsidering that Shanghai is a coastal city local residents believe that they should never suffer from IDDwith high iodineenriched aquatic products consumption However it may not be true to rely on consumption ofiodineActually the environmental levels of iodine in Shanghaiare deficient μgL [] Furthermore based on theresearch initiated by Shanghai Municipal CDC iodizedsalt contributed of the total dietary iodine inShanghai[] Aquatic products which residentsthought to be rich in iodine accounted for only ofthe total dietary iodine []Thus iodized salt is still themain source foriodine supplementation in coastalpopulationsDKD wascommonlydefinedby ADA asœUACR‰¥30mgg or eGFR60 mlmin per m2 []We found that ID was associated with elevated UACRand DKD The mechanism underlying the association ofID with DKD is not yet fully understood Deficiency ofiodine could reduce thyroid hormone production andelevate TSH It has been reported that lower free triiodothyronine and elevated TSH have significant associationwith risk for albuminuria in T2DM [ ] Moreoverinadequate iodine intake is significantly correlated withan increase in oxidative stress [] Recent evidence hasshown thattumorinflammatory cytokinessuch as 0cChen Nutrition Metabolism Page of necrosis factoralpha and interleukin1 play a pivotalrole in the pathogenesis of DKD [] Therefore we suppose the possible mechanism may be via inflammatoryresponseIn view of the evergrowing prevalence of T2DM andDKD all over the world successive intervention in thislarge population can have important impact on publichealth ID unlike most micronutrient deficiencies is notrestricted to people in developing countries with poordiets Since salt iodization is simple effective and inexpensive the best strategy to control ID is addition ofiodine into salt in nearly all countries [] Monitoringiodine situation of people with diabetes is of critical significance and education programs to diabetes especiallywomen with high academic background may also include information of adequate iodine intake in our clinical practice Our study shed light on the possiblebeneficial effect of iodine supplementation in reducingalbuminuria in T2DM which warrants further investigation in welldesigned randomized controlled trialOur study benefited from its welldefined communitybased participants with a relatively large sample sizeSecond regarding the novelty our study is the first toprovide iodine status and linked iodine insufficiency toan increased risk of albuminuria in people with diabetesThird we used ICPMS to detect UIC in the presentanalysis which was considered as the goldstandardmethod [] There were also some limitations weshould acknowledge First no causal relationship couldbe determined due to the crosssectional design of thestudy and thus our findings need to be validated by longitudinal prospective studies Second although UIC isrecommended by the WHOICCIDDUNICEF for evaluation of iodine status at the population level and widelyused in largescale epidemiological studies [ ] thesingle spot urine measurement may not accurately assesslongterm iodine status at the individual level Actuallyinter and intraindividual variability exists in UIC []However the application of a large sample size from to subjects per subgroup may counteract thebias related to the use of only one casual urine sample[] Future followup studies collecting 24h urine specimens twice are needed to replicate the present resultsConclusionA large proportion of diabetic patients in downtownShanghai consumed noniodized salt and had ID IDmay increase the risk of DKD independent of thyroidfunction in diabetic patients Maintaining USI at an appropriate level is indispensable for diabetic patients Cohort and intervention studies as well as basic researchexploring the effect and mechanism of iodine supplementation on renal function are warrantedAbbreviationsT2DM Type Diabetes Mellitus DKD Diabetic kidney disease DR Diabeticretinopathy ID Iodine deficiency UIC Urinary iodine concentrationTSH Thyroidstimulating hormone BMI Body mass index OR Odds ratioCI Confidence interval UACR Urinary albumintocreatinine ratioeGFR Estimated glomerular filtration rateAcknowledgementsThe authors thank Xiaojin Wang and Bingshun Wang from the Departmentof Biostatistics and Shanghai Jiaotong University School of Medicine for dataprocessingAuthors™ contributionsYL and NW designed the study CC YC HZ FX BH WZ YW HWand NW conducted the research CC and YC analyzed the data and wrotethe manuscript CC and YC contributed equally The final manuscript wasread and approved by all authorsFundingThis study was supported by National Natural Science Foundation of China Science and Technology Commission of ShanghaiMunicipality the Fourth Round of ThreeYear Public HealthAction Plan of Shanghai by the Shanghai Municipal Commission of Healthand Family Planning 20164Y0079 Municipal Human Resources DevelopmentProgram for Outstanding Young Talents in Medical and Health Sciences inShanghai 2017YQ053 Fundamental research program funding of NinthPeople™s Hospital affiliated to Shanghai Jiao Tong university School of MedicineJYZZ099 Shanghai Sailing Program 20YF1423500Availability of data and materialsThe datasets during andor analyzed during the current study are availablefrom the corresponding author on reasonable requestEthics approval and consent to participateEthical approval was obtained from the Ethics Committee of Shanghai NinthPeople™s Hospital Shanghai Jiao Tong University School of Medicine Writteninformed consent was received from all participantsConsent for publicationNACompeting interestsNo potential conflicts of interest relevant to this were reportedReceived May Accepted August ReferencesKahm K Laxy M Schneider U Rogowski WH Lhachimi SK Holle R Healthcare costs associated with incident complications in patients with type diabetes in Germany Diabetes Care “Chen C Chen Q 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"Identifying somatic mutations is critical for cancer genome characterization and for prioritizing patient treatment. DNA whole exome sequencing (DNA-WES) is currently the most popular technology; however this yields low sensitivity in low purity tumors. RNA sequencing (RNA-seq) covers the expressed exome with depth proportional to expression. We hypothesized that integrating DNA-WES and RNA-seq would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method called UNCeqR that detects somatic mutations by integrating patient-matched RNA-seq and DNA-WES. In simulation the integrated DNA and RNA model outperformed the DNA-WES only model. Validation by patient-matched whole genome sequencing demonstrated superior performance of the integrated model over DNA-WES only models including a published method and published mutation profiles. Genome-wide mutational analysis of breast and lung cancer cohorts (n = 871) revealed remarkable tumor genomics properties. Low purity tumors experienced the largest gains in mutation detection by integrating RNA-seq and DNA-WES. RNA provided greater mutation signal than DNA in expressed mutations. Compared to earlier studies on this cohort UNCeqR increased mutation rates of driver and therapeutically targeted genes (e.g. PIK3CA ERBB2 and FGFR2). In summary integrating RNA-seq with DNA-WES increases mutation detection performance especially for low purity tumors. cover-date 2014 INTRODUCTION Somatically acquired sequence mutations (nucleotide substitutions insertions and deletions) fuel the initiation and progression of cancer (1). Knowledge of mutations in patient specimens informs therapeutic management (23) and in large patient cohorts provides the basis to assess recurrently altered genes that may drive molecular pathogenesis (14“5). DNA whole exome sequencing (DNA-WES) is currently the popular technology to sequence cancer genomes and has led to an abundance of discoveries in many cancer types (46“8). However detecting somatic mutations by DNA-WES with high sensitivity and specificity remains a challenge (79“10) as evidenced by validation rates of 73% in repeated sequencing and by large inter-rater disagreement among different groups analyzing the same sequencing data (710). The biggest challenge is high quality mutation detection in low purity tumors (2911) which are prevalent in widespread cancer types such as breast and lung (12). Advances in somatic mutation detection could improve cancer genome characterization and lead to new diagnostic and therapeutic targets. Somatic mutation detection is dependent on tumor features the sequencing technology and the method of statistical modeling (8“913“17). To detect somatic mutations algorithms compare tumor and patient-matched germline sequencing based on a variety of models (46“7913“17). A tumor's degree of normal contamination and clonal heterogeneity decrease tumor purity. Low purity affects the fraction of mutated DNA observed out of all DNA at a genomic site the mutant allele fraction (MAF) (812). MAF is not often 100% can be slightly above zero in low purity tumors and varies across the genome depending on the prevalence of clones possessing a given mutation and on copy number alterations (7912). DNA-WES targets roughly 200 000 exonic regions and in practice can yield depths of 100X or greater over targeted regions (46). DNA-WES has limitations including variable capture-efficiency and incomplete exome coverage (718). In cases of high MAF mutation detection is straightforward as only a small number of reads are needed to detect the mutation with confidence. The combination of low depth and low MAF make mutation detection very difficult because of low statistical power a result of the scant sample size in which to observe and detect the low prevalence mutation. Increased mutation detection sensitivity and specificity could be achieved by statistical improvements by increasing sequencing quantity or by increasing sequencing quality. In cancer profiling projects such as The Cancer Genome Atlas (TCGA) (46) and in clinical sequencing (219) DNA-WES is utilized for mutation detection while RNA sequencing (RNA-seq) (20) is performed for gene expression fusion transcript and splicing analyses. Beyond those applications RNA-seq provides an observation of the underlying tumor DNA sequence via transcription and can be used to detect sequence variants (21). In fact we have previously used RNA-seq to confirm mutations from DNA-WES (4). A few earlier studies have used RNA-seq alone for genome-wide identification of somatic mutations (22“25) and germline variants (2627). However RNA-seq has challenges including dependency on gene expression which limits the genes that can be measured for sequence mutations and quality control requirements which when not considered result in abundant false positive variants (112128“30). For these reasons RNA-seq has not been the standard for somatic mutation detection. Herein we posed the original hypothesis that integrating patient-matched tumor RNA-seq and tumor DNA-WES would enable superior mutation detection versus DNA-WES alone. We developed a first-of-its-kind method UNCeqR that simultaneously analyzes DNA-WES and patient-matched RNA-seq to detect somatic mutations genome-wide. UNCeqR was applied to large breast and lung cancer cohorts and evaluated with respect to simulation and whole genome sequencing validation. Subsequently genome-wide analysis of UNCeqR mutations led to novel discoveries in tumor genomics. MATERIALS AND METHODS Data sources DNA-WES and RNA-seq alignments in BAM (31) format for 176 lung squamous cell carcinoma cases and for 695 breast cancer cases were acquired from TCGA at ://cghub.ucsc.edu (Supplementary Table S1). RNA-seq were paired 50 nt read from Illumina HiSeq aligned by MapSplice (432). DNA-WES were paired 76“100 nt reads from Illumina Genome Analyzer aligned by BWA (33). All lung and breast cancer cases had germline DNA-WES tumor DNA-WES and tumor RNA-seq and were referred to as the triplet cohorts. A subset of 12 lung and 91 breast tumors also had germline RNA-seq available and were referred to as the quadruplet cohorts. DNA whole genome sequencing (DNA-WGS) was acquired from TCGA for tumors in this cohort (breast: n = 43 lung: n = 17) which consisted of BWA alignments of paired 100 nt reads. Exonic coordinates were extracted from the TCGA Genome Annotation File (http://tcga-data.nci.nih.gov/docs/GAF/GAF.hg19.June2011.bundle/outputs/TCGA.hg19.June2011.gaf) and padded with 10 flanking positions for a total of 222 055 exons. Published mutations (lung: LUSC_Paper_v8.aggregated.tcga.somatic.maf breast: genome.wustl.edu_BRCA.IlluminaGA_DNASeq.Level_2.5.1.0.somatic.maf) expression subtypes DNA copy number calls and tumor purity calls (12) were obtained when available from TCGA. Numerical purity calls of 1 with an incongruent ˜Low purity™ categorical call were censored. Sequencing quality filtering The high quality data filter applies to alignments and genomic positions similar to earlier studies (914). High quality sequenced bases from tumor alignments had base quality ?20 and occurred in a parent alignment with the following properties: mapping quality ? 20 sum of reference mismatches insertions and deletions ?2 a proper pair orientation not a marked duplicate or qc-failure not within the terminal two bases and the singular best alignment. All bases from germline alignments were accepted. High quality genomic positions were those with germline depth ?10 tumor high quality depth ?5 in RNA or DNA no homopolymer > 4 on either side of the site proportion of high quality bases ?0.25 in RNA or DNA and without an insertion or deletion event at 10% allele fraction within 50 positions in germline sequencing. The high quality data filter was applied prior to detecting to tumor variant alleles. The high quality variant filter passes DNA or RNA variant alleles without significant strand bias compared to germline alleles (chi-square P < 0.01) with at least one read on both strands for indel variants with major variant allele prevalence (the proportion of major variant reads out of all variant reads) ?0.75 and a MAD of distance to the end of its aligned read sequence ?1. Somatic mutation detection The UNCeqR algorithm detected somatic mutations within exons based on input of tumor and patient-matched germline sequence alignments. The algorithm applied the following steps to each genomic site within exons: filter for high quality data;identify germline alleles from germline reads that have at least 2% allele prevalence; add population polymorphisms and mapping artifact alleles to germline alleles (see following section ˜Population polymorphisms and mapping artifacts™).Using tumor sequences: let g be the number of reads matching germline allelesdetermine most frequent allele that does not match germline alleleslet k be the number of reads with this major variant allelelet n = k + g.If major variant allele is insertion or deletion re-align nearby indel alleles: scan 20 neighboring sites to find site s with maximum k and same major variant alleleif current site is not s. Move major variant read count from current site to s by incrementing k at s and decrementing g at s by current site's major variant read count.Continue to next site.If high quality variant filter is passed apply statistical test otherwise P = 1 if k = 0 else P = NA.. A set of mutation detection models applied the algorithm with different inputs and statistical models. UNCeqRDNA takes tumor DNA-WES as input and models the corresponding read counts by a beta-binomial distribution. For a variant site with read count \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$k_{{\rm DNA}}$\end{document} the P-value to assess whether this variant allele is a somatic mutation was calculated by \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{equation*} P_{{\rm DNA}} = 1 - \sum\limits_{i = 0}^{k - 1} {\left( {\begin{array}{*{20}c} {n_{{\rm DNA}} } \\ i \\ \end{array}} \right)} \frac{{B\left( {i + \alpha _{{\rm DNA}} n_{{\rm DNA}} - i + \beta _{{\rm DNA}} } \right)}}{{B(\alpha _{{\rm DNA}} \beta _{{\rm DNA}} )}} \end{equation*} \end{document}where B is the beta function and ?DNA and ?DNA are parameters of the null distribution where the variant allele is not a somatic mutation. Specifically ?DNA and ?DNA are estimated using randomly sampled sites until 50 000 have passed the high quality data filter in both tumor DNA-WES and tumor RNA-seq. In real data analysis these sampled sites may include real somatic mutations and thus the estimates of ? and ? are conservative which may lead to conservative P-value estimates. However based on mutation rates reported in prior studies (8 mutations per 1 000 000 sites (4)) less than one mutation is expected in these sampled sites and thus our estimates of ? and ? would be good approximations of the estimates from a set of non-somatic mutation sites. The UNCeqRRNA model is identical to UNCeqRDNA substituting tumor RNA-seq for tumor DNA-WES. The UNCeqRMETA model combines P-values from UNCeqRDNA and UNCeqRRNA if RNA and DNA have the same major variant allele irrespective of filtering; otherwise the UNCeqRMETA P-value is set to that of UNCeqRDNA. In effect this condition precludes sites with only RNA variant evidence that are suggestive of RNA-editing (3435) from being called somatic mutations. UNCeqRMETA combines P-values by the Stouffer method (36“38) with weights of the root of their sample size (read depth at the site) as follows: \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{} \begin{eqnarray*} &&P_{{\rm META}} = 1 - \\ &&\varPhi\left({\frac{{\varPhi ^{ - 1} \left( {1 - P_{{\rm DNA}} } \right)\sqrt {n_{{\rm DNA}} } + \varPhi ^{ - 1} \left( {1 - P_{{\rm RNA}} } \right)\sqrt {n_{{\rm RNA}} } }}{{\sqrt {n_{{\rm DNA}}^{} + n_{{\rm RNA}}^{} } }}} \right) \end{eqnarray*}\end{document}where \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} is the standard normal cdf and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi ^{ - 1}$\end{document} is the inverse of \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\varPhi$\end{document} i.e. the quantile function of the standard normal distribution. If the RNA major variant equals the DNA major variant and PDNA = NA PMETA is set to PRNA. DNA and RNA variant read counts among putative false positives were unassociated supporting the usage of Stouffer's method (Supplementary Figure S1). Due to possible ambiguity around insertions and deletions (˜indels™) between DNA and RNA alignments high quality variant sites with an insertion or deletion major variant allele in one alignment and with the same variant allele (insertion or deletion) occurring within 20 sites as the major variant allele in the other alignment were merged to have the same genomic position prior to statistical testing. This indel merge allowed indel variants sites between DNA and RNA that represent the same variant to be recorded at the same site and allowed UNCeqRMETA to combine this DNA and RNA evidence despite slightly different representation in the sequence alignments. UNCeqR software consisted of modified samtools (31) Perl R and VGAM (39). The total number of applied statistical tests is reported in UNCeqR output to provide interested users the possibility of multiple testing adjustment. Population polymorphisms and mapping artifacts Population-level polymorphisms were acquired from dbSNP common version 137 via the UCSC genome browser (40). Variant alleles caused by ambiguous mapping artifacts were calculated by BlackOps (41) using 2 × 50 paired-end reads aligned by MapSplice. UNCeqR was applied to 45 TCGA RNA-seq of matched normal tissue specimens (not part of the lung or breast cohorts) to detect non-reference sequence variants representing further germline polymorphic and alignment artifact alleles. These alleles always augmented germline genotype in UNCeqR thus preventing somatic mutation detections with these alleles even if unobserved in a given germline sequencing. Mutation annotation and analysis Sequence mutations were annotated with a gene a predicted transcript and protein alteration using Annovar (version 8/23/13) (42) and RefSeq gene models. Non-silent mutations referred to non-silent substitution insertion and deletion mutations within translated regions and splice-site mutations. MAFs were compared by one-sided Fisher's exact tests on mutant versus germline read counts with significant results having false discovery rate < 5%. Sequence alignments were visualized using the Integrative Genomics Viewer (43). Germline variant analysis Patient germline variants relative to the reference genome were detected in germline DNA-WES and patient-matched germline RNA-seq using UNCeqRMETA without population polymorphism or mapping artifact allele augmentation P ? 1.1e?9. Germline variant allele fractions were defined and compared between DNA and RNA using the procedure described for somatic mutations. Simulation analysis A novel simulation strategy was followed (diagrammed in Supplementary Figure S2). Using chromosome 2 simulated tumor genomes were generated by randomly sampling 500 sites from exons to define positive mutation sites while the remainder of exon sites served as negative mutations. For the positive sites mutant alleles (substitution insertion or deletion) were randomly sampled at rates 90 5 and 5%. For insertion and deletion alleles allele lengths of 1“6 were randomly sampled at rates 602095 5 and 1%. Positive mutations were spiked into germline DNA-WES and RNA-seq sequencing by editing a specified MAF of read alignments overlapping the site producing simulated tumor alignments. ˜V™ characters were used for substitutions and insertions to avoid overlap with germline genotype. Simulated tumor alignments contained a subset of the total positive mutations because the alignment may have minimal or zero depth at some positive sites reflecting reality that a sequencing technology does not cover every site in the genome at high depth and enabling simulated mutations to occur at RNA-seq and DNA-WES uniquely covered sites. Original tumor sequencing served as simulated germline sequencing. Simulated germline sequencing contained the original somatic mutations which had the effects of expanding germline genotype with additional alleles and not triggering variant detection. UNCeqR models were applied to these simulated data. Limiting to sites with at least a germline depth of 10 model detections were compared to the truth to define receiver operating characteristic (ROC) curves (44). A pair of models was compared by their difference in area under the curve over the false positive rate range of 0 to 1 × 10?5. A P-value was defined using a distribution of differences in area under the curve calculated from 100 permuted models in which the rank of the discrimination threshold (i.e. P-value) between the models at each genomic site was randomly shuffled. Mutation detection by other programs Strelka v2.0.8 (17) was executed on tumor and germline DNA-WES using recommended settings for BWA alignments (strelka_config_bwa_default.ini) DNA-WES (isSkipDepthFilter = 1) and filtering (passed). SNVMix2 (13) was executed upon RNA-seq using default settings. Validation analysis Within exonic regions true positive and false positive mutation detections were defined using patient-matched DNA-WGS alignments based on a published procedure for exome mutation validation (4). Tumor and germline DNA-WGS BAM files were downloaded from ://cghub.ucsc.edu. Specifically tumor and germline DNA-WGS were interrogated at each predicted mutation using samtools (31) with no filtering. True positive mutation predictions met one of two conditions: (1) germline depth ? 10 and read count of predicted mutant allele ?1 in tumor and zero in germline; or (2) germline depth ?10 proportion of mutant allele in germline sequencing not significantly > 2% (proportions test P > 0.25) and proportion of mutant allele in tumor significantly greater than in germline (proportions test P < 0.05). Otherwise false positive mutation predictions had germline DNA-WGS depth ?10 and had depth in tumor DNA-WGS providing ?80% power to detect the mutant allele based the predicted MAF. Power was estimated by a binomial distribution a null probability of 3 × 10?3 an alpha of 0.05 the "

"These alleles always augmented germline genotype in UNCeqR thus preventing somatic mutation detections with these alleles even if unobserved in a given germline sequencing. Mutation annotation and analysis Sequence mutations were annotated with a gene a predicted transcript and protein alteration using Annovar (version 8/23/13) (42) and RefSeq gene models. Non-silent mutations referred to non-silent substitution insertion and deletion mutations within translated regions and splice-site mutations. MAFs were compared by one-sided Fisher's exact tests on mutant versus germline read counts with significant results having false discovery rate < 5%. Sequence alignments were visualized using the Integrative Genomics Viewer (43). Germline variant analysis Patient germline variants relative to the reference genome were detected in germline DNA-WES and patient-matched germline RNA-seq using UNCeqRMETA without population polymorphism or mapping artifact allele augmentation P ? 1.1e?9. Germline variant allele fractions were defined and compared between DNA and RNA using the procedure described for somatic mutations. Simulation analysis A novel simulation strategy was followed (diagrammed in Supplementary Figure S2). Using chromosome 2 simulated tumor genomes were generated by randomly sampling 500 sites from exons to define positive mutation sites while the remainder of exon sites served as negative mutations. For the positive sites mutant alleles (substitution insertion or deletion) were randomly sampled at rates 90 5 and 5%. For insertion and deletion alleles allele lengths of 1“6 were randomly sampled at rates 602095 5 and 1%. Positive mutations were spiked into germline DNA-WES and RNA-seq sequencing by editing a specified MAF of read alignments overlapping the site producing simulated tumor alignments. ˜V™ characters were used for substitutions and insertions to avoid overlap with germline genotype. Simulated tumor alignments contained a subset of the total positive mutations because the alignment may have minimal or zero depth at some positive sites reflecting reality that a sequencing technology does not cover every site in the genome at high depth and enabling simulated mutations to occur at RNA-seq and DNA-WES uniquely covered sites. Original tumor sequencing served as simulated germline sequencing. Simulated germline sequencing contained the original somatic mutations which had the effects of expanding germline genotype with additional alleles and not triggering variant detection. UNCeqR models were applied to these simulated data. Limiting to sites with at least a germline depth of 10 model detections were compared to the truth to define receiver operating characteristic (ROC) curves (44). A pair of models was compared by their difference in area under the curve over the false positive rate range of 0 to 1 — 10?5. A P-value was defined using a distribution of differences in area under the curve calculated from 100 permuted models in which the rank of the discrimination threshold (i.e. P-value) between the models at each genomic site was randomly shuffled. Mutation detection by other programs Strelka v2.0.8 (17) was executed on tumor and germline DNA-WES using recommended settings for BWA alignments (strelka_config_bwa_default.ini) DNA-WES (isSkipDepthFilter = 1) and filtering (passed). SNVMix2 (13) was executed upon RNA-seq using default settings. Validation analysis Within exonic regions true positive and false positive mutation detections were defined using patient-matched DNA-WGS alignments based on a published procedure for exome mutation validation (4). Tumor and germline DNA-WGS BAM files were downloaded from ://cghub.ucsc.edu. Specifically tumor and germline DNA-WGS were interrogated at each predicted mutation using samtools (31) with no filtering. True positive mutation predictions met one of two conditions: (1) germline depth ? 10 and read count of predicted mutant allele ?1 in tumor and zero in germline; or (2) germline depth ?10 proportion of mutant allele in germline sequencing not significantly > 2% (proportions test P > 0.25) and proportion of mutant allele in tumor significantly greater than in germline (proportions test P < 0.05). Otherwise false positive mutation predictions had germline DNA-WGS depth ?10 and had depth in tumor DNA-WGS providing ?80% power to detect the mutant allele based the predicted MAF. Power was estimated by a binomial distribution a null probability of 3 — 10?3 an alpha of 0.05 the observed depth in DNA-WGS and an alternate probability of the predicted DNA MAF. The number of true positives and false positives were tabulated at each model discrimination threshold i.e. P-value or score. The step function of these points (number of false positives versus number of true positives) generated a performance curve in absolute counts that is equivalent to a ROC curve without the denominators of total positives and negatives which were constant and unknown for the validation cohort. Between models performance curves were compared by area under the curve from 0 to 3000 false positives and by the number of true positives (proportional to sensitivity) at fixed numbers of false positives (proportional to 1 ? specificities) of 250 500 and 1000). P-values were calculated to provide evidence for the change in area under the curve and sensitivity estimates using permutation (see ˜Simulation analysis™ methods). RESULTS Mutation detection models Existing methods to detect somatic mutations are based on either DNA sequencing alone or on RNA sequencing alone and do not integrate more than one type of sequencing (913“17). In order to test whether integrating DNA-WES and RNA-seq enables superior somatic mutation detection versus the current standard of DNA-WES alone a new method was developed called UNCeqR. UNCeqR contains different models for detecting somatic mutations based on different sequencing input and statistical modeling. Briefly UNCeqRMETA integrates tumor DNA-WES and RNA-seq UNCeqRDNA uses tumor DNA-WES and UNCeqRRNA uses tumor RNA-seq. UNCeqR software is available at http://lbg.med.unc.edu/tools/unceqr. Evaluation in simulated tumor sequencing To test our hypothesis that somatic mutation detection based on integrated RNA-seq and DNA-WES is superior to that based on DNA-WES alone simulated tumor genomes were generated so that the entire genome space is a completely defined truth of positive and negative somatic mutations. In brief for each patient's sequencing 500 mutant sites were sampled for each site a mutant allele was randomly sampled and then aligned reads in the real RNA-seq and DNA-WES were edited to have the mutant allele at a rate of a fixed MAF (Supplementary Figure S2). By using real sequencing as the basis of the simulation authentic sequencing depths random errors (sequencing and alignment) and patients™ germline variants were preserved. Sequencing from the lung cancer quadruplet cohort was used for simulation. Patients™ DNA-WES and RNA-seq had large and similar numbers of sequenced nucleotides (DNA-WES median: 10.6 billion RNA-seq median: 10.2 billion; Kruskal-Wallis P = 0.54) indicating no significant imbalance in total sequencing. UNCeqR models were applied to the simulated tumor sequencing and detected mutations were compared against the truth by receiver operating characteristic curves. In simulations with a 10% MAF (A) the UNCeqRMETA model had significantly superior performance over UNCeqRDNA (difference in area under the curve P < 0.01); in other words UNCeqRMETA achieved a greater true positive rate (greater sensitivity) at the same false positive rate (same specificity) than UNCeqRDNA. In simulations with a 20% MAF (B) UNCeqRMETA continued to be superior to UNCeqRDNA (difference in area under the curve P < 0.01) although the gain in 20% MAF simulations was less (roughly 50% less) than the gain in 10% MAF simulations. This demonstrates that adding RNA-seq improved sensitivity particularly when the mutation signal that is MAF was low. UNCeqRMETA and UNCeqRDNA had large and clear superior performance to UNCeqRRNA which incurred false positives at a higher rate. Alternative ways to integrate RNA and DNA (taking the union or intersection of UNCeqRDNA and UNCeqRRNA) were both inferior to UNCeqRMETA (Supplementary Figure S3). Therefore in simulation UNCeqRMETA achieved superior performance over UNCeqRDNA with the largest gains occurring in mutations with low MAF. . Mutation detection performance in simulated tumor genomes. Model performance is displayed as receiver operating characteristic curves. Sensitivity plateaus below 1 because simulated mutations include sites with zero tumor sequencing depth in DNA and/or RNA (see ˜Simulation analysis™ methods). Validation by whole genome sequencing To validate the superior performance of integrated DNA-WES and RNA-seq mutation detection (UNCeqRMETA) over DNA-WES only detection (UNCeqRDNA) tumor and germline whole genome DNA sequencing (DNA-WGS) was used as an independent measure of truth for evaluating DNA-WES and RNA-seq mutation detections. Following a published validation procedure (4) mutation detections were interrogated in patient-matched DNA-WGS to determine if a mutation detection was a true positive that is present in the tumor specimen and absent from the germline specimen or false positive that is absent from the tumor specimen or present in the germline specimen. For each mutation model true positives and false positives were summed at each discrimination threshold (e.g. P-value) to generate a performance curve by which true positive rates could be compared at the same false positive rates (see methods for further description). These curves demonstrated that UNCeqRMETA achieved overall superior performance than UNCeqRDNA (difference in area under the curve P < 0.01) and at fixed false positive thresholds (250 500 and 1000) thus validating the result from simulated tumor genomes (Figure 2). Therefore in real tumor sequencing integrated DNA and RNA mutation detection by UNCeqRMETA outperformed DNA-only mutation detection. Figure 2. Validation of mutation detection by whole genome sequencing. The number of true positives and false positives of mutation detection models are plotted as step functions. At fixed false positive totals (250 500 or 1000) each pair of models was compared for differences in number of true positives (*). The published mutation set (46) did not include mutation rankings and was not amenable to rank-based statistical analysis. Other models displayed overall reduced performance relative to UNCeqRMETA and UNCeqRDNA. As another DNA-only control a leading (45) DNA-WES mutation caller from Illumina Strelka (17) was run on the same DNA-WES. Strelka exhibited inferior performance overall smaller true positive rates at fixed false positive rates and never achieved the sensitivity of UNCeqRMETA or UNCeqRDNA (Figure 2). Strelka had greater sensitivity than UNCeqRMETA or UNCeqRDNA at the highest extreme of specificity; however at UNCeqR's minimum false positive rate Strelka's sensitivity was only ?70% of either UNCeqR model. Providing another DNA-only control previously published mutations of this cohort made by heterogeneous pipelines (46915“16) had reduced sensitivity than UNCeqRMETA and UNCeqRDNA at the same false positive rate (256 false positives). At this false positive rate indel mutation detections were rare in all models (maximum 1.7%) with UNCeqRMETA and UNCeqRDNA having no significant difference in indel precision (number of true positives divided by the sum of false positives and true positives 92 and 96% respectively) but both having greater indel precision than Strelka (83%) and previously published mutations (82%) (proportions test P < 0.001). Taking the union or intersection of UNCeqRDNA and UNCeqRRNA had higher false positive rates and inferior performance than UNCeqRMETA or UNCeqRDNA (Supplementary Figure S4A). Integrating Strelka with an RNA-seq mutation detector SNVmix did not result in superior performance versus Strelka UNCeqRDNA or UNCeqRMETA (Supplementary Figure S4A). Providing a separate source of validation UNCeqRMETA detected nearly all mutations that were published as validated by targeted resequencing within this cohort (up to 97% depending on the model threshold; Supplementary Figure S5). Repeating this analysis with a slightly increased true positivity definition minimum two confirming tumor WGS DNA reads maintained all findings listed above (Supplementary Figure S4B). Increased mutation signal in RNA-seq To analyze integrated mutation detection across larger cohorts UNCeqR was applied to the lung and breast triplet cohorts (n = 871) and using model thresholds with the same empirically estimated specificity (500 false positives in DNA-WGS validation sequencing marked as triangle point in Figure 2 UNCeqRMETA P-value ? 1.1 — 10?9 UNCeqRDNA P-value ? 9.3 — 10?9)."

" the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour and patient characteristics treatment efficacy and tolerability and quality of life better patient selection might lead to improved outcomesmethods this post hoc exploratory analysis examined the effect of prognostic factors on outcomes in the randomized double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with mcrc refractory to standard chemotherapies recourse trial patients were redivided by prognosis into two subgroups those with metastatic sites at randomisation low tumour burden and ‰¥ months from diagnosis of metastatic disease to randomisation indolent disease were included in the good prognostic characteristics gpc subgroup the remaining patients were considered to have poor prognostic characteristics ppcresults gpc patients n386 had improved outcome versus ppc patients n414 in both the trifluridinetipiracil and placebo arms gpc patients receiving trifluridinetipiracil n261 had an improved median overall survival vs months hr ci to p00001 and progression free survival vs months hr ci to p00001 than ppc patients receiving trifluridinetipiracil n273 improvements in survival were irrespective of age eastern cooperative oncology group performance status ecog ps kras mutational status and site of metastases at randomisation in the trifluridinetipiracil arm time to deterioration of ecog ps to ‰¥ and proportion of patients with ps0“ discontinuing treatment were longer for gpc than for ppc patients vs months and vs respectively low tumour burden and indolent disease were factors of good prognosis in late line mcrc with patients experiencing longer progression free survival and greater overall survivalintroductioninclusion of new therapeutic options into the current treatment landscape in metastatic colorectal cancer mcrc has led to an increased survival in the last couple of key questionswhat is already known about this subject –º the choice of treatment in patients with metastatic colorectal cancer mcrc is generally influenced by tumour characteristics and patient factors as well as treatment characteristics such as tolerability efficacy and quality of life effects trifluridinetipiracil is indicated in pretreated patients with mcrc based on results of the pivotal randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial which demonstrated significantly improved overall survival os compared with placebo with a manageable safety profilewhat does this study add –º in recourse classification of patients as having good prognostic characteristics gpc defined as those with low tumour burden metastatic sites at randomisation and less aggressive disease ‰¥ months from diagnosis of first metastasis at randomisation identified a subgroup of patients with improved os and progression free survival with trifluridinetipiracil compared with patients with poor prognostic characteristics treated with trifluridinetipiracil and gpc patients treated with placebohow might this impact on clinical practice –º low tumour burden and indolent disease were shown to be factors of good prognosis in late line mcrc with these patients experiencing longer time on treatment and greater os this suggests that these patients could be candidates to receive further lines of therapy post trifluridinetipiracildecades1“ first line treatment of patients typically involves the use of vascular endothelial growth factor vegf or epidermal growth factor receptor egfr targeted agents eg bevacizumab cetuximab panitumumab to fluoropyrimidine based fluorouracil or tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesscapecitabine chemotherapy regimens depending on the presence or absence of ras mutation positive disease2 in the usa immunotherapies nivolumab±ipilimumab or pembrolizumab are also recommended for the treatment of patients with mismatch repair deficient or microsatellite instability high disease4 in the second line setting vegf targeted treatments eg aflibercept ramucirumab can also be used in combination with chemotherapy2 the optimal chemotherapeutic regimen for use beyond third line remains unclear where resistantrefractory disease and residual toxicity potentially limit the treatment options with only two possible candidates at present5the general condition and performance status of a patient are strong prognostic and predictive factors for mcrc treatment2 fitter patients are typically assigned to a more intensive treatment approach ie a combination of “ cytotoxic agents with a biological agent than less fit patients2 the choice of treatment in the metastatic setting is generally influenced by tumour characteristics tumour burden localisation and biology patient characteristics age eastern cooperative oncology group performance status ecog ps an function and comorbidities and treatment characteristics efficacy toxicity profile administration and quality of life qol effects2the proportion of patients with mcrc receiving active treatment decreases from line to line leaving more than half of patients who received an active treatment in the first line without treatment in the third line setting even in randomised clinical trials in folfiri plus cetuximab versus folfiri plus bevacizumab as first line treatment for patients with metastatic colorectal cancer only of patients reached third line6 data from the usa indicate that only of patients receiving a first line of treatment move into the second line move to the third line and only will receive a fourth line of treatment7 being unable to receive a subsequent line of treatment therefore appears to have a negative impact on the patient™s survivalis trifluridinetipiracil ftdtpi lonsurf indicated for the treatment of adult patients with mcrc who have been previously treated with or are not considered candidates for available therapies including fluoropyrimidine based oxaliplatin based and irinotecan based chemotherapies anti vegf agents and anti egfr agents for eligible patient ras wild type combination of tipiracil hydrochloride with the nucleoside metabolic inhibitor trifluridine improves its bioavailability by inhibiting its catabolism by thymidine phosphorylase8 the relatively limited non haematological toxicity of trifluridinetipiracil makes it a good option in the third line and refractory settings2 in the pivotal phase iii randomised double blind phase study of trifluridine tipiracil ftdtpi plus best supportive care bsc versus placebo plus bsc in patients with metastatic colorectal cancer refractory to standard chemotherapies recourse trial conducted in patients with mcrc eligible for treatment in the third line and beyond treatment with trifluridinetipiracil versus placebo extended overall survival median os vs months hr p0001 and progression free survival median pfs vs months hr p000110 this effect was shown in all subgroups regardless of age ecog ps “ geographical region race and kras mutational status10 furthermore trifluridinetipiracil was well tolerated with few serious adverse events aes reported haematological toxicities were the most frequently observed aes10 also time to deterioration of ecog ps to ‰¥ was significantly improved median vs months hr p000110 with of patients treated with trifluridinetipiracil remaining at ps “ at discontinuation11 remaining at ecog ps “ is important as it could allow patients to further benefit from subsequent therapy and potentially extend their survival in recourse and of patients treated with trifluridinetipiracil remained alive at and months respectively in the refractory setting in the post hoc analysis described here we set out to explore other factors that could extend survival in the recourse population for the purposes of our exploratory analysis we defined the characteristics of good prognosis as low tumour burden metastatic sites by response evaluation criteria in solid tumors recist evaluation at randomisation and less aggressiveindolent disease ‰¥ months from diagnosis of first metastasis to randomisation which are known to be strong prognostic factors in patients with mcrc with good ecog ps12 our ultimate aim is to explore how clinicians can better predict individual treatment outcomes and support treatment selection through the continuum of carematerials and methodsstudy design and patientsthe study design and methodology of the recourse trial clinicaltrials gov number nct01607957 have been previously published10 in brief recourse was a phase iii randomised double blind placebo controlled study comparing the efficacy and safety of trifluridinetipiracil plus best supportive care with those of placebo plus best supportive care10 this study included patients with metastatic biopsy provendocumented adenocarcinoma of the colon or rectum who were previously treated with ‰¥ standard chemotherapy regimens or who had tumour progression within months of their most recent chemotherapy or who had clinically significant aes precluding readministration of standard chemotherapies patients were randomised to trifluridinetipiracil mgm2 two times a day on days “ and “ every weeks or matching placebo10 randomisation was stratified according to kras mutation status wild type vs mutant time from diagnosis of first metastasis to randomisation vs ‰¥ months and geographical region japan vs usa european union and australia10 all patients had adequate an function and were ecog ps tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0cof “ at inclusion10 the primary endpoint of the study was os and secondary endpoints included pfs objective response rate clinical benefit rate and safety10patient subgroupsin examining the effects of prognostic factors on treatment outcomes in the current analysis several subgroups of recourse patients were considered patients from recourse n800 were divided according to good prognostic characteristics gpc and poor prognostic characteristics ppc good prognosis was considered to be defined by low tumour burden metastatic sites by recist tumour evaluation at randomisation and less aggressiveindolent disease ‰¥ months from diagnosis of first metastasis to randomisation12 of the gpc subgroup n386 patients received trifluridinetipiracil and received placebo the remaining patients were included in the complementary ppc subgroup n414 of these received trifluridinetipiracil and received placeboanalysis outcomesos and pfs in the gpc subgroup were compared with those in the ppc subgroup these subgroups were then analysed according to other tumour and patient characteristics that is metastatic site at randomisation for those sites present in of the population liver lung lymph or peritoneum ecog ps vs kras mutation status wild type vs mutant and age vs ‰¥ years os and pfs with trifluridinetipiracil were compared with placebo and were analysed according to prognostic subgroups within each of the two arms finally the effect of prognostic classification of patients on ecog ps deterioration was analysed for all patients and subgroupsstatistical methodsdemographic and baseline characteristics of patients were summarised by treatment arm and subgroups using descriptive statistics n mean sd median minimum and maximum andor frequency distributions as appropriatethe differences in os pfs and time to ecog ps deterioration between trifluridinetipiracil and placebo patients or between subgroups of patients in a specific arm of treatment were assessed using the stratified log rank test stratification factors used for the randomisation from a cox proportional hazards model for each arm or each subgroup survival was summarised using kaplan meier curves and was further characterised in terms of the median with the corresponding two sided cisresultspatientsbaseline patient demographics and clinical characteristics were generally similar between gpc and ppc patients table in the trifluridinetipiracil arm slight imbalances were seen in ecog ps more gpc than ppc open accesspatients had an ecog ps of and kras status more gpc than ppc patients were kras wild type also more gpc than ppc patients had received ‰¥ prior regimens among the ppc group treated with trifluridinetipiracil of patients had ‰¥ months from diagnosis of first metastasis to randomisation but had ‰¥ metastatic sites and of patients had metastatic sites but months from diagnosis of first metastasis similar differences were observed in the placebo arm with the exception of kras status which was comparable in the gpc and ppc subgroupstreatmentamong trifluridinetipiracil treated patients those in the gpc group received more treatment cycles mean sd compared with patients in the ppc group mean sd online supplementary table s1 a higher proportion of gpc patients than ppc patients receiving trifluridinetipiracil had a dose delay vs respectively or dose reduction vs respectively which is consistent with a longer duration of treatment online supplementary table s1 however median dose intensity in the first four cycles was high ‰¥ and did not differ markedly between the groups cycle in the gpc group and the ppc group cycle and respectively cycle and respectively cycle and respectivelythe effect of good versus poor prognosis classifications on survivalsurvival curves for the gpc versus ppc subgroups are shown in figure median os was longer in the gpc subgroup than the ppc subgroup for both trifluridinetipiracil vs months hr ci to p00001 figure 1a and placebo vs months hr ci to p00001 figure 1b rates of month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively and month os and in gpc and and in ppc for trifluridinetipiracil and placebo respectively were also higher in gpc subgroups compared with ppc subgroups median pfs with trifluridinetipiracil was also longer in the gpc subgroup versus the ppc subgroup vs months hr ci to p00001 respective values for gpc versus ppc in the placebo arm were versus months hr p00699 pfs at and months in the ppc subgroup was and for trifluridinetipiracil and and for placebo respectively in the gpc subgroup these were and with trifluridinetipiracil and and with placebo respectivelyeffects of good prognostic factors on the relative efficacy of trifluridinetipiracilmedian os was prolonged with trifluridinetipiracil versus placebo in both subgroups but to a greater tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable baseline patient demographics and clinical characteristics according to prognosistrifluridinetipiracilplacebogpc subgroup n261 ppc subgroup n273 gpc subgroup n125ppc subgroup n141 females male asian other years to years ‰¥ yearsmedian age yearspatient age n gender n ethnicity n ecog ps n kras status n time since diagnosis of metastasis n number of prior regimens n number of metastatic sites n site of metastatic lesion n   primary site of disease n liver lung lymph peritoneum ‰¥ “ ‰¥ mutant wild type months ‰¥ months colon rectum defined as metastatic sites and ‰¥ months since first metastasis only those in more than of the intent to treat population are included liver lung lymph and peritoneumecog ps eastern cooperative oncology group performance status gpc good prognostic characteristics ppc poor prognostic characteristicsextent in the gpc subgroup than in the ppc subgroup figure 2a similarly median pfs was prolonged with trifluridinetipiracil versus placebo in both subgroups with the greatest magnitude of benefit observed in the gpc patients figure 2banalysis of prognostic factorsthe effect of various prognostic factors on median os and pfs is shown in table their effect on month and month os and month month and month pfs is shown in online supplementary tables and for both tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessfigure overall survival os for the good prognostic characteristics gpc and poor prognostic characteristics ppc subgroups in patients receiving a trifluridinetipiracil or b placebo ap0001 one sided bp0001 two sided ftdtpi trifluridinetipiracil mos median overall survival nr not reachedtrifluridinetipiracil and placebo the gpc subgroup had better median os and pfs than the ppc subgroup irrespective of patient age ‰¥ vs years ecog ps vs kras mutation status mutant vs wild type and liver metastases yes vs nowhen analysing the gpc subgroup the absence of liver metastasis at randomisation n153 representing of the gpc and of the intent to treat population was found to be the best factor of prognosis further information on this group of patients is available in online tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accessa overall survival os b progression free survival pfs and c time to eastern cooperative oncology group figure performance status ecog ps ‰¥ with trifluridinetipiracil versus placebo in the good prognostic characteristics gpc n386 and poor prognostic characteristics ppc n414 subgroups ftdtpi trifluridinetipiracil mos median overall survivalsupplementary table s4 and online supplementary figures s1 s3 among gpc patients treated with trifluridinetipiracil median os was months longer in patients with no liver metastases compared with those with liver metastases vs months table the month os rate in gpc patients treated with trifluridinetipiracil was in those without liver metastases and in those with liver metastases corresponding month os rates in these groups were and respectively online supplementary table s2 median os was also longer in patients with no liver metastases compared with those with liver metastases in the trifluridinetipiracil ppc subgroup vs months and both the gpc and ppc subgroups of the placebo arm vs months and vs months respectively table in the group of ppc patients treated with trifluridinetipiracil the month and month os rates were and respectively in those without liver metastases compared with and respectively in those with liver metastases online supplementary table s2 for the trifluridinetipiracil and placebo arms patients with baseline ecog ps had higher median os compared with ecog ps patients in both the gpc and ppc subgroups table in the trifluridinetipiracil arm age or ‰¥ years and kras status did not seem to affect the treatment outcome table similar results were found for pfs with an effect for all trifluridinetipiracil gpc and ppc subgroups with median pfs values ranging from to months table among gpc patients treated with trifluridinetipiracil the month pfs rate was in those with no liver metastases compared with in those with liver metastases corresponding month pfs rates in the ppc group of patients treated with trifluridinetipiracil were and respectively online supplementary table s3 no such effect was observed in the placebo arm with values ranging “ months whatever the prognosis at the outset for almost all subgroups median tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0ctable the effect of various prognostic factors on median overall survival os and progression free survival pfsnumber of patientsftdtpi placebomedian survival monthshr cinumber of patientsftdtpiplacebomedian survival monthshr ciopen accessosgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgrouppfsgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroupgpc subgroupppc subgroup vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs no liver metastasesn97n56n35n21no lung metastasesn89n25n54n24no lymph metastasesn208n93n116n53no peritoneal metastasesn242n119n203n100ecog ps0n158n77n143n70age yearsn137n72n163n76kras wild typen142n61n120n70 vs vs vs vs vs vs vs vs vs vs vs vs vs vs to to to to to to to to to to to to to to to to to to to to to to to to to to to to liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age‰¥ yearsn124n53n110n65kras mutantn119n64n153n71liver metastasesn164n69n238n120lung metastasesn172n100n219n117lymph metastasesn53n32n157n88peritoneal metastasesn19n6n70n41ecog ps1n103n48n130n71age ‰¥ yearsn124n53n110n65kras mutantn119n64n153n71 vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to vs vs to to good prognostic characteristics gpc were defined as metastatic sites at randomisation and ‰¥ months from first metastasis to randomisationftdtpi trifluridinetipiracil ppc poor prognostic characteristics ecog ps eastern cooperative oncology group performance statustabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0copen accesstable effects of prognostic classification of patients on eastern cooperative oncology group performance status ecog psmedian time to deterioration to ecog ps ‰¥ monthsftdtpiplaceboitt population n80011good prognosis patients n386poor prognosis patients n414ftdtpi trifluridinetipiracil itt intent to treatpfs was longer and all hrs favoured treatment with trifluridinetipiracil table effects of prognostic classification of patients on ecog psdata relative to the effect of ecog ps are presented in table the proportion of gpc patients treated with trifluridinetipiracil with an ecog ps of “ at treatment discontinuation was among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of “ at treatment discontinuation similarly among gpc patients with an ecog ps of at baseline had not deteriorated beyond a ps of “ at treatment discontinuation the median time to deterioration of ecog ps to ‰¥ in patients receiving trifluridinetipiracil was months in the gpc subgroup and months in the ppc subgroup figure 2ctolerability and safetythe most common aes in patients receiving trifluridinetipiracil were nausea anaemia neutropenianeutrophil count decrease diarrhoea fatigue and reduced appetite online supplementary table s5 the most common grade ‰¥ aes experienced by patients receiving trifluridinetipiracil were haematological anaemia neutropenianeutrophil count decrease white blood cell count decrease there was no evidence of a higher incidence of aes in patients with ppc versus gpc in the group receiving trifluridinetipiracil but there was a trend towards a higher incidence of aes in placebo recipients with ppc compared with gpc online supplementary table s5discussionthe results of our analysis show that patients in the gpc subgroup consistently performed better than those in the ppc subgroup in both the trifluridinetipiracil and placebo arms within the same subgroups patients treated with trifluridinetipiracil performed better than placebo trifluridinetipiracil has consistently been shown to provide a significant survival benefit to patients with mcrc refractory to standard therapy with a well tolerated safety profile in three large scale randomised clinical trials10 “ a previous subanalysis of recourse showed that trifluridinetipiracil was more effective than placebo in patients irrespective of region age racialhr ci to to to p valueecog ps “ at treatment discontinuation ftdtpiplaceboethnic differences or kras mutation status17 in the current analysis further categorisation of patients as having good prognosis using the criteria of metastatic sites by recist tumour evaluation at randomisation and ‰¥ months from diagnosis of first metastasis to randomisation12 identified a subgroup of patients with improved os and pfs with trifluridinetipiracil compared with poorer prognosis patients ie those with ‰¥ metastatic sites and months from first metastasis pfs and os were also improved in gpc patients treated with trifluridinetipiracil compared with gpc patients who received placebopatients with gpc received more cycles of treatment than patients with ppc because progression was delayed in this group which may have contributed to the better survival outcomes the difference cannot be explained by a difference in dose intensity since this was high and similar in both the ppc and gpc subgroups of patients receiving trifluridinetipiracil in addition there was no evidence for higher toxicity in the ppc than the gpc group in fact the haematological aes occurred at a slightly higher rate in gpc patients than in ppc patients who received trifluridinetipiracil which probably reflects a longer exposure to treatment in the gpc group more patients in the gpc than in the ppc subgroup had dose delays which suggests that grade ‰¥ haematological aes were appropriately managed during treatmentit is thought that the availability of more treatment options for mcrc has contributed to an improvement in os over the last years3 indeed a retrospective study in elderly patients aged ‰¥ years a patient population more prone to comorbidities poor performance status and the development of treatment related toxicity reported a correlation between os and the number of treatment lines received18 thus maintaining the general condition and performance status of a patient throughout the continuum of care is of great importance especially beyond the second line to ensure patients remain fit with good qol5 our analysis showed that the majority of patients in the gpc subgroup discontinued treatment with an ecog ps of “ at the time of disease progression suggesting that these patients could be candidates to receive further lines of therapy post trifluridinetipiracil this is important when sequencing through the continuum of care this is in line with other tabernero a0j et a0al esmo open 20205e000752 101136esmoopen2020000752 0canalyses indicating preservation of health related qol on treatment of patients with mcrc with trifluridinetipiracil19 while the post hoc nature of this analysis limits it to an exploratory analysis the relatively large number of patients analysed make these data a good tool to estimate the expected outcomes when treating patients with refractory mcrc with trifluridinetipiracil the smaller size of some of the subgroups may limit the s that can be drawn thus preventing an evaluation of other parameters that might impact on outcomes such as lactate dehydrogenase levels the exact definition of good and poor prognostic factors12 may require further validation in a prospective cohortthe current analysis shows that compared with poor prognosis patients treated with either trifluridinetipiracil or placebo and good prognosis patients treated with placebo patients with gpcs treated with trifluridinetipiracil adequate an function ecog ps “ metastatic sites by recist tumour evaluation at randomisation and ‰¥ months from diagnosis of first metastasis have an increased survival in terms of median os and month and month survival rates treatment with trifluridinetipiracil is effective and provides the majority of patients the opportunity to maintain ecog ps and the possibility to receive further treatment options through the continuum of careauthor affiliations1vall d™hebron institute of oncology uvic ucc medical oncology vall d'hebron hospital barcelona catalunya spain2vall d™hebron institute of oncology uvic ucc iob quironmedical oncology vall d'hebron hospital barcelona catalunya spain3medical oncology ospedale policlinico san martino istituto di ricovero e cura a carattere scientifico per l'oncologia genova liguria italy4department of medical oncology university hospital centre besançon besancon bourgogne franche comté france5kashiwa national cancer center hospital east kashiwa chiba japan6department of medical oncology dana farber cancer institute boston massachusetts usa7centre of excellence methodology and valorization of data centex mvd institut de recherches internationales servier suresnes france8global medical affairs les laboratoires servier sas suresnes île de france france9digestive oncology ku leuven university hospitals leuven leuven flanders belgiumacknowledgements the authors would like to thank andrea bothwell who wrote the first draft of this manuscript on behalf of springer healthcare communications this medical writing assistance was funded by institut de recherches internationales servier suresnes francecontributors jt and srmv contributed to the conception and design of the study all authors were involved in the acquisition analysis and interpretation of data and in writing andor revising drafts of the manuscript all authors have read and approved the final draft of the manuscript and accept responsibility for the finished article and the decision to submit the manuscript for publicationfunding the recourse study was funded by taiho oncology and taiho pharmaceutical co this analysis was funded by servier in partnership with taihocompeting interests jt has received personal fees from array biopharma astrazeneca bayer ag beigene boehringer ingelheim chugai genentech open accessgenmab as halozyme imugene limited inflection biosciences limited ipsen kura oncology eli lilly and company merck menarini merck serono merrimack pharmaceuticals merus molecular partners novartis peptomyc pfizer pharmacyclics proteodesign sl rafael pharmaceuticals f hoffmann la roche sanofi seattle genetics servier symphogen taiho pharmaceutical vcn biosciences biocartis foundation medicine haliodx sas pharmaceuticals and roche diagnostics ga has had an advisory role or received honoraria or travel grants from hoffmann la roche merck serono amgen sanofi bayer servier and bristol myers squibb afs has had an advisory role for amgen bayer celgene roche merck serono sanofi and servier and has attended a speakers™ bureau for amgen astrazeneca bayer bristol myers squibb celgene lilly merck serono roche sanofi and takeda evc has received research funding from amgen bayer boehringer ingelheim celgene ipsen lilly merck merck kga novartis roche sanofi and servier and has attended advisory boards for astellas astrazeneca bayer bristol myers squibb celgene lilly merck sharp dohme merck kgaa novartis roche and servier cb has attended advisory boards for roche servier and sanofi and has received a research grant from roche ao has received honoraria from ono bms chugai taiho eisai and amgen and has received research funding from bristol myers squibb an immediate family member of ao has been employed by celgene rjm declares no conflicts of interest lv and srmv are employees of servierpatient consent for publication not requiredethics ap

EndocrinesyPostoperative vocal fold dysfunction in covid19 era are we still intime for a recoveryElena Bonati Elena Giovanna Bignami2 Paolo Del Rio1Received May Accepted July Springer ScienceBusiness Media LLC part of Springer Nature To the EditorThe novel coronavirus COVID19 is a highly contagious zoonosis produced by SARSCoV2 which arose inChina and spread all over the world transmitting from manto man through respiratory secretions In March itwas defined by the World Health anization WHO as apandemic to underline its spread and severityHealthcare professionals are one of the categories most atrisk of contracting the infection in particular when theiractivity involves the direct management of the patient™sairways Among these categories we can count anesthetistshead and neck surgeons otolaryngologists maxillofacialsurgeons ophthalmologists and dentists For these reasonsthe latest evidencebased recommendations for otolaryngology and head and neck surgery practice suggest thathealthcare facilities should prioritize urgent and emergencyvisits and procedures until this condition stabilizes ceasingelective care []Nevertheless oncological surgical activity althoughslowed down did not stop in most hub hospitals Regardingthyroid cancer thyroid surgery is complex and the rate ofnerve damage is still considerable Immediate postoperativevocal fold rate is “ in our case study and decrease to“ after months Postoperative dysphonia can becaused by several factors other than nerve damage such astracheal intubation or scarring in the thyroid lodge It istherefore important to identify the cause of vocal corddysfunction and treat it correctly at the right time If anunilateral vocal fold paresisparalysis is diagnosedthetreatment consist in improving the speech while in case of Elena Bonatiebonati86gmailcom General Surgery Unit Department of Medicine and SurgeryParma University Hospital Parma Italy Unit of Anesthesiology Department of Medicine and SurgeryParma University Hospital Parma Italybilateral vocal fold paresisparalysis respiratory obstructionalso needs to be urgently treated Fortunately we havebroughtthe incidence of this last and most dangerouscomplication to at our Clinic since the introduction in of the routine use of intraoperative neuromonitoringduring thyroidectomyThe latest guidelines published by the Americanin March Association of Endocrine Surgeonsrecommend laryngeal examination in patients with knownor suspected new recurrent laryngeal nerve dysfunctionafter thyroidectomy for additional evaluation and possibletreatment with a speech pathologist According to theAmerican Academy of Otolaryngology”Head and NeckSurgery they assert that early referral “ weeks postsurgery to a laryngologistin combination with earlyintervention results in superior voice outcomes since theideal time for vocal fold augmentation is months afterthyroidectomy []A metaanalysis about therapy for vocal fold paresisparalysis after thyroidectomy concluded that the timingof therapy for unilateral vocal fold paralysis after thyroidectomy has a significant impact on the effect sizebeing significantly greater if therapy is performed within months This may be explained by progressive atrofolds and disappearance of nervephy offunction so that vocalfold movements cannot berecovered []the vocalPatients who underwent thyroid surgery from February and who had experienced a vocal fold disfunctionVFD were unable to undergo a laryngoscopy nor muchless a speech therapy according to health measuresnecessary to contain the spread of the virus This unfortunately causes a progressively reduced possibility ofrecovery increasing the specific morbidity related to surgery for thyroid cancer in this period The only indicationthat we can give to patients is the rest of the voice to avoidthe establishment of compensation mechanisms worseningthe clinical picture waiting to be able to resume the correcttreatment 0cTherapeutic diagnostic pathways in the COVID19 erahave become difficult and dangerous logarithms that mustconsider the need for patient care and the possibility oftreatments delay in safety but also the risk of contagion ofthe patientsleast protection ofhealthcare personnel The hospital setup has been significantly changed and much of the economic structuraland human health resources have been dedicated to themanagement of the COVID pandemicthemselves and notIn parallel with the COVID19 emergency we areexperiencing another health emergencythe one thatinvolves the management of nonCOVID19 patients Evenin the second phase of the pandemic only urgent healthservices are provided A reanizing effort within theindividual healthcare companies is required to guaranteetreatment even for nonCOVID19 patientsMoreoverThe COVID19 pandemic highlighted the limits andweaknesses of our health system and now that the correctprotocols for the protection of healthcare personnel havebeen described allthe all healthcare companies shouldequip their staff with the appropriate materials such as N95masks hair cover protective coverall gown gloves faceshields goggles and shoe covers In the face of higherexpenses this would allow the resumption of activitiesminimizing the risk of an increase in the rate of infectionroutine health practices must be reconsidered preferring less invasive techniques in order toscreen patients who need secondlevel examination Evenif not used yet in our hospital transcutaneous laryngealultrasonography is a valid noninvasive and painlessalternative method in the assessment of vocal cords It hasbeen demonstrated in a recent prospective multicentricstudy that it has concordance with laryngoscopy in themajority of cases and so it can be a valid alternative asfirstline exam for vocalfold examination pre andpostoperativelyEndocrineFinally the growing use of virtual platforms for the needof social distancing could encourage their application evenin healthcare services that can be performed by teleconference such as speech therapyWe can assess that COVID19 pandemic is causingdirect morbidity and mortality and even a related one dueto missed or delayed treatment of multiple nonCOVID19diseases The delivery of the health service should beimproved and the health system itself must be modernizedto adapt to new needsCompliance with ethical standardsConflict of interest The authors declare that they have no conflict ofinterestPublisher™s note Springer Nature remains neutral with regard tojurisdictional claims in published maps and institutional affiliationsReferences LP Kowalski A Sanabria JA Ridge WT Ng R de BreeA Rinaldo RP Takes A A Mkitie AL Carvalho CR Bradford V Paleri DM Hartl V Vander Poorten IJ Nixon C PiazzaPD Lacy JP Rodrigo O GuntinasLichius WM MendenhallA D™Cruz AWM Lee A Ferlito COVID19 pandemic effectsand evidencebased recommendations for otolaryngology and headand neck surgery practice Head Neck 101002hed26164 KN Patel L Yip CC Lubitz EG Grubbs BS Miller W ShenP Angelos H Chen GM Doherty TJ Fahey 3rd E KebebewVA Livolsi ND Perrier JA Sipos JA Sosa D StewardRP Tufano CR McHenry SE Carty The American Associationof Endocrine Surgeons Guidelines for the definitive surgical management of thyroid disease in adults Ann Surg e21“e93 X Chen P Wan Y Yu M Li Y Xu P Huang Z Huang Typesand timing of therapy for vocal fold paresisparalysis after thyroidectomy a systematic review and metaanalysis J Voice “ 0c'

inanic arsenic ias is the chemical form of as commonlyfound in drinking water atsdr and in some foodscubadda chronic exposure to ias has been associatedwith risk of skin bladder lung and liver cancers iarc aswell as with other diseases naujokas including diabetes maull cardiovascular abhyankar moon saquib states respiratorysanchez and neurological diseases caito andaschner parvez humans and most other mammalian species have developed a mechanism for detoxifying iasthat involves the sequential conversion of ias to monomethylasmas and mas to dimethylas dmas thomas both methylation steps are catalyzed by orthologs of a singleenzyme arsenic oxidation state methyltransferase as3mtlin the methylation of ias by as3mt not onlyaddress correspondence to miroslav st½blo department of nutritionuniversity of north carolina at chapel hill chapel hill nc usa telephone email styblomeduncedu or beverly hkoller department of genetics university of north carolina at chapel hillchapel hill nc usa telephone emailbkolleremailuncedusupplemental material is available online 101289ehp6943this document was reviewed by the center for computational toxicology andexposure office of research and development us environmental protectionagency and approved for publication approval does not signify that thecontents reflect the views of the agency nor does mention of trade names orcommercial products constitute endorsement or recommendation for usethe authors declare they have no actual or potential competing financialinterestspublished august received february revised july accepted july note to readers with disabilities ehp strives to ensure that all content is accessible to all readers however some figures and supplementalmaterial published in ehp s may not conform to standards due tothe complexity of the information being presented if you need assistanceaccessing content please contact ehponlineniehsnihgov our staï¬will work with you to assess and meet your accessibility needs within working dayspromotes wholebody clearance of as drobn¡ hughes but also produces methylated intermediates that contain highly reactive and toxic trivalent as asiii watanabe andhirano masiii and dmasiii unlike their pentavalent counterparts masvand dmasv exceed ias in potency as cytotoxinsgenotoxins and enzyme inhibitors thomas hencemasiii and dmasiii may be critical determinants of toxic and carcinogenic eï¬ects associated with chronic exposure to ias alteredcapacity to methylate ias has been linked to an increased risk ofdiseases associated with ias exposure ahsan pierce vahter in most mammals the gene encoding as3mt is present asa single copy ˆ¼ kb from the gene encoding bloc1 relatedcomplex subunit borcs7 to date the methylation of ias isthe only known function of as3mt however recent studies haveidentified the as3mtborcs7 locus as conferring risk for schizophrenia duarte compared with healthy individualsexpression of borcs7 and of the humanspecific splicing variantof as3mt as3mtd2d3 has been found to be consistently higher inthe brains of patients with schizophrenia li notablyexpression levels of as3mt and borcs7 were found to be weaklycorrelated li suggesting that the two genes may sharetranscriptional regulatory elements although published datashowed that ias exposure can aï¬ect as3mt expression in micest½blo the role of ias exposure in the expression ofborcs7 or as3mtd2d3 which is thought to lack ias methylationactivity has never been studiedlaboratory studies of the mechanistic basis of iasassociateddiseases have been hindered by substantial diï¬erences between laboratory animals and humans in their capacity to metabolize anddetoxify ias rats unlike humans sequester significant portions ofingested ias in erythrocytes in the form of dmasiii lu mice the species most commonly used in mechanistic studies diï¬erfrom humans displaying very high rates of as methylation thisspecies diï¬erence is associated with faster rates of urinary clearanceof methylated metabolites and the predominance of dmas as themain urinary metabolite of ias in mice vahter interspeciesdiï¬erences in as metabolism may contribute to difficultiesenvironmental health perspectives august 0cencountered replicating some of the eï¬ects of ias exposure reportedin humans including the carcinogenic eï¬ects in laboratory micethus producing an ias methylation phenotype in mice that resembles the phenotype found in humans could create a better animalmodel to study the role of ias metabolism in adverse health eï¬ectsof chronic ias exposure to generate such a model we have humanized the entire borcs7as3mt locus in 129s6 mice by syntenicreplacement in this process the segment of mouse dna carryingthese two genes was removed and replaced with the syntenic regionof human dna in mouse embryonic stem es cells using homologous recombination this ensured that the junctions between thehuman and mouse dna are known to the base pair level mice weregenerated from the es cells with the expectation that they wouldexpress the human as3mt and borcs7 proteinsthe present study describes in detail the creation of thehumanized mouse strain expression of human as3mt andborcs7 in tissues of the humanized mice and metabolism ofias in these mice after a single dose and during a subchronic exposure to ias in drinking watermethodsrationale for humanizing the borcs7as3mt locuswe chose to excise the 61kb segment of mouse dna carrying theas3mt and borc7 genes and replaced it with the correspondingpromoter of as3mt abuts59kb segment of human dna the untranslated region of borc7 and the weak correlation inthe the expression pattern of the genes suggests possible shared transcriptional regulatory elements li in addition the current human transcript databases for example the university ofcalifornia santa cruz genome browser ucsc lists a putative readthrough borcs7as3mt transcript lacking the finalexon of borcs7 and the initial exon of as3mt this suggests thepossibility that at least some as3mt activity may be linked to transcription driven by the borcs7 promoter thus the inclusion ofborcs7 in the humanized region increases the likelihood that theelements directing the tissue species diï¬erences in the expressionof as3mt are included in the humanized regionassembly of borcs7as3mt displacerthe borcs7as3mt displacer construct was assembled using astandard recombineering approach figure the mouse arms ofhomology were derived from the 129s7ab22 bmq bac librarysource bioscience plc nottingham uk the short homologyarm was derived from bac bmq455l14 and the long arm ofhomology from bac bmq345l20 the segment of humangenomic dna containing the borcs7 and as3mt loci wasderived from the human tile path bac rp11753c18 bacpacresources center children™s hospital oakland research instituteoakland ca the single nucleotide polymorphisms snps previously associated with interindividual diï¬erences in ias metabolism apata or schizophrenia risk li which were included in the this borcs7as3mt segment aredescribed in table s1 a dna segment bp extendingfrom chr1946683032 to were deleted from the mousegenome and replaced with a 59261bp segment of human dnaextending from chr10102845563 to the haplotypeof the as3mt gene carried in the displacer construct is 1a wood the resistance marker gene used for selection of escells in which the displacer construct underwent genomic integration consisted of a phosphoglycerate kinase pgkneo cassetteflanked by mutant loxp sites the marker gene can be excised leaving only a nonfunctional lox site in its placeall polymerase chain reactions pcrs carried out during thedisplacer assembly used taq polymerase bio basic with an initial denaturation temperature of °c for s followed by cycles of denaturation for s at °c annealing for s at a°c and extension at °c with an extension time of min per bp of product length all redet recombination reactionswere carried out using a commercially available kit gene bridgescat no k001 all predesigned primers used during the construction of the displacer vector were from sigmaaldrich and arelisted in table s2 in descriptions of specific assays each of theseprimers is listed by the corresponding number as stated above the short arm of the displacer construct wasderived from the bmq455l15 bac library this was accomplished by first replacing the borcs7as3mt region of the bac withan ampicillin resistance marker by redet recombination theampicillin resistance marker was amplified from the pbluescriptii sk cloning vector stratagene primers and the homology arms referred to as the red arm primers and and the ds arm primers and respectively were generated by pcr amplification ofsegments of the bmq455l15 bac the homology arms werefused to the ampicillin resistance gene by overlap pcrabhuman locuscyp17a1borcs7as3mtmouse locuscyp17a1borcs7as3mt kb deletedcnnm2cnnm2cborcs7 as3mt displacerddisplaced mouse locuscyp17a1floxedneofloxedneoborcs7as3mtcnnm2homologousrecombinationborcs7as3mtcnnm2 kb insertedfigure scheme for humanization of the mouse borcs7as3mt locus a human borcs7as3mt locus showing the relative positions of the borcs7 andas3mt genes as well as the closest flanking genes b mouse borcs7as3mt locus with flanking genes c schematic of the borcs7as3mt displacer construct d humanized locus prior to cremediated marker excision the names of human genes are capitalizedenvironmental health perspectives august 0cwas used to insert the human borcs7as3mt segment and neomycin resistance cassette by redet recombination into thebmq354l20 bac carrying the short arm and ampicillin genesimultaneously displacing the ampicillin gene this reactionyielded the final borcs7as3mt displacer vector which wasdigested with noti to release the bac backbone before transfection into es cellsgeneration of mouse es cellsthe parental es cell line phnx43 used in these studies was generated as follows male and female 129s6svevtac mice taconicbioscience were mated females were checked every morning forcopulatory plugs indicative of successful mating three days laterfemales were euthanized by lethal carbon dioxide co2 exposurefollowed by physical euthanasia and the 35d embryos blastocysts were collected by flushing the uterus with co2independentmedium gibco cat no supplemented with bovine serum albumin sigmaaldrich cat no a3675 individualembryos were collected from the lavage fluid with a pipette andplaced in 2cm2 wells that had been seeded h earlier withprimary mouse embryo fibroblasts mefs gibco cat nogsc6005m blastocysts and mefs were cultured in gibcoknockout„¢ ko medium cat no supplementedwith esqualified fetal bovine serum fbs gibco cat no and 2mm lglutamine gibco cat no after “ d in culture when the inner cell mass of the embryosreached ˆ¼ mm in diameter the embryos were removed and disrupted with a pipette and placed in a new well seeded with mefsthis process of serial expansion of a single inner cell mass wascontinued until a single inner cell mass was expanded to at least colonies of cells cells were then further expanded by disruptionand exposure to trypsin for min at °c after expansionand cryopreservation in dimethyl sulfoxide sigmaaldrichcat no d2660 a sample of the cell line was subjected to karyotype analysis in karyologic inc rtp nc the cell line used inthis study had a xy normal male mouse karyotypeexpression of human borcs7as3mt in mouse es cellssinglecell suspensions of the parental es cell were prepared bytreatment with trypsin as described above in a volume of ml — cells were electroporated in the presence of lgof dna the apparatus used was a btx electro cell manipulator with cuvette btx cheshire analytical the settingsused were v lf r8 ohms after electroporation the cells were distributed onto seven 100mm tissue cultureplates corning cat no in gibco ko medium supplemented with esqualified fbs gibco cat no and mm lglutamine gibco cat no after hselection was initiated with the addition of geneticin„¢ final concentration mgml gibco cat no after d individual neomycin resistant colonies became visible cells fromˆ¼ colonies were pooled and used for rna isolation to confirmthat the human genes were expressed see details below once thiswas established individual colonies from the remaining plateswere transferred individually by pipette to a 96cell plate each colony was trypsinized and a portion used for pcr analysis to identify those in which the dna had integrated by homologousrecombination as described below the remaining cells wereallowed to grow and cultures carrying a targeted allele as determined by the assay detailed below were transferred sequentially to and 60cm2 tissue culture plates at this point some cellswere cryopreserved while a portion of each was used for karyotypeanalysis and to confirm the expression of human as3mt by probebased droplet digital pcr ddpcr using commercially availablethe displacer short arm together with the inserted ampicillingene was subcloned from the modified bmq455l15 bac into aplasmid vector by redet recombination the plasmid vectorwas prepared by ligation of four pcr products the vector backbone carrying a cole1 origin of replication and a spectinomycingene was derived by amplification of the pfloxxerx plasmid previously assembled in koller™s laboratory see table s3 primers and the bmq455l15 bac was used as atemplate for the other three pcrs us arm primers arm primers and andand short ds arm primers and all pcr productswere gel purified on a agarose gel using a commerciallyavailable kit qiagen cat no digested with bbsi nebcat no r3539s and mlui neb cat no r0198s and columnpurified qiagen cat no fifty nanograms of each pcrproduct was combined and ligated neb cat no m0202s in a20ll reaction for h at °c the ligation reaction was heatinactivated at °c for min and then ll of the reaction wastransformed into chemically competent e coli dh5afcellszymo research cat no t3002 according to the manufacturer™s directions and plated on luria broth agar plates supplemented with spectinomycin at lgml the resulting plasmidvector was digested with mlui and used for redet recombination with the modified bmq455l15 bac the resulting plasmidwas in turn digested with bbsi to release the displacer short armand ampicillin resistance gene flanked by the us and dshomology arms this bbsi restriction fragment was introducedinto the bmq345l20 bac by redrecombination to create abac vector in which the ampicillin resistance marker wasflanked by the displacer arms of homologythe segment of the human borcs7as3mt locus included inthe displacer vector was prepared for excision from the rp11753c18 bac in two steps in the first step a neomycin resistancegene was inserted into the bac upstream of the borcs7 gene toaccomplish this the neo gene flanked by mutant loxp sites wasligated to homology arms referred to as blackred and purplerespectively the neomycin resistance gene was excised from thevector psfiijt15neojtz17sfii previously assembled in koller™slaboratory see table s4 by digestion with sfii neb cat nor0123s the blackred homology arm was amplified primers and using the bmq455l15 bac as a templateand the purple homology arm was amplified primers and using the rp11753c18 bac as a template all fragments were gel purified as described above and the blackred andpurple homology arm pcrs were digested with bgli neb catno r0143s and then column purified as described above theloxpflanked neomycin resistance gene was ligated to the homology arms as described above in a 20ll reaction containing a totalof lg of dna with the three fragments at an equimolar ratiotwo microliters of the ligation reaction was used for a redetreaction with the rp11753c18 bac in the second step an ampicillin gene was inserted downstream of the as3mt gene in therp11753c18 bac carrying the neomycin resistance marker toaccomplish this homology arms referred to as bluegreen andyellow respectively were added to the ampicillin resistance genethe bmq455l14 bac was used as the template for the bluegreen pcr primers and pbluescript ii sk was used as the template for the ampicillinyellow pcr primers and the two pcr products were gel purifiedand then combined in an overlap pcr primers and the resulting pcr product was gel purified and ngwas used for a redet reaction with the rp11735c18 bac carrying the neomycin resistance cassetteapproximately ng of the modified rp11753c18 bacwas digested with mlui in a 20ll digest and ll of the digestenvironmental health perspectives august 0cprimers applied biosystems hs00960526 here a 20ll reaction mixture was pipetted into a dg8„¢ cartridge along with llof droplet generation oil for probes bio rad and loaded into theqx200 droplet generator bio rad according to the manufacturer™s instructions after droplet generation the droplets weretransferred to an eppendorf twintec® 96well plate and placed in ac100 touch thermal cycler bio rad using the manufacturer™srecommended cycling conditions after cycling the plate was readin the qx200 droplet reader bio rad quantasoft„¢ softwareversion a portion of each of the es colonies ˆ¼ cells was disruptedby incubation for min at °c in lysis buï¬er consisting of trisedta [ mm trisma base thermo fisher scientific cat nobp152 mm ethylenediaminetetraacetic acid edta thermofisher scientific cat no volvol triton„¢ x100millipore sigma cat no and mgml proteinase kroche cat no ] lysates from all colonies werescreened for homologous recombination by pcr using primersscreen f and see table s4 with gxl polymerase takarabio according to the manufacturer™s suggested protocol the pcrprogram used was °c initial denaturation for s followed by cycles with s denaturation at °c s annealing at °c minat °c and a final 10min extension at °c pcr products wereanalyzed by agarose gel electrophoresis agarose bio basiccat no d0012 in — trisacetateedta running buï¬er the sizeof the pcr product was determined by comparison with 2logladder neb cat no n3200ltwentyseven clones with homologous recombination wereidentified and were subjected to further analysis because of thishigh recombination frequency it was not necessary to use clustered regularly interspaced short palindromic repeats and crisprassociated protein 9crisprcas9 to stimulate recombinationevents rna was prepared from pools of the cells and examinedfor the expression of the human genes rna was also preparedfrom a number of the individual clones after expansion brieflycells were lysed directly in the culture dish and homogenized byrepeated pipetting rna was isolated using rna bee or stat60teltest according to the manufacturer™s instructions rna yieldand quality were assessed using a nanodrop thermofisher one to two micrograms total rna was reverse transcribedwith the high capacity reverse transcription kit appliedbiosystems expression of the human as3mt was examined byqualitative pcr qpcr on a c100 touch thermal cycler biorad using commercially available primers applied biosystemshs00960526generation of mice expressing human borcs7as3mtfor injection into blastocysts the es cells carrying the humanborcs7as3mt locus were again trypsinized to generate singlecell suspensions collection of recipient blastocysts blastocystinjection and transfer to pseudopregnant dams was carried out bythe unc chapel hill animal models core following proceduresdescribed by longenecker and kulkarni and by koller to maintain the mutation on the 129s6svevtac background the chimeric mice were bred to 129s6svevtac micetaconic bioscience the resulting f1 mice were identifiedby taq as wildtype wt homozygotes for the mouse as3mtborcs7 locus wtwt or heterozygous wths or homozygoushshs for the human as3mtborcs7 locus the primers usedwere common and endo for the endogenous locus and common anddisplaced for the displaced locus see table s2 the pcr assayswere carried out using taq polymerase bio basic with an initialdenaturation temperature of °c for s followed by cycles ofdenaturation for s at °c annealing for s at a °c andextension at °c for s with a final extension of min the f2generation”consisting of hshs homozygotes hswt heterozygotes and wtwt homozygotes”was produced by mating of f1hswt heterozygotes the hshs hswt and wtwt oï¬springfrom the f2 generation were housed together one litter per cagecunder controlled conditions with 12h lightdark cycle at ± and ± relative humidity the parental mice and mice in thef1 and f2 generations were fed purina labdiet 5v5r and drankdeionized water ad libitumall proceduresinvolving mice were approved by theuniversity of north carolina institutional animal care and usecommittee 0ecollection of tissues for mrna expression analysistissues for mrna expression analysis were collected from f2hswt mice both males and females measurement of rnaexpression in hswt mice which were heterozygous for thehuman and mouse gene allowed direct comparison of expressionin the same tissuerna sample minimizing the eï¬ects of physiological variation between animals or quality of sample whichcould result in subtle diï¬erences in rna quality and cdnaformationmice were euthanized by exposure to co2 followed by physical euthanasia the following tissues were collected whole brainparts of brain cortices pons medulla hippocampus cerebellumpituitary spinal cord adrenals liver spleen kidney duodenumjejunum colon stomach uterus ovaries testes heart and skinfrom back of neck neuronal and glial cells were also dissectedbrain parts and spinal cord were collected from male mice at dof age neuronal cells were isolated from embryonic day e17embryos and glial cells from postnatal day p1 pups all othertissues were collected from to 12wkold mice three mice wereused for each tissue blood was collected from lethally anesthetized mice via cardiac puncture with an edtacoated syringe justprior to thoracotomy to isolate mononuclear cells from blood ml of whole blood were layered onto ml of histopaque® sigma and centrifuged at room temperature for min with nobrakes at — g the upper layer was discarded and the interfacecontaining the mononuclear cells was transferred to a 15ml conical tube the cells were then washed twice with phosphatebuï¬ered saline and then lysed with rna beeneuronal cell isolation and culture brains were harvestedfrom eight e17 embryos and placed in a petri dish containinghanks™s balanced salt solution hbss gibco meninges wereremoved cortices dissected and placed in a new petri dish containing hbss cortices were transferred to a 15ml conical tube containing ml hbss and centrifuged at — g for min at °cthe tissue was digested with ml tryple express gibco catno and dnase i roche cat no for min at °c the digested cortices were centrifuged at — gfor min at °c the tryple dnase i was aspirated and thecortices were washed for min with ml fbs to inactivate thetrypsin the tissue was centrifuged again and resuspended in complete dulbecco™s modified eagle medium dmem gibco with fbs vwr glutamax gibco with penicillinstreptomycingibco and 2mercaptoethanol sigma the cortices were titurated times with a 10ml pipette and then times with aflamedtip pasteur pipette the cells were then passed through a100lm cell strainer into a clean conical tube cells were then pelleted by centrifugation at — g for min the cell pellet wasresuspended in ml complete dmem and plated in mm culture dishes coated with lgml poly dlysine sigma — cells per dish after h 1lm cytosine bdarabinofuranosidearac sigma cat no c6645 was added using a mediachange without exposing the cells to air the cells were harvestedfor rna isolation on day environmental health perspectives august 0cmixed glial cell culture brains were harvested from six p1pups and placed in a dish containing hbss meninges wereremoved cortices dissected and placed in a conical tube containing cold dmem cortices were centrifuged at — g for min at°c and media replaced with ll cold dmem the corticeswere titurated times with a flamedtip pasteur pipette coated inserum the cells were then passed through a 100lm cell strainerinto a fresh conical tube cells were pelleted by centrifugation at — g for min the cell pellet was resuspended in ml complete dmem and ml of the suspension was plated on three100mm culture plates the medium was changed h later andthen daily the cells were harvested for rna on day isolation of adrenal cortex and medulla to obtain samplesenriched for each of these two distinct functional regions of thegland the adrenal cortex was removed from medulla of male andfemale adrenal gland by microdissection enrichment was quantitated by qpcr analysis of genes known to be expressed in onlyone of these two regions chgb in the medulla and cyp11b1 inthe cortex the human protein atlas using appliedbiosystem taqman probes mm00483287 and mm01204952respectively rna was isolated from cells and tissues using themethod described for es cells however the tissues were first homogenized in stat60 in a desktop homogenizer fastprep mp bio using ceramic beadsanalysis of as3mt as3mtd2d3 borcs7 as3mt andborcs7 expression in collected tissues and cellsboth ddpcr and qpcr were used to detect and quantify as3mtas3mtd2d3 borcs7 as3mt and borcs7 mrna the specificmethod is indicated in each figure legend ddpcr is minimallyinfluenced by diï¬erences in the efficiency of the mouse and humanprimer sets quan and therefore provides a means ofdirect comparison of expression between the endogenous mouselocus and the humanized locus the distribution of the as3mttranscript in the tissues was compared with as3mt protein distribution described by the human protein atlas for the human tissues sybr green qpcr was used to compare rna expression inwhole brain cortex hippocampus pituitary cerebellum pons medulla spinal cord glial cells neurons adrenals ovaries testesheart kidney liver colon jejunum spleen and blood ddpcr wasused to assess rna expression in es cells whole brain liver adrenals spinal cord ovaries testes spleen and heart for all assays“ lg total rna was reverse transcribed with the high capacityreverse transcription kit applied biosystemsfor sybr green qpcrthe relative expression of fulllength as3mtthe d2d3 variant and the borcs7as3mtfusion transcript were assessed on a quantstudio6 flex„¢ realtime pcr system applied biosystems sybr green reactions were run using itaq universal sybr green supermixbio rad nm of each primer and ngll of cdnaprimers used for quantification of as3mt isoforms were d2d3fand d2d3r for the d2d3 isoform and fullf and fullr for thefulllength isoform see table s2 for analysis of borcs7as3mt readthrough transcripts expression in mouse tissues lgrna was reverse transcribed and sybr green quantitative pcrwas run using primers gaacagtcatcggatctacagga3and and itaq sybrgreen universal supermix bioradgaacagtcatcggatctacagga3for ddpcr absolute concentration of as3mt as3mt borcs7and borcs7 was acquired using the ddpcr supermix forprobes no dutp bio rad and taqman gene expressionassays as3mt mm00491075_m1 as3mt hs00960526_g1borcs7 mm01205060_m1 and borcs7 hs00376014_m1all from applied biosystems the ddpcr analysis was carriedout as described aboveevaluation of the metabolism of a single dose of iasthe metabolism of ias was examined in to 22wkold hshsmale n and female n f2 oï¬spring and their wtwtmale n and female n littermates the mice weregiven a single dose of ias sodium arsenite pure sigmaaldrich in deionized water diw by gavage lg askg ofbody weight immediately after dosing each mouse was placedin a metabolic cage mousecage and urine and feces were collected in 24h intervals for d during these d all mice drankdiw ad libitum the mice were fasted during the first h buthad free access to purified laboratory diet envigo teklad catno ain93g during the second and the third days our published data showed that ias content in this type of diet rangesfrom to ˆ¼ lg askg douillet huang murko the 24h urine and feces samples werefrozen in dry ice and stored at ˆ’c 0eevaluation of the metabolism of ias during subchronicexposureafter collection of urine and feces following the singledoseadministration the hshs and wtwt mice were again cagedtogether one litter per cage and maintained on the purified dietand diw for wk to allow for clearance of as from the bodywhich was confirmed by measuring total as tas levels inurine see the œresults section for details both wt andhshs mice now to 27wkold were then exposed to iassodium arsenite pure sigmaaldrich in drinking water lg asl for wk spot urine samples ˆ¼ to llwere collected weekly body weights were recorded before andafter the exposure after wk all mice were sacrificed by cervical dislocation without anesthesia and tissues were collectedincluding the liver kidneys pancreas spleen heart lung adrenals kidneys bladder visceral fat calf muscle testes or ovaries and cecum and colon all tissues were flashfrozen in dryice and stored at ˆ’c 0eanalysis of as species in urine feces and tissuesspeciation analysis of as was carried out in 24h urines and fecescollected after the single dose of ias and in spot urine samplescollected during subchronic ias exposure the speciation analysis was also performed in livers and kidneys collected after subchronic exposure at sacrifice ten percent homogenates of liverand kidney were prepared in icecold diw wtvol usingwheaton potterelvehjem“style tissue grinders with a ptfepestle and wheaton overhead stirrer apparatus dwk lifesciences feces were snap frozen in liquid nitrogen and pulverized to powder using a steel mortar and pestle placed in dry icethe powder was mixed with 2n ultrapure phosphoric acid thermo fisher and digested in a mars5 microwave cemcorp for h at °c this method eliminates the biologicalmatrix but does not alter as speciation currier the digestates were neutralized by sodium hydroxide sigmaaldrich to ph the urines tissue homogenates and neutralized digestates were treated with lcysteine sigmaaldrich at room temperature for h to reduce pentavalent asspecies to their trivalent counterparts prior to the analysiscurrier the cysteinetreated samples were analyzed by hydridegeneration atomic absorption spectrometrycoupled with a cryotrap hgctaas as previously describedcurrier hern¡ndezzavala this analysis determined the concentrations of ias mas and dmas tasconcentration in urine feces and tissues was calculated as thesum of ias mas and dmas for assessment of the efficiencyof ias metabolism after a single oral dose the amount of tasenvironmental health perspectives august 0cwas expressed as the percentage of the dose by comparing theamount of elemental as administered as ias to each mousewith the amount of tas in 24h urine and feces samples collected from that mousethe instrumental limits of detection lod for ias mas anddmas using this method are and pg as respectivelyhern¡ndezzavala an imputed value of was usedfor measurements below the lod hgctaas is also capableof detecting and quantifying trimethylarsine oxide tmasohern¡ndezzavala a product of ias metabolism byrat as3mt waters however because tmaso israrely detected in human urine and because tmaso was not aproduct of ias methylation by recombinant human as3mt inour published study ding we did not includetmaso analysis in this studystatistical analysisoneway analysis of variance with studentnewmankeuls ortukey™s multiple comparison posttest was used to assess diï¬erences in gene expression and in the concentrations and proportions of as species among wtwt and hshs mice student™s ttest was applied for comparison of mouse and human geneexpression in a single tissue of hswt mice and for comparisonof as species concentrations or proportions between male andfemale mice and between hshs and wtwt mice of the samesex the statistical analyses were performed using prism software graphpad software diï¬erences with p were considered statistically significantresultsexpression of borcs7 and as3mt in hswt miceexpression of as3mt and borcs7 were directly compared withthose of endogenous mouse genes in tissues collected from f2male hswt mice that carried one copy of the mouse locus andone copy of the human locus figure 2a using both conventionalqpcr and when appropriate ddpcr expression of humanas3mt and borcs7 was easily detected in all tissues expressingthe corresponding mouse genes although the level of expressionof the human and mouse genes diï¬ered figure 2bc notable inthis analysis was

colorectal cancer crc is a malignant tumor in the gastrointestinal tract and it arises from theinner wall of the large intestine the colon crc is the third most common cancer worldwideaccounting for roughly million new cases per year and ˆ¼ deaths per year whichmakes it the fourth most common cause of cancerrelated death globally and remains a hugechallenge “ in order to identify eï¬ective molecular targets for crc diagnosis and potentialedited byzhonghua taofudan university shanghai cancercenter chinareviewed byshengli liuniversity of texas health sciencecenter at houston united statesrossano lattanziouniversity of studies g d™annunziochieti and pescara italycorrespondencejianhua wangwangjianhuaman163comyansong pudoctor_puyahoocom these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived september accepted june published august citationliu y cao j zhu yn ma ymurtaza g li y wang jh and pu ys c1222c deletion in exon ofabl1 is involved in carcinogenesisand cell cycle control of colorectalcancer through irs1pi3kaktpathway front oncol 103389fonc202001385frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerinterventions for crc therapy indepth studies on the regulatorymechanism of crc progression should be conductedthe pathogenesis of crc accompanies with genetic orepigenetic changes numerous genes and pathways such aswnt tgf egfr“ras erk“mapk pi3k and p53 havebeen demonstrated to be associated with crc “ abl1a protooncogene of cabl encodes a nonreceptor tyrosinekinase plays an important role in carcinogenesis regulatingcell adhesion proliferation diï¬erentiation and apoptosis studies have characterized abl1 as an oncogene thatpromotes breast cancer cell proliferation and induces anchorageindependent growth under p53 deficiency in breast cancercells “ craig reported that inhibition of abl1by imatinib reduced the proliferation of lymphoma cells andprevented tumor formation in mice however the roleand mechanism of abl1 in crc development and progressionremain largely unclearthe aim of this study was to elucidate the role of abl1using highthroughput dna sequencing technology to obtaininformation on colon cancer gene mutation we analyzedthe variation in the expression of abl1 among patients withcrc and in crc celllines we additionally determinedthe eï¬ect of downregulating abl1 on the proliferation cellcycle progression and apoptosis of crc cells further theeï¬ects of knockout of abl1 in tumor and the molecularmechanisms of activated and suppressed downstream signalingpathways were assayed to elicit the mechanisms involved incrc carcinogenesismaterials and methodsadmitted atclinicalpathological data ofpatients and samplesthefortyeight patients with crc wereshaanxi people™s hospitalshaanxi china colorectalcancer was confirmed by histopathology or biopsy basedon which thesubjectswere evaluated formalxed paraffinembedded ffpetissues were used as the study material tumor contentsin the ffpe tissues werethedepartment of histopathology shaanxi people™s hospitaland only ffpe tissue blocks with tumor contentwere qualified the study was approved by the ethicscommittee of shaanxi people™s hospital written informedconsent was obtained from allsubjects participating inthis studythethoroughlychecked atcell culture and rna interferencecrc cell lines ncm460 lovo sw620 sw480 and hct116were purchased from the cell bank of the chinese academyof sciences shanghai china the cells were cultured inrp1640 medium supplemented with heatactivated fetalbovine serum gibco gaithersburg md and penicillinstreptomycin gibco at —¦c with co2abl1 protein phosphatase catalyticsubunit alphappp3ca and tgf1 knockdown kd lentiviruses weregenerated using pfugwgfprnai vector by insertingshabl1 shppp3ca and shtgf1 sequence empty pfugwgfp vector was used as vector control shctrl in crc cells or ncmice the rnai sequence of abl1 ppp3ca and tgf1 were²cgttctatatcatcactga3² ²atatacgcgttctgaatactt3² ²gattatcga catggagctg3² respectivelysw480 and hct116 cells were plated in a 24wellplate andincubated at —¦c with co2 for h a multiplicity ofinfection of was added to infect sw480 and hct116 cellsovernight the infection medium was then replaced with normalcomplete growth medium cells without infection were used ascorresponding controlsproliferation and colony formation assayproliferation rate was determined using bromodeoxyuridinebrdu cell proliferation elisa kit abcam boston ma theoptical density of each sample was measured at nm using asynergy h1 microplate reader biotek winooski vtfor the clonogenic assay sw480 and hct116 cells wereplated onto 6wellplate and incubated in culture medium for days the cells were then fixed with pfa and stained with crystal violet sigmaaldrich st louis mo for h at roomtemperature the total number of colonies was counted wheneach clone contained more than cells size “ mmflow cytometric analysisfor cell cycle analysis cells were fixed in pfa for minat —¦c and treated with propidium iodide pi µgmlsigmaaldrich at room temperature for min in dark atotal of cells were analyzed by flow cytometry using abd facscalibur system becton“dickinson el paso tx thedistribution of cell cycle phases was estimated using modfitlt in mac v30 software apoptosis was further determinedby annexin v fitcconjugated thermo fischer scientificmiami ok and pi staining cells were immediately counted byflow cytometrydna extraction and sequencingfortyeight specimens of colorectal cancer tissue with clinicalliver metastases were collected dna was isolated using allprep dna ffpe kit qiagen germantown md genomic dnawas extracted by fully automated purification using promegamaxwell promega madison wi the dna concentrationwas measured fluorimetrically using the qubit dna highsensitivity kit thermo fisher scientific waltham ma anion torrent semiconductor chip sequencer was used to sequencecommon gene mutations in the tumorsin vivo studybalbc nude mice female aged weeks were purchased fromshanghai ling chang biological technology co ltd shanghaichina the mice were housed in spflevel laboratories with freeaccess to food and water and accommodated for week prior toany experiments the animal study was performed in accordancewith iacuc guidelines shabl1hct116 kd or shctrlhct nc cells — µl were subcutaneously injected tothe left flank of the mice at day posttransplantation micewere sacrificed and tumors were excised and weighed the tumorfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancervolume was calculated using digital calipers with the followingformula tumor volume volume length width22ingenuity pathway analysisto elucidate the role and action mechanism of abl1 incrc after abl1 kd high throughput realtime pcr arraywas performed by shanghai genechem co ltd shanghaichina and the data were analyzed using ingenuity pathwayanalysis ipa software to elucidate the aï¬ected molecules andsignal pathwaysimmunohistochemistryfor crc 180point tissue microarray hcolade180sur07which contained crc tissuesfrom patients and thecorresponding adjacent tissues table s4 was purchased fromshanghai outdo biotech co ltd shanghai china briefly thetissue microarray block was constructed by embedding a singletissue core mm in diameter was taken from each region informalxed paraffinembedded crc or adjacent tissue blockusing a tissue microarrayer beecher instruments silver springmd usa and was set to a blank recipient block predrilled with mm holesthe tissue microarray blocks and paraffinembedded tumorsections were cut into 7µm sections for immunohistochemicalihc analysis slides were deparaffinized and rehydrated aspreviously described followed by antigen retrievalincitrate buï¬er mm citric acid tween ph for min in —¦c water bath after washing with pbsslides were incubated with pbst with bovine serumalbumin sigmaaldrich for h slides were then incubatedovernight at —¦c with antiabl1 antibody ab15130abcam cambridge ma and developed using mouse andrabbit specific hrpdab detection ihc kit ab64264 abcamfollowing the manufacturer™s instructionsthe selection of cutoï¬ value to dichotomize the expressionlevels of abl1 was based on previously reported method []briefly the high expression level of abl1 was defined from twocriteria dab staining showed equal or darker color comparedto positive control the population of abl1positive cells washigher than all cases were independently evaluated anddiagnosed by two senior pathologists y m and l y who wereblinded to the pathologic diagnosis cases with any disagreementwere reviewed simultaneously by the original two pathologistsand a senior pathologist j w until they reach a consensuswestern blotthe western blotting assay was performed by wellestablishedprotocols as previously described primary antibodiesused in this study were antiabl1 antibody ab85947abcam cambridge ma antibcl2 antibody bcl10c4biolegend san diego ca antibclxl antibody sc santa cruz biotechnology dallas tx antibaxantibody 2d2 biolegend antiactin antibody 2f11 biolegend antigapdh antibody ff26af9biolegend antip27 antibody sc56338 santa cruzbiotechnology anticyclind1 antibody sc8396 santacruz biotechnology antiirs1 antibody ab52167abcam antiakt2 antibody ab175354 abcam antippp3ca antibody ab52761 abcam antitgf1antibody ab92486 abcam antimap2k2 antibody sc81473 santa cruz biotechnology antipi3kp11aantibody ab151549 abcam secondary antibodiesused were antimouse igg hrpconjugated secondary antibody sc516102 santa cruz biotechnology and antirabbitigg hrpconjugated secondary antibody sc2357 santacruz biotechnologyreverse transcriptionpolymerase chainreactionthe mrna level was measured using realtime polymerasechain reaction briefly total rna was extracted from culturedcells using trizol reagent thermo fisher scientific andcdna synthesis was performed using the quantitect reversetranscription kit qiagen the primers used were as followsabl1andantisense ²acaccctcccttcgtatctcag3² gadphsense ²tgacttcaacagcgacaccca3² antisense ²caccctgttgctgtagccaaa3² the realtime pcr wascarried out by using rt2 sybr rcid13 green qpcr mastermixesqiagen according to the manufacturer™s instructions all pcrswere performed in triplicate 01 01ct method was used to calculatethe relative expression levels²catcacgccagtcaacagtct3²sensestatistical analysisstatistical analyses were performed using spss spss incchicago il χ 2test was used to investigate the possiblerelationships between abl1 expression and clinic pathologicalcharacteristics mann“whitney utest was used to compare thediï¬erence in abl1 protein expression between paired coloncancer and adjacent normal colon tissues survival analysiswas performed using the kaplan“meier curve and the logranktest the values are expressed as mean ± sd comparisonsbetween two groups were conducted using student™s ttest allexperiments were carried out in triplicate results with p were considered statistically significantresultshighly expressed abl1 in crc tissue isassociated with poor clinical outcometo verify the existence of abl1 in crc tissues we comparedcrc tissues and their adjacent noncancerous tissues by ihcstaining our results showed that the immunostaining of abl1was significantly higher in crc tissues compared with adjacentnormal colon tissues p figures 1a“d table ourwestern blot and realtime pcr results confirmed the muchhigher expression level of abl1 in crc tissues compared tonormal tissues figures 1ef similarly the expression of abl1was significantly increased in diï¬erent crc cell lines comparedwith that in the normal colon cell line ncm460 figures 1ghremarkably the expression of abl1 was significantly p increased in the advanced stages stage iiiiiiv of crccompared with the early stages stage i and noncancerousfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 is highly expressed colorectal cancer tissue and cell lines a“d representative ihc staining of abl1 in normal ab and crc cd tissuesac x bd x e western blot analysis of abl1 expression in crc tumor t and adjacent normal n tissues f realtime pcr analysis of abl1expression in crc tumor crc and adjacent normal n tissues p g western blot analysis of abl1 expression in crc cell lines h realtime pcranalysis of abl1 expression in crc cell lines relative expression was normalized to ncm460 cells p i realtime pcr analysis of abl1 expression incrc tumors at different clinical stages relative expression was normalized to adjacent normal n tissues p table expression of abl1 in colon cancer and adjacent tissuestable c1222c deletion in exon of abl1variablesnoexpression levelspvariablesexpression levelsor95 cipneglowhighno no mutation mutationnormalcolon cancergenderfemaleadjacent normal colon tissues neg negativetissues figure 1i with a median followup of monthsranging from to months our survival analysis showed thatthe patients with high abl1 expression death had a lowersurvival rate compared to patients with low abl1 expression death p figure s5 these results suggested thatabl1 is a potential oncogene abl1 and that its expression waspositively associated with the clinical stage in patients with crcc1222c deletion in exon of abl1 inrelation to the tnm stageprevious studies have shown the mutations of the abl1 geneare of major clinical relevance to study the possiblemutations in patients with crc we performed dna sequencingour results indicated that a mutation of abl1 was presentin males and females of patients with crc females and males this mutation occurred in exon ofthe abl1 gene and all mutations were found to be deletion““ maletnm stage““ofthe c1222c nucleotide sequence within this exon theincidence rate of mutation was in females and malestable figure additionally the analysis of patients withmutations and the corresponding stages revealed that patientswere in stages “ and in stages “ the tnm stage was asignificant risk factor for c1222c deletion the results suggestedthat c1222c deletion is involved in crc carcinogenesisinterference of abl1 decreased theproliferation and enhanced the apoptosisof sw480 and hct116 cellsto further investigate the role of abl1 in crc carcinogenesiswe used lentivirus vector to downregulate abl1 expression insw480 and hct116 cells after infection both cell lines showedfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure structural domain and c1222c deletion in exon of the abl1 genefigure depletion of abl1 decreases the proliferation of crc cells a representative fluorescent images showing gfppositive cells after infection images weretaken under x b western blot detects the abl1 expression in cells transfected infected with shctrl or shabl1 c representative images of colony formationassay using crc cells infected with shctrl or shabl1 cells without infection was used as control con d quantification of colony numbers data are shown asmean ± sd p compared with control e brdu assay detects the proliferation of sw480 and hct116 cell lines infected with shctrl or shabl1 cellswithout infection was used as control con p compared with control“ of average gfppositive rate figure 3a our westernblot results showed a significant decrease of abl1 protein levelfigure 3b indicating a successful downregulation after rnainterference to evaluate the proliferation of crc cells afterabl1 depletion we performed a clonogenic assay figure 3ccompared with the control group the number of clones inthe shabl1 group was obviously decreased figure 3d ourbrdu proliferation assay confirmed that the proliferation offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 causes cell cycle arrest and apoptosis in sw480 and hct116 cells ab representative cell cycle analysis of sw480 a andhct116 b infected with shctrl or shabl1 cells without infection was used as control con c quantification of cell cycle distribution in g1 s g2m phases p p compared to control group d western blot analysis of p27 and cyclin d1 expressions in crc cells infected with shctrl or shabl1 cellswithout infection was used as control configure depletion of abl1 increases the apoptosis of crc cells flow cytometry detected apoptosis of sw480 a and hct116 b cells infected with shctrl orshabl1 cells without infection was used as control con c quantification of apoptotic cells data are shown as mean ± sd p compared with controld western blot analysis of bax bclxl and bcl2 expression in crc cells infected with shctrl or shabl1 cells without infection was used as control conshabl1 cells was obviously reduced compared with that ofthe control cells figure 3e additionally our flow cytometryresults showed more cells were arrested in s phase afterabl1 depletion while fewer cells in g2m phases were foundfigures 4a“c indicating downregulation of abl1 inhibitedcell cycle progression of crc cells to validate these data wedetected p27 a negative regulator of cell cycle progression andfound its expression was significantly increased in cells infectedwith shabl1 vector figure 4d on the contrary cyclin d1 wasdecreased in abl1depleted crc cells figure 4dnext we performed flow cytometric analysis to examinethe apoptosis of abl1depleted crc cells figures 5ab wefrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure depletion of abl1 inhibits crc tumor growth in vivo a representative image of mice injected with hct116 cells infected with shctrl nc or shabl1kd b representative tumor images showing depletion of abl1 decreased the size of crc tumors body weight c tumor volume d and tumor weight e weremeasured at day after inoculation p compared to nc groupfound downregulation of abl1 significantly increased apoptosisin crc cells as compared with the control group p figure 5c the expression of the apoptosisrelated proteinbcl2associated x bax was obviously increased while bcelllymphomaextralarge bclxl and bcell lymphoma bcl2were remarkably decreased in the shabl1 group figure 5dtaken together these results suggest depletion of abl1 increasesthe apoptosis of crc cellsdepletion of abl1 inhibited crc tumrowth in vivoin order to examine the involvement of abl1 in regulatingcrc tumor growth we inoculated hct116 cells infected withshctrl nc group or shabl1 kd group into balbc nudemice figure 6a as shown in figure 6b the tumor growth wasremarkably inhibited in the kd group compared with the ncgroup interestingly we also observed a significant bodyweightincrease in the kd group figure 6c as compared to controlabl1 depletion caused a significant reduction in tumor volumefigure 6d the average tumor weight in kd xenografts wasobviously lower than that in the nc group ± mgvs ± mg p figure 6e taken together ouranimal experiments demonstrated that abl1 knockdown couldinhibit crc tumor growth in vivoabl1 interference inhibited tgf1 via thepi3kaktirs1 pathway and ppp3cato elucidate the molecular pathways regulated by abl1 in crcwe performed high throughput pcr array from xenografts inabl1 kd or nc mice our ipa results identified upregulatedgenes and downregulated genes in xenografts from kdmice compared with those from nc mice further analysisrevealed that these diï¬erentially expressed genes were involvedin multiple biological functions and pathogenesis of multiplediseases figure s1as shown in figures s2 s3 and tables s1“s3 the tgfand pi3kakt pathways were inhibited by depletion of abl1the associated molecules of the two pathways including tlr4akt2 il4r camk2d ppp3cb map2k2 pdia3 irs1itpr3 abl1 atf4 ppp3ca ca2 cpt1a csrp1 ctsv fn1lamp2 ptgs2 runx2 s100a4 and spp1 were mapped withabl1 gene to show a predicted interaction network figure 7ato verify this interaction we examined the levels of proteins intgf and pi3kakt pathways in xenografts of nc and kd micefigure 7b our western blot results showed that knockdown offrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure abl1 interacts with pi3kakt and tgf1 pathways a molecule network generated by ipa showing interactions among abl1 pi3kakt and tgf1pathways up and downregulated genes are shown in red and green respectively b western blot analysis of predicted interactive proteins in xenografts fromabl1 knockdown kd or control nc mice the gene of œ is detected by multiple probes and it is statistically significantabl1 significantly decreased irs1 akt2 ppp3ca and tgf1 expression while did not change the expression of map2k2compared with those in the nc groupto further verify the involvement of tgf1 in the regulationof pi3kakt pathway we generated a tgf1depletion hct cell line by lentivirus infection figure 8a our western blotresult showed that the expression of tgf1 was significantlydownregulated after infection figure 8b as expected thekey proteins in pi3kakt pathways were deactivated upontgf1depletion figure 8c including irs1 phosphopi3kand akt according to the findings in the previous study thedownregulated gene ppp3ca found in abl1 kd mouse isinvolved in the regulation of the pi3kakt pathway wenext investigated the interaction between ppp3ca and abl1by establishing a ppp3ca knockdown cell line figures 8dewe found the depletion of ppp3ca significantly decreasedthe expression of abl1 figure 8f to validate the regulatoryrole of ppp3ca we also examined the abl1 expression inppp3ca overexpressed cells and found the expression of abl1was elevated by upregulated ppp3ca figure s4 indicatingppp3ca is a positive regulator of abl1discussionas a ubiquitously expressed nonreceptor tyrosine kinase abl1has been reported to be associated with glioblastoma and breastcancer in this study we examined the role of abl1 incrc progression the results indicated that abl1 might play animportant role in crc which is associated with the mutation andexpression of the abl1 genewe found the expression of abl1 was remarkably elevatedin crc tissues and cell lines figure which is correspondedto the survival rate among patients with crc figure s5indicating abl1 is a potential oncogene in crc “moreover the mutation of abl1 was also elevated in crcpatients based on the results from previous studies the mutationrate of the abl1 gene is relatively higher in men than in womenpatients with crc worldwide in a previous study theabl1 gene was found to be mutated in of patients withcrc at sir ganga ram hospital delhi india in the presentstudy the mutation rate was much higher in crcpatients accepted in our hospital the diï¬erent races of patientsfrom diï¬erent regions of the world reported in the two studiesfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancerfigure tgf1 knockdown deactivates pi3kakt pathway in hct116 cells a representative images of hct116 cells infected with gfpcontaining lentivirusexpressing shrna against tgf1 gfp positive lentivirus with scramble shrna was used as control images were captured under 200x magnification bknockdown of tgf1 was confirm by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading control cwestern blot examined irs1pi3kakt pathway in hct116 cells after knockdown of tgf1 d western blot examined irs1pi3kakt pathway in hct116 cellsinfected with gfpcontaining lentivirus expressing shrna against ppp3ca gfp positive lentivirus with scramble shrna was used as control e representativefluorescent images of hct116 cells at h postinfection images were captured under 200x magnification f expression of abl1 after ppp3ca knockdown wasdetermined by western blot proteins were extracted from hct116 cells at h postinfection actin was used as loading controlcould be a possible reason causes the diï¬erence of abl1 mutationrates “ gene mutations are often involved in tumorigenesisthe clustered deletions were found in abl1 notch1 retstk11 gna11 and jak3 genes in crc melanoma and nonsmall cell lung cancers additionally the abl1 mutationdata in tcga showed that an average mutation rate of abl1is in coad patients the high mutation rate isconsistent with the findings in this study that abl1 mutationcorrelates with the oncogenesis of crc to the best of ourknowledge the present study presents a novel mutation inexon in which c1222c deletion occurred this deletion wasrelatively higher in female patients than in male patients thehigher distribution of this deletion at the higher tnm stage inpatients with crc suggests that this deletion might be relatedto tumorigenesis of crc however further investigation withlarger sample size is needed to elucidate the relationship andmechanism between c1222c deletion and crc progressionto determine the role of abl1 in crc progression wedownregualted abl1 expression in crc cell lines and foundthat the cell cycle was arrested at s phase figure theseobservations are consistent with previous reports thatthenumber of cells in s phase was increased when abl1 wasinhibited by imatinib or sti571 in u2os hela and a549 cells it is wellreported that p27 inhibits g1s transition ofthe cell cycle while cyclin d1 is a key regulator of cell entryinto the s phase allowing cells to enter the s phase smoothlyfrom the g1 phase our study provides direct evidencethat abl1 interference increased p27 expression and decreased incyclin d1 expression figure which is similar to the increasedp27 expression and decreased cyclin d1 expression found incells treated with nilotinib an abl1specific inhibitor however previous studies showed that when abl1 expressionwas inhibited by nilotinib the number of cells in the g0g1 phasewas increased while the number of cells in s and g2m phases wasdecreased which is contradictory to the finding in this studythat downregulation of abl1 arrested crc cells at s phase thismight be due to the varied function of abl1 in diï¬erent tissuesand cell types abl1 controls cell apoptosis via downstream moleculessuch as puma bax and p73 as well as by changingfrontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancermembrane potential depletion of abl1 inducedapoptosis of crc cells observed in this study is consistentwith the findings ofthese studies studies have reportedthat abl inhibitor danusertib treatment significantly decreasedthe expression of bclxl and bcl2 while increasing theexpression of bax similarly we found increasedbax expression and decreased levels of bcl2 and bclxl after downregulation of abl1 in crc cells figure indicating abl1 is involved in the regulation of apoptosis incrc cellsinvolved in cell proliferation migrationfurthermore information obtained using the ipa indicatedthe expressions of numerousthat after abl1 knockdowngenesinvasiondiï¬erentiation death and survival were aï¬ected figures s1“s3 tables s1“s3 the results demonstrated that abl1 mightplay a pivotal role in crc progression especiallythe tgf and pi3kakt pathways were inhibited afterabl1 interferenceit has been reported that abl1 regulates tgf signaling which is associated with tumor progression by modulatingangiogenesis in crc resulting in poor prognostic outcome“ studies have demonstrated thattreatment withan abl1 inhibitor significantly reduced the tgf level similarly we found the expression of tgf1 wassignificantly inhibited after abl1depletion figure thisindicated that abl1 is a positive regulator of tgf signalpathways as one of the tgfaï¬ected downstream signals thepi3kakt pathway plays a crucial role in tumorigenesisitassociated withproliferation apoptosisinvasion and metastasis of cancercells insulin receptor substrates irs including irs1and irs1 are a downstream messenger of the pi3k pathway our study provides novel evidence that abl1 mightinteract with tgf1 via pi3kaktirs1 that is involved incrc progressionexpression of proteinsregulatestheca2aandcalcineurincalmodulindependentserinethreonine protein phosphatase has been reported topromote intestinal tumor development and crc tumorigenesis the expression of calcineurin a specifically increases inhuman crc cell lines in the present study we foundthat ppp3ca which is also known as an alpha isoform ofthe calcineurin catalytic subunit was inhibited afterknockdown of abl1 figure 7b this finding provides novelevidence that abl1 might interact with the ppp3ca oncogenein crc carcinogenesisconclusionin conclusion we found a high level of abl1 expression incrc tissue and cells which was associated with the tnmstages a novel mutation of c1222c deletion in exon of theabl1 gene was found and was associated with the crc stagedepletion of abl1 decreased the growth of crc cell lines bothin vitro and in vivo by inhibiting tgf pathway these resultsdemonstrated novel understandings of the function of abl1during the progression of crc thus provides a clinically viablestrategy for crc therapydata availability statementthe raw data supporting the conclusions of this will bemade available by the authors without undue reservationethics statementthe studies involving human participants were reviewed andapproved by ethics committee of shaanxi people™s hospital thepatientsparticipants provided their written informed consent toparticipate in this study the animal study was reviewed andapproved by ethics committee of shaanxi people™s hospitalauthor contributionsall authors have a significant scientific contribution to all aspectsof this studyfundingstudy wasthissupported by national natural sciencefoundation of china”youth projects grant no shaanxi natural science foundation grant nos 2015jq8321and 2019jm547 shaanxi innovative talents cultivate programgrant no 2017kct28 and operating expenses of basicscientific research project of xi™an jiaotong university grantno xzy012019112supplementary materialthe supplementary materialonline202001385fullsupplementarymaterialfor this can be foundhttpswwwfrontiersins103389foncatreferences ouerhani s bougatef k soltani i elgaaied ab abbes s menif s theprevalence and prognostic significance of kras mutation in bladder cancerchronic myeloid leukemia and colorectal cancer mol biol rep “ 101007s1103301325128 brenner h kloor m pox cp colorectal cancer lancet “ 101016s0140673613616499 navarro m nicolas a ferrandez a lanas a colorectal cancer populationscreening programs worldwide in an update world j gastroenterol “ 103748wjgv23i203632 favoriti p carbone g greco m pirozzi f pirozzi re corcione fworldwide burden of colorectal cancer a review updates surg “ 101007s133040160359yjauhri m bhatnagar a gupta s shokeen y minhas s aggarwal stargeted molecular profiling of rare genetic alterations in colorectalcancer 101007s1203201608202sequencing med oncolnextgenerationusing peng x luo z kang q deng d wang q peng h foxq1mediates the crosstalk between tgfbeta and wnt signaling pathways inthe progression of colorectal cancer cancer biol ther “ frontiers in oncology wwwfrontiersinaugust volume 0cliu abl1 is involved in carcinogenesis of colorectal cancer rossner f gieseler c morkel m royer hd rivera m blaker h uncoupling of egfrras signaling and nuclear localization of ybx1in colorectal cancer oncogenesis 5e187 101038oncsis alpay k farshchian m tuomelainhibition ofsiljamaki ecancer9e105526 101371 pone0105526cells highly sensitivealetj sandholm j aittokallio krenderscabl kinaseactivityto mitoxantrone plos one tian xq guo ff sun df wang yc yang l chen sl downregulationof znf278 arrests the cell cycle and decreases the proliferation of colorectalcancer cells via inhibition of the erkmapk pathway oncol rep “ 103892or20176031 udden sm moritafujimura y satake m ikawa s cabl tyrosinefate “kinase modulates p53dependent p21 induction and ensuing celldecision in response to dna damage cell signal 101016jcellsig201310005 cai s cheng x liu y lin z zeng w yang c eya1 promotes tumorangiogenesis by activating the pi3k pathway in colorectal cancer exp cell res “ d

"purpose to assess the efficacy and safety of recombinant human endostatin in combination with radiotherapy rtor concurrent chemoradiotherapy ccrt in patients with locally advanced nonsmall cell lung cancer lansclcmethods we searched eligible literature in available databases using combinations of the following search termslung cancer endostatin or endostar radiotherapy or radiation therapy or chemoradiotherapy the inclusion criteriawere prospective or retrospective including singlearm studies that evaluated the efficacy and safety of endostatinplus radiotherapy ert or concurrent chemoradiotherapy ecrt in patients with lansclc primary outcomesincluded the following objective response rate orr local control rates lcr overall survival os progressionfreesurvival pfs and adverse events aes tests of heterogeneity sensitivity and publication bias were performedresults a total of patients with lansclc from studies were enrolled including six prospective trials andone retrospective study the pooled median pfs was months overall months in the ecrt group and months in the ert group pooled median os and orr were months and overall months and in the ecrt group and months and in the ert group respectively the incidences of major grade ‰¥ aes for all patients subgroups of ecrt and ert were vs vs for radiation pneumonitis vs vs for radiation esophagitis vs vs for leukopenia vs vs for neutropeniaand vs vs for anemias combined endostatin with rt or ccrt is effective and well tolerated in treating lansclc and lesstoxicities occur further validation through prospective randomized control trials is requiredkeywords chemoradiotherapy endostatin nonsmall cell lung cancer radiotherapy correspondence ma_jt126comdepartment of oncology shengjing hospital of china medical universityshenyang china the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0czhang radiation oncology page of atthe time ofintroductionlung cancer is the most common cancer type worldwide and nonsmall cell lung cancer nsclc is the mostcommon form “initialdiagnosis approximately onethird of patients withnsclc present with locally advanced nsclc lansclc furthermore about of lansclcsare unresectable and chemoradiotherapy crt was therecommended standard care for these patients [ ] nosignificant progress in the treatment of lansclc wasmade for many years until the pacific study confirmedthat consolidation therapy with durvalumab a monoclonal antibody that blocks interactions of programmed celldeath ligand with the pd1 receptor further improvedsurvival following crt [“]previous studies indicated that a hypoxic tumor microenvironment contributes not only to resistance of tumorcells to chemoradiation but also promotes metastasis and tumor oxygenation is essential for effective application of radiotherapy rt or crt thereforenovel treatments that enhance radiosensitivity by improving the hypoxic microenvironment are urgentlyneeded prior to the findings of the pacific study researchers explored whether patients with lansclccould benefit from antiangiogenic drugs combined withrt or crt however earlier studies showed that administration of bevacizumab along with thoracic rt ledto a high incidence of pulmonary toxicity including radiation pneumonitis hemoptysis and tracheoesophagealfistulaein patients with stage iii nsclc [ ]therefore concurrent bevacizumab with thoracic rt isunlikely to be further pursued as a treatment option forstage iii nsclcpreclinical studies have demonstrated that endostatina broadspectrum angiogenesis inhibitoris able tonormalize tumor vasculature alleviate hypoxia and increase tumor sensitivity to radiation [ ] severalstudies have indicated enhanced efficacy and tolerabletoxicity of endostatin combined with thoracic rt orcrt for patients with lansclc [“] howeverthe reported studies to date are mostly retrospective orsingle arm studies with limited patient enrolment in thepresent study we performed a pooled analysis to assessthe clinical efficacy and safety of endostatin combinedwith rt or concurrent chemoradiotherapy ccrt inpatients with lansclcmaterials and methodssearch strategywe conducted a systematic search for available sboth in published and forms of pubmed ovidweb of sci embase google scholar cochranelibrary chinese national knowledge infrastructure andwanfang databases the finalliterature search wasperformed on june using the following searchterms œlung cancer and endostatin or endostarand radiotherapy or radiation therapy or chemoradiotherapy manual updates of s presented tillthe meetings such as american society of clinicaloncology european society for medical oncologyworld conference of lung cancer and americansociety for therapeutic radiology and oncology wereadditionally performedstudy selection and search strategystudies that met the following inclusion criteria were included in the pooled analysis prospective or retrospective including singlearm studies that evaluated theefficacy and safety of endostatin plus radiotherapy ertor concurrent chemoradiotherapy ecrt in patientswith lansclc studies with primary outcomesreporting at least one of the following endpoints objective response rate orr progressionfree survival pfsand overall survival os and local control rates lcror adverse events aes based on common terminologycriteria for adverse events version or numberof cases included for study was ‰¥ s or s were written in english after the selectionprocess the remaining titles and s were screenedfor relevance independently by two authors fulltext s and meeting s were finally reviewed for allstudies that met the inclusion criteriadata extraction and quality assessmentdata were extracted independently by two reviewersaccording to the inclusion criteria discrepancies wereresolved by discussing with a third reviewer each reviewer extracted data including author name the publication years of the studies number of patients patientcharacteristics treatment regimen radiotherapy dosagethe method of endostatin administration orr pfs oslcr and aes the jadad scale and newcastleottawa scale were used to assess the quality of theincluded studiesstatistical analysisstatistical analyses were conducted using comprehensive metaanalysis version software biostat incnj usa for dichotomous variables such as os ratespfs rates orr lcr and aes we calculated the rawproportion of events divided by the total number ofclinically evaluable patients additionally we calculatedweighted pooled rates of events by the number of clinically evaluable patients using a random effects model toaccount for heterogeneity in study size and the large variations in proportion median pooled weighted os andpfs were calculated with descriptive statistics subgroup 0czhang radiation oncology page of analysis was performed per type of treatment regimenert or ecrtpublication bias and sensitivity analysisthe potentialfor publication bias in reported orrvalues was assessed by funnel plots with the appropriateaccuracy intervals sensitivity analyses were performedfor the results for orr based on the leaveoneoutapproachresultsliterature searchfigure depicts a flowchart of the literature searchprocedure overall records were identified using thesearch strategy and records excluded after screeningthe titles and s among the remaining potentially relevantfour were excluded due toendostatin administration via arterial infusion or discontinuation of endostatin in the first cycle during rtfinally seven studies [ “]involving patients were pooled for analysisstudiesincluded studies and patient characteristicsthe characteristics of the selected studies are summarized in table the included studies comprised threeprospective cohort studies three singlearm prospectivestudies and one singlearm retrospective study followup data were available for five studies with a medianfollowup period between and months in total evaluable patients in four studies received endostatincombined with ccrt ecrt and evaluable patientsin three studies received endostatin combined withsingle rt ert patients received a total dose of “gy in “ fractions for “ weeks howeverthemethods of endostatin treatment differed among studiesincluding continuous intravenous pumping civ ofendostatin mgm2day over days administrationof endostatin mgm2day over h for days atweeks and or via an endostatin intravenous dripiv mgday for days per weeks etc almost allincluded patients had unresectable lansclc at thetime of study entry the median patient age ranged from to yearspooled orr and lcrpooled orr and lcr data are summarized in table the pooled overall orr for the seven studies was i2 confidence intervalfig 2a ci “ i2 fig 2b inthe ert group and ci “ i2 fig 2c in the ecrt group higher orr was observedin the ert group compared with the rt alone group vs respectively[ci] “only two studies in which the treatment regimenswere ecrt and ert reported lcr data the pooled and 2year lcr rates were ci “i2 fig 2d and ci “ i2 fig 2e respectivelyfig overview of study search and selection 0czhang radiation oncology page of table characteristics of the included studiesstudystudy typeendpointspublishedyearjiang zhai sun bao tang prospectivecohort studysinglearmprospectivestudysinglearmprospectivestudysinglearmprospectivestudysinglearmretrospectivestudyno ofcases 2yr os rate 2yr lcr osorr aes 3yr pfsosrate pfsosorr aestreatmentregimenertradiationdose gyecrt“orr pfs os aesecrt“os 3yrpfsos rate andlcr pfs orr aesecrt“pfs os orrecrt“wen chen prospectivecohort studyprospectivecohort studyorr pfs 1yros rateertorr pfs os aesert““endostatin usage mgday iv for days during thefirst week of rttotal duration ofendostatin days× cycles mgm2day civ for days beforethe beginning of rt and then repeatedat week and during rt mgm2day iv for days per weeks during rt days× cycles days× cycles mgm2day iv for days before thebeginning of rt and then repeated atweek and during rt mgm2day iv over h per day for days or civ for days at week and endostatin administrated week prior to crt mgday iv during the first threeweeks of rt mgday iv for days per threeweeks during rt day× cycles days× cycles days× cycles day× cyclesos overall survival pfs progressionfree survival orr objective response rate lcr local control rate aes adverse events ert endostatin combined withradiotherapy ecrt endostatin combined with concurrent chemoradiotherapy yr year rt radiotherapy iv intravenous injection civ continuousintravenous pumpingpooled survivalthe pooled survival data are summarized in table only two studies in ecrt group reported pfs ratesthe pooled and 3year pfs rates were ci “ i2 fig 3a ci “ i2 fig 3b and ci “i2 fig 3c respectivelyfour studies documented the 1year os rate three the2year os rate and two the year os rate the overallpooled and 2year os rates were ci“ i2 fig 4a and ci“ i2 fig 4b respectively based onstratification by treatment regimens the pooled and 3year os rates in the ecrt group were ci “ i2 fig 4c ci“ i2 fig 4d and ci “ i2 fig 4e the pooled 1year os rate in theert group was ci “ i2 six of the included studies had recorded median pfsvalues patients received ecrt in four of these studiesand ert in the remaining two studies with only threeof the above studies recording both the pfs value and ci accordingly pooled median pfs was calculatedby a weighted average of the single study median the pooled median pfs was recorded as monthsoverall months in the ecrt group and months in the ert groupos data and ci were reported in four studiesthe overall pooled median os was months ci “ i2 months ci “ i2 in the ecrt group and months ci “ i2 in the ert groupradiationsafetythe most common aes documented in the five selectedstudies including patients were radiation pneumonitisandanemia additionally nauseavomiting neutropenia andleukopenia were three commonly observed aes in threeofthe above four studies pooled data on aes aresummarized in table thrombocytopeniaesophagitisradiation pneumonitis and esophagitisthe pooled frequencies of any grade and grade ‰¥ radiation pneumonitis were and overall and in the ecrt group and and in theert group respectively the pooled frequencies of anygrade and grade ‰¥ radiation esophagitis were and overall and in the ecrt group and and in the ert group respectivelyhematological toxicitymore than of grade ‰¥ hematological toxicities inall patients were neutropenia leukopenia and anemiawith incidences of and respectivelythe pooled rates were vs vs and vs respectivelyin the ecrt and ertgroups rates of thrombocytopenia of grade ‰¥ were and for all patients ecrt and ert groupsrespectively 0czhang radiation oncology page of table pooled efficacy of endostatin combined with radiotherapy or chemoradiotherapyendpointsno of casesresponse rateno of studiesgrouporr 1yr lcr 2yr lcr progressionfree survivalmedian pfs months1yr pfs rate 2yr pfs rate 3yr pfs rate overall survivalmedian os months1yr os rate 2yr os rate 3yr os rate overallecrtertoveralloveralloverallecrtertecrtecrtecrtoverallecrtertoverallecrtertoverallecrtecrtweighted pooled data 95ci “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “os overall survival pfs progressionfree survival orr objective response rate lcr local control rate ert endostatin combined with radiotherapy ecrt endostatincombined with concurrent chemoradiotherapy yr yearother toxicitiesseveral other toxicitiesincluding nausea arrhythmiafatigue hemorrhage and hypertension were additionallyreported table all of above aes incidences ofgrade ‰¥ were less than for either all patients or forany of subgroups only one study reported ae ofhypertension in which patients received ecrt with afrequency of in any grade and in grade ‰¥ respectivelypublication bias and sensitivity analysispublication bias was assessed for orr according tobegg™s test and no significant publication bias wasobserved fig besides results of sensitivity analysisby omitting one study at a time did not substantiallychange the overall resultsdiscussioncrt plus consolidation durvalumab is now consideredstandard of care for inoperable stage iii nsclc but theoptimalthe sequence andimmunotherapy and even anticombination of crttreatmentstrategiesforangiogenic therapy are still being studied although datafrom prospective phase iii randomized control studiesevaluating the efficacy and safety of endostatin combinedwith rt or ccrt for patients with lansclc arelacking our pooled analysis indicates that endostatincombined with ccrt or rt presents a promising treatment modality in treatment of lansclc subgroups ofecrt and ert have similar efficacy and survival benefitbut patients in the ert subgroup had lower rates oftoxicitysince tumor angiogenesis has been identified as acritical step in growth and metastasis of malignant solidtumors antiangiogenesis strategies have become established as an effective therapeutic approach [“]vascular endothelial growth factor vegf a specificand potent angiogenic factor contributes to the development of solid tumors by promoting angiogenesis severalantivegf or antivegfreceptor vegfr strategieshave been developed to dateincluding neutralizingantibodies to vegfvegfr soluble vegfrvegfrhybrids and receptor tyrosine kinase inhibitors [“]chemotherapy combined with antiangiogenic drugs 0czhang radiation oncology page of fig pooled orr for all patients a ert b and ecrt c groups pooled lcr for all patients 1year lcr d and 2year lcr e orr objectiveresponse rates ert endostatin combined with radiotherapy alone ecrt endostatin combined with concurrent chemoradiotherapy lcr localcontrol rates[“] including bevacizumab a vegfa monoclonalantibody recombinant human endostatin and ramucirumab a vegfr monoclonal antibody has led tosignificantly prolonged survival compared with chemotherapy alone and is currently approved by the usfood and drug administration fda andor chinafda for first or secondline treatment of advancednsclcsolid tumors generally have characteristics of hypoxiaand exhibit resistance to radiation to some extentleading to failure of local control therefore attempts toincrease the sensitivity of rt via tumor oxygen enrichment present a novel direction for research [ ]one of the most common factors causing hypoxia isinadequate vascular supply of the tumor and thus sufficient blood vessel supply in the tumor microenvironment may be essential to improve the tumor radiationresponse for patients treated via rt recombinanthuman endostatin is an endogenous broadspectrumangiogenesis inhibitor produced by proteolytic cleavageof collagen xviii that is suggested to interfere with theproangiogenic action of growth factors such as basicfibroblast growth factor and vegf preclinical studieshave shown that recombinant human endostatin couldtransiently œnormalize the tumor vasculature to enhance efficiency of oxygen delivery and sensitivity to radiation treatment [ ] our pooled data indicate thatcombination of endostatin and rt with or withoutchemotherapy leads to better response rate local controlrate and survival demonstrating superior short andlongterm survival benefits which are not inferior to theresults of previous randomized controlled trials rctsof ccrt summarized in table [ “]although rtog trial showed a superior medianos of months patients in this study had stageiiia disease in contrast more than patientsin our pooled analysis had stage iiib disease whichmay be one ofthe factors contributing to survivaldifferences in a phase ii trial involving unresectable stage iiib patients endostatin combined withccrt resulted in a median os of months ineach of the rcts listed in table over of patientshad a performance status ps score of however inour pooled analysis only of patients had a psscore of in a phase ii trial involving only of patients with a ps score of endostatin combined withccrt resulted in median pfs and os of monthsand months respectively 0czhang radiation oncology page of fig pooled pfs rates for ecrt group 1year a 2year b and 3year pfs rates c pfs progressionfree survival ecrt endostatin combinedwith concurrent chemoradiotherapyrecently the pacific study conducted in patientswith unresectable stage iii nsclc showed a significantsurvival advantage with durvalumab consolidation therapy after ccrt achieving a 3year os rate of inthe durvalumab group versus in the controlgroup based on this study national comprehensivecancer network guidelines have recommended this regimen as standard treatment for unresectable stage iiinsclc however the optimal sequence and combination of crtrt and immunotherapy are being studied results from several phase ii trials such as thedeterred and etop nicolas studies have indicated that concurrent crt with checkpoint inhibitorsicis atezolizumabnivolumab for the treatment of advanced nsclc might be feasible and has no significantadded toxicities over historical rates [ ] currentlymany ongoing phase iiiii clinical trials such as pacific2 nct03519971 keynote799 nct03631784ea5181 nct04092283 checkmate73l nct04026 etc are evaluating the optimal treatment strategiesof immunotherapy“radiotherapy combinationsalthough ccrt plays an indispensable role in thetreatment of unresectable stage iii nsclc some patients especially the elderly or those with poor performance status who cannot tolerate toxicity induced bychemotherapy have to receive sequential crt or evenrt alone [ ] our pooled analysis indicated thatpatients treated with endostatin in combination with rtalone have comparable pfs vs months os vs184 months and orr vs to thoseadministered endostatin with ccrt in addition pooledorr data from the three prospective cohort studies 0czhang radiation oncology page of fig pooled 1year a and 2year os rates b for overall patients pooled os rates for the ecrt group 1year c 2year d and 3year e osrate os overall survival ecrt endostatin combined with concurrent chemoradiotherapyshowed that patients subjected to endostatin combinedwith rt had higher orr vs comparedwith the rt alone patient group therefore combinationtherapy of rt and endostatin may be a promising strategy for lansclc patients with poor ps who cannottolerate chemotherapyof note the duration and intervals of endostatin andradiotherapy combinations differed in clinical trials andmay affect the outcomes as shown in table resultsfrom preclinical studies showed that endostatin treatment could transiently normalize the tumor vasculatureby reducing microvessel density and increasing pericyticcoverage of the vessel endothelium thereby providing atime window about week to enhance the sensitivityto rtthus rt delivery in this period resulted inmaximal antitumor outcomes [ ] ct perfusionimaging and hypoxia imaging suggested that the œtimewindow was within about week after administrationduring which endostatin improved blood perfusion anddecreased hypoxia of lung cancer these studiesprovide an important experimental basis for combiningendostatin with radiotherapy within the time window of days range “ after endostatin administration inaddition given the short halflife of endostatin in vivociv is considered a better delivery route to maintain asteady concentration and may improve its efficacy [“] a recent study which compared the outcomesof two phase ii trials that involved different administration routes of endostatin combined with ccrt showedthat endostatin at mgm224 h civ for daysachieved higher and 5year os rates andsafety than endostatin at mgm2day iv for daystherefore administration of mgm224h civ for days per weeks from week prert to the end of rtcould be a preferred scheme on the basis of the currentstudies however the optimal duration and intervals ofendostatin administration require further investigationin our pooled analysis we observed that grade ‰¥ aesin the ecrt group were similar to those caused byccrt reported previously summarized in table indicating that addition of endostatin to ccrt did notobviously increase the main aes the pooled incidencesof grade ‰¥ radiation pneumonitis and radiation esophagitis were and respectively analogous toprevious findings importantly compared with the ecrtgroup significantly lower rates of grade ‰¥ aes were observed in the ert group such as radiation pneumonitis vs radiation esophagitis vs nauseavomiting vs thrombocytopenia vs neutropenia vs anemia vs and leukopenia vs our pooled analysis has severallimitations firstlyfour in seven included studies belonged to singlearmtrial and lacked a comparative control group and 0czhang radiation oncology page of table pooled adverse events of endostatin combined with radiotherapy or chemoradiotherapyeventsgradeincidence cioverall “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “nrnrnrnrecrt group “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ “ert group “ “ “ “ “ “ “ “ “ “nrnrnrnrradiation pneumonitisradiation esophagitisneutropenialeukopeniaanemiathrombocytopenianauseavomitingarrhythmiafatiguehemorrhagehypertensionall‰¥all‰¥all‰¥all‰¥all‰¥all‰¥all‰¥all‰¥all‰¥all‰¥all‰¥ert endostatin combined with radiotherapy ecrt endostatin combined with concurrent chemoradiotherapy nr not reportedfig funnel plot of publication bias for orr orr objective response rates 0czhang radiation oncology page of table the efficacy of concurrent chemoradiotherapy in previously reported phase iiiii randomized controlled trialslcr study1yrnrpfs rate 2yr1yrnrnros rate 1yrmosmonthsmpfsmonthsnrorrrtog crt regimen2yrnumberrt vprtog proclaim cams wjog5008l rt epldr pchdr pcldr pc cethdr pc cetrt pprt eprt eprt pcrt sprt vpnrnrnrnrnrnrnrnrnrnrnrnrnrnrnrnrnrnr2yrnrnrnrnrnrnroverallnrnrnrnrnrnrnrnrcrt chemoradiotherapy rt radiotherapy ldr low dose radiation hdr high dose radiation vp vinblastine plus cisplatin ep etoposide plus cisplatin pc paclitaxelplus carboplatin cet cetuximab pp pemetrexed plus cisplatin sp s1 plus cisplatin nr not reported pfs progressionfree survival mpfs median progressionfreesurvival os overall survival mos median overall survival orr objective response rate lcr local control rate yr yearanother three of the studies were prospective cohort trials with a comparative control group they were of nonrandom design and lacked sufficient data to facilitateeffective analysis secondly heterogeneity of the doseregimen or endostatin usage between studies was nottaken into consideration resulting in unstable mergedfindings thirdlythatendostatin combined with rt alone is comparable toendostatin with ccrt in terms of orr lcr andsurvival however the differences in efficacy and safetybetween the two treatment methods remain to be established further welldesigned prospective randomizedthe currentsuggestresultscontrolled clinical trials are warranted to reach definitivesincreasing interest has emerged in studying the feasibility of combined radiotherapy antiangiogenic agentsand icis current evidence suggests that antiangiogenicagents have the potential for increasing the response toimmunotherapy by modulating the tumor microenvironment tme the impower150 study identified thesynergic effect of antiangiogenic agents plus immunotherapy in which patients in the atezolizumab plusbevacizumab and paclitaxelcarboplatin abcp groupachieved survival advantage over those in the bevacizumab plus paclitaxelcarboplatin bcp group similarlytable adverse events of concurrent chemoradiotherapy in previously reported phase iiiii randomized controlled trialsstudycrt regimenleukopeniaallnr‰¥neutropeniaallnr‰¥nrthrombocytopeniaallnr‰¥anemiaallnrradiationpneumonitis ‰¥‰¥all nrradiationesophagitis ‰¥allnrnrnrnrnrnrnrnrnr nrnrrtog rt vprt eprtog ldr pchdr pcnrldr pc cet hdr pc cet proclaim rt ppcams rt eprt eprt pcwjog5008l rt sprt vpcrt chemoradiotherapy rt radiotherapy ldr low dose radiation hdr high dose radiation vp vinblastine plus cisplatin ep etoposide plus cisplatin pc paclitaxelplus carboplatin cet cetuximab pp pemetrexed plus cisplatin sp s1 plus cisplatin nr not reported 0czhang radiation oncology page of preclinical study showed that endostatin plus antipd1also exerted a synergic effect on tumor growth in murinemodels of lewis lung carcinoma by improving the tmeand inducing autophagy an ongoing clinical trialnct04094909 is investigating the efficacy and safety ofendostatin combined with chemotherapy and pembrolizumab as firstline therapy in patients with advanced ormetastatic nsclc despite the lack of clinical trials involving the combination therapy of endostatin icis andrtcrt the synergic effect between endostatin andicisrt will provide a potential way to improve clinicalbenefits for these patients when compared with currentstandard treatmentbased on this pooled data analysis adding recombinanthuman endostatin to radiotherapy or concurrent chemoradiotherapy is an effective and less toxic method for thetreatment of patients with unresectable lansclc wesuggest that concurrent administration of endostatin andcrt or rt presents a promising treatment approach forsome patients in the era when crt plus durvalumab hasbecome the current standard of care for patients whocannot tolerate ccrt and icis endostatin combinedwith rt alone may be a good alternative but for thosepatients who can tolerate ccrt but cannot tolerateicis addition of endostatin to ccrt may become amore effective treatment strategy highquality prospective studies are needed to validate this suggestion giventhe synergistic antitumor effect of antiangiogenic agentsand rticis on lung cancer triple or quadruple combination therapy of endostatin icis and rtcrt forpatients with inoperable stage iii nsclc might becomea potential strategy in the future however multiplechallenges regarding this combination remain to beaddressed before it can be applied to clinical practiceabbreviationsaes adverse events ccrt concurrent chemoradiotherapy ci confidenceinterval civ continuous intravenous pumping crt chemoradiotherapyecrt endostatin plus concurrent chemoradiotherapy ert endostatin plusradiotherapy fda food and drug administration icis immune checkpointinhibitors iv intravenous injection lansclc locally advanced nonsmallcell lung cancer lcr local control rate nsclc nonsmall cell lung cancerorr objective response rate os overall survival pfs progressionfreesurvival ps performance status rcts randomized controlled trialsrt radiotherapy tme tumor microenvironment vegf vascular endothelialgrowth factor vegfr vascular endothelial growth factor receptoracknowledgmentsnoneauthors™ contributionsall authors read and approved the final manuscript prior to submission chand jm conceived and designed the project sz ls and lh performed theproject sz analyzed the data and wrote the paper jm was the seniorauthor who oversaw the projectfundingthis study was supported by grants from the talent project of shengjinghospitalavailability of data and materialsthe authors declare that all data generated or analyzed during this study areincluded in this ethics approval and consent to participateall analyses were based on previously published studies and hence noethical approval and patient consent were requiredconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived november accepted august referencesbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “ wistuba ii gelovani jg jacoby jj davis se herbst rs methodological andpractical challenges for personalized cancer therapies nat rev clin oncol“yang p allen ms aubry mc wampfler ja marks rs edell es clinicalfeatures of primary lung cancer patients experience at mayo clinicfrom to chest “auperin a le pechoux c rolland e curran wj furuse k fournel p metaanalysis of concomitant versus sequentialradiochemotherapy in locally advanced nonsmallcell lung cancer jclin oncol “curran wj jr paulus r langer cj komaki r lee js hauser s sequential vs concurrent chemoradiation for stage iii nonsmall cell lungcancer randomized phase iii trial rtog j natl cancer inst “criss sd mooradian mj sheehan df zubiri l lumish ma gainor jf costeffectiveness and budgetary consequence analysis of durvalumabconsolidation therapy vs no consolidation therapy afterchemoradiotherapy in stage iii nonsmall cell lung cancer in the contextof the us health care system jama oncol “uemura t hida t durvalumab showed long and durable effects afterchemoradiotherapy in stage iii nonsmall cell lung cancer results of thepacific study j thorac dis 201810suppl 9s1108“s12antonia sj villegas a daniel d vicente d murakami s hui r overallsurvival with durvalumab after chemoradiotherapy in stage iii nsclc nengl j med “tomadasu i dasu a karlsson m the relationship between temporalvariation of hypoxia polarographic measurements and predictions oftumour response to radiation phys med biol “seo y yan t schupp je colussi v taylor kl kinsella tj differentialradiosensitization in dna mismatch repairproficient and deficient humancolon cancer xenografts with 5iodo2pyrimidinone2²deoxyribose clincancer res “ ansiaux r baudelet c jordan bf crokar

" fasassociated factor faf1 has been implicated in parkinson™s disease pd and activates the celldeath machinery in the cytosol however the presence of extracellular faf1 has not been studiedmethods serumfree conditioned medium cm from faf1transfected shsy5y cells was concentrated andanalyzed by western blotting exosomes were isolated from cm by ultracentrifugation and analyzed by westernblotting electron microscopy and nanop tracking analysis soluble faf1 from cm was immunodepleted usingantifaf1 antibody transmission of secreted faf1 was examined by transwell assay under a confocal microscopecminduced cell death was determined by measuring propidium iodide pi uptake using a flow cytometerresults faf1 was secreted from shsy5y cells via exocytosis and brefeldin a bfaresistant secretory pathwaysfurthermore faf1 was secreted as a vesiclefree form and a genuine exosome cargo in the lumen of exosomes inaddition faf1 increased the number of exosomes suggesting a regulatory role in exosome biogenesis extracellularfaf1 was transmitted via endocytosis to neighboring cells where it induced cell death through apoptotic andnecrotic pathwayss this study presents a novel route by which faf1 induces neuronal death through celltocelltransmissionkeywords faf1 secretion exosome vesiclefree form celltocell transmission cell death cells secrete proteins harboring signal peptides throughthe classical secretory pathway via the endoplasmicreticulum ergolgi complex from which vesicles withcargo proteins move toward and fuse with the plasmamembrane and subsequently export cargos to the extracellular space however proteins lacking signal peptides are secreted via alternative nonclassical secretorypathways these pathways are classified as vesicularand nonvesicular secretory pathways some proteins correspondence eunheecnuackr gyeongrin park and bokseok kim are considered cofirst authorsdepartment of biological sciences chungnam national university daehakro yuseonggu daejeon south koreaare secreted via extracellular vesicles including exosomesand other vesicles of various sizes [ ] alternativelyother proteins are secreted through membrane poresand atpbinding cassette abc transporters althoughthe exact mechanisms of nonvesicular secretory pathways are elusive [ ]exosomes which are nanosized membrane vesicles “ nm in diameter secreted into the extracellular environment by various cell types are associated with intercellular communication with neighboring cells and play arole in a myriad of pathological functions in diseases including cancer cardiovascular and neurodegenerative diseasestheextracellular matrix and promote metastasis angiogenesisin particular exosomes[“]remodel the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cpark cell communication and signaling page of thrombosis and tumor cell proliferation in cancer exosomes in cardiovascular disease display proangiogenesis procoagulant and proinflammatory effects on thevessel walls in neurodegenerative diseases exosomesare potential sources of key pathogenic proteins such astau amyloid prion and αsynuclein [“]inflammation triggersin addition to their vesiclemediated secretion manyproteins are secreted through nonvesicular mechanismsfor instance the proteins such as tau and fibroblastgrowth factor are recruited to the plasma membrane toform lipidic pores and are subsequently secreted [ ]in additionthe secretion ofinterleukin1 and transglutaminase through pores fibroblast growth factor translocates across the plasmamembrane via poorly understood mechanisms includingthe membrane release complex upon stress proteinssuch as hydrophilic acylated surface protein b are secretedby abc transporters proteins secreted via nonvesicular secretory pathways are advantageous over cargo proteins in vesicles as immunotherapeutic targets because ofthe antibody accessibility of the extracellular spacefasassociated factor faf1 is involved in diverse biochemical processes including cell death inflammation cellproliferation and proteostasis [“] consistent with itsdiverse functions faf1 has been implicated in certain diseases first faf1 plays a tumor suppressive rolethrough activation of the apoptotic machinery and nfκbsuppression [ ] faf1 also suppresses tumor metastasis via tgf signaling moreover faf1 expressionis downregulated in various cancers including lung colonliver prostate brain ovarian and breast cancers second faf1 is involved in parkinson™s disease pd as it iscolocalizes with αoverexpressed in pd patientssynuclein and acts as a substrate of parkin furthermorefaf1 mediates the degeneration of dopaminergic neuronsthrough apoptosis and parthanatos [“]faf1 is an intracellular protein present mainly in thecytosol to date faf1 secretion has not yet been reportedherein we uncovered for the first time that faf1 is secreted via an ergolgiindependent pathway specificallyfaf1 is secreted through exosomal and nonvesicular pathways in shsy5y cells in addition faf1 augments thenumber of exosomes suggesting the involvement of faf1in exosome biogenesis furthermore extracellular faf1moves into neighboring cells via pinocytosis and clathrinmediated endocytosis transmitted faf1 induces cell deathvia apoptosis and necrosis collectivelythese resultspresent a novel measure by which faf1 propagates itsdeath function through celltocell transmissionmethodsreagents and antibodiesthe following reagents and antibodies used in this studywere purchased commercially tnfα from abfrontierseoul south korea zietdfmk caspase8 inhibitormouse antiha antibody and rabbit antigm130 antibody from abcam cambridge uk ²²dichlorofluorescin diacetate dcfhda hydrogen peroxide h2o21methyl4phenylpyridinium mpp propidium iodidepi polydlysine brefeldin a bfa gw4869 monensin cycloheximide chx necrostatin1 nec1 dpqproteinase k pk dynasore heparin heparinase iiimouse antiactin and mouse antiflag antibody fromsigmaaldrich saint louis mo usa ²6diamidino2phenylindole dapi horseradish peroxidase hrpconjugated antimouse antibody and hrpconjugatedantirabbit antibody from thermo fisher scientific incrockford il usa mouse antiflotillin1 from bd biosciences san jose ca usa bafilomycin a1 mouseantialix antibody mouse antigalactosidase antibody401a rabbit anticalregulin antibody mouse anticd63 antibody mouse antiparkin antibody and mouseantifaf1 antibody from santa cruz biotechnologydallas tx usa mouse antihsc70 antibody andmouse antihsp90 antibody from enzo life sciencesfarmingdale ny usa and zvadfmk zvad fromcalbiochem darmstadt germanycell culture and transfectionshsy5y mef hek293 raw2647 hela panc1mia paca2 and mcf7 cells were maintained in dulbecco™s modified eagle™s medium dmem welgenedaegu korea containing fetal bovine serum fbsatlas biologicals fort collins co usa and antibioticantimycotic gibco brl grand island neusa unless otherwise specified cells were transfectedwith the indicated plasmids using bio t manvillescientific inc manville nj usa following the manufacturer™s protocol rat midbrain cultures derived frompostnatal day were prepared using standard procedures briefly material dissected form the ventral portion of the midbrain was cleaned free of meningealtissue minced and enzymatically dissociated in a mixture of papaindnase sigmaaldrich dissociated cellswere plated onto aminecoated 6well plates bd biosciences cells were maintained in neurobasal mediumgibco with b27 serumfree supplements gibco mm lglutamine after days of culture the cells wereinfected using aav1 adenoassociated virus 1hfaf1viral vectors moi of and sitedirected mutagenesisthe sirnaresistant parkin construct was generatedusing quikchange sitedirected mutagenesis kit stratagene la jolla ca usa the primers were as follows²gagctgagaaacgactggactgtgcagaattgtg3²²gggaaggagctgagaaacgattgcactgtgcaga ² 0cpark cell communication and signaling page of rna interferenceall small interfering rnas sirnas against parkin andscrambled rna scrna were purchased from bioneerdaejeon south korea the sequences of the sirnasused in this study were as follows sirna against parkin²ugaggaaugggacugu3² thescrna orsirna were transfected into shsy5y cells using lipofectamine rnai max thermo fisher scientific according to the manufacturer™s instructionspreparation of conditioned mediumto prepare conditioned medium cm cells transfectedwith the indicated plasmids were cultured in mmdiameter dishes in dmem containing fbs after h the cells were switched to serumfree dmem forthe indicated times here we used serumfree mediumto avoid interference from albuminenriched fbs thenthe cm was collected and centrifuged at ¨¯g for min and ¨¯g for min to remove cellular debrisfor western blot analysis the cm was concentratedusing kda or kda cutoff amicon ultra filtersmillipore billerica ma usa at ¨¯g for min or minwestern blot analysiscells were harvested washed twice with pbs and lysedwith mammalian lysis buffer [ mm triscl ph mm nacl mm edta nonidet p40 mmphenylmethylsulfonylfluoride] then the protein concentrations were quantified by using a biorad proteinassay kit biorad hercules ca usa after quantification samples were boiled in × protein sample buffer[ mm triscl ph glycerol sds mercaptoethanol] then samples were electrophoresedby sdspage and transferred to nitrocellulose membranes ge healthcare maidstone uk the membranes were blocked with skim milk in pbs with tween20 pbst and incubated with the indicatedprimary antibodies overnight after their washing withpbst the membranes were incubated with secondaryantibodies immunoblot signals were measured by usingchemiluminescent detection lab frontier anyangkoreachx chase assayshsy5y cells were transiently transfected with the indicated plasmids for h after transfection the cells wereswitched to serumfree dmem containing chx sigmaaldrich μgml for the indicated of times subsequently the medium was concentrated with kda cutoff amicon ultra filters millipore and analyzed bywestern blot analysispurification of exosomeswe followed a previously described protocol with somemodifications briefly cells were transfected withthe indicated plasmids for h and incubated in dmemcontaining exosomedepleted fbs system biosciences palo alto ca usa for h the cm was thencollected and subjected to sequential centrifugation at¨¯g for min ¨¯g for min and ¨¯g for min at °c to remove cellular debris using a beckmancoulter optima l90 k ultracentrifuge with a type 41tirotor the supernatant was then spun down at ¨¯gfor min the pellet was resuspended in pbs and thenspun again at ¨¯g for min at °c finally thepellet was resuspended in pbs or radioimmunoprecipitation assay ripa buffer sigmaaldrichin addition to their isolation via ultracentrifugationwe isolated exosomes using exoquicktc system biosciences according to the manufacturer™s instructionstreatment of vesicles with na2co3to separate integral membrane proteins and luminalproteins purified exosomes were treated with mmna2co3 ph for min at °c as previously described after centrifugation at ¨¯g for minintegral proteins remained in the pellet fraction whileluminal proteins remained in the supernatant fractionthe pellet fractions were resuspended in ripa buffersigmaaldrich and the supernatants were collected ina separate tube for western blot analysisproteinase k digestionpk sigmaaldrich was added to the samples at a finalconcentration of μgml then the samples were incubated at °c for min and mm phenylmethylsulfonyl fluoride was added to inhibit the activation of pkfollowed by the addition of protein sample bufferimmunodepletion of cmfifty microliters of protein gconjugated dynabeadsthermofisher scientific was incubated overnight withmouse monoclonal antibody against faf1 final concentration of or μgml before its addition to cm theantibodydynabeads complex was incubated with mlof cm at °c overnight after the complex was removedusing a magnet the immunodepleted cm was concentrated using a kda cutoff amicon ultra filter andused for western blot analysis for flow cytometryunconcentrated immunodepleted cm was applied to recipient cellsflow cytometryto evaluate cell death we measured pipositive cellstaining by using a guava easycyte flow cytometermillipore briefly cells were switched to serumfree 0cpark cell communication and signaling page of dmem or neurobasal medium for the indicated timeadditionally to measure recipient cell death shsy5yand rat primary neuronal cells were treated with cmfrom donor cells for the indicated times the cells were μgml and evaluatedharvested stained with piusing a guava easycyte flow cytometer following whichthe results were quantified using incyte softwaremilliporeconcentrated cm treatmentshsy5y cells donor cells were plated on mm tissueculture dishes and transfected with gfpvector or gfpfaf1 plasmid at h after transfection the cells wereincubated in serumfree medium for h after the cmwas concentrated by using kda cutoff amicon ultrafiltersthe concentrated cm was dissolved in newserumfree medium that was applied to recipient cellson polyllysinecoated coverslips in 12well plates for hpropagation assayshsy5y cells donor cells were plated on mm tissueculture dishes donor cells were transfected with gfpvector or gfpfaf1 plasmids at h after transfectionthe cells were collected and replated in cell culture inserts polycarbonate membrane μm pore size corning kennebunk me usa at a density of ¨¯ cellsshsy5y cells recipient cells were plated at a densityof ¨¯ cells on polydlysinecoated coverslips in well plates after h the cultures were combined suchthat the donor cells were in the insert and separatedfrom recipient cells plated on a coverslipconfocal microscopycells were fixed with paraformaldehyde for minthe cisgolgi were stained with gm130 after the nucleiwere stained with dapi for min the coverslips weremounted onto microscope slides using fluorescencemounting medium dako carpinteria ca usa andanalyzed using a zeiss lsm laser scanning confocalmicroscope carl zeiss oberkochen germanyelectron microscopyfor transmission electron microscopy tem sampleswere prepared using the exosometemeasy kit containing a formvarcarboncoated em mesh gridwash buffer and em solution bio mountain viewca usa the pellets from the ml of cm vector orfaf1transfected cells obtained by ultracentrifugationwere resuspended in μl of pbs μl of which was applied to the grid all samples were prepared followingthe manufacturer™s instructions for immunoem thepellets were first fixed with μl of paraformaldehyde and glutaraldehyde sigmaaldrich overnightat °c then the fixed exosome solution was transferredto grids and subsequently treated with m glycinefor min to quench free aldehyde groups after blocking with pbs containing bsa for min the gridswere incubated for h with the indicated antibodies diluted in pbs containing bsa atroomtemperature after three washes with pbs containing bsa the grids were incubated for h with the secondary antibody antimouse igg conjugated to nmgoldroomtemperature three washes to eliminate secondary antibody were followed by incubation with em solution anda wash step samples were viewed under a talos f200xtransmission electron microscope fei hillsboro orusa operated at kv and images were capturedwith a ceta m pixel cmos camera feisigmaaldrichatpsnanop tracking analysisfollowing isolation by differential ultracentrifugation orexoquicktc system biosciences the exosome pelletswere resuspended in μl of pbs then μl of theexosome solution was diluted in pbs to a total volumeof ml the samples were analyzed by nanoptracking analysis using a nanosight ns300 malvernpanalytical ltd malvern uk equipped with a nmlaser to accurately identify the vesicles the detectionthreshold was set at the number of vesicles in eachsample represents the number of ps per ml ofmedium cells were counted using a muse count viability kit millipore on a muse cell analyzer milliporecaspase3 activity assayshsy5y cells were treated with cm from faf1transfected cells plus caspase8 inhibitor or tnfα abfrontier plus chx sigmaaldrich at the indicated concentrations for the indicated times then caspase3 activity was measured by using a caspase3 colorimetricassay kit biovision milpitas ca usa in accordancewith the manufacturer™s protocol the absorbance at nm was measured with the use of a victor microplate reader perkinelmer norwalk ct usasignalp41we investigated the presence of a signal peptide in faf1using signalp41 httpwwwcbsdtudkservicessignalp41 with secretogranin1 used as a positive controlas it contains a signal peptidestatistical analysesexperiments were independently carried out three timesn all the data are expressed as the mean ± standarddeviation sd statistical comparisons were performedusing student™s ttest or oneway analysis of varianceanova followed by tukey™s hsd post hoc analysis 0cpark cell communication and signaling page of using spss software statistics version ibm incchicago il usa statistical significance was established when the pvalue was lower than resultsfaf1 is secreted via nonclassical exocytosisaccording to the csf proteome resource faf1 is detected in the cerebrospinal fluid and plasma additionalfile this study aimed to determine whether faf1 isalso secreted at the cellular level and investigate faf1secretion in shsy5y human neuroblastoma cellsbecause faf1 is a deathpromoting protein sublethal experimental faf1 transfection conditions wereused to exclude cellular debris due to death additionalfile fig s1a and b the faf1 transfection conditionsin shsy5y cells under which faf1 secretion but notcell death occurs were determined by pi stainingfollowed by flow cytometry cells were transfected with3xflagfaf1 plasmid at a sublethal dose for h andsubsequently moved to serumfree medium faf1 wasdetected in the serumfree medium at h and accumulated henceforth indicating that faf1 is secreted in atimedependent manner fig 1a this finding suggeststhat faf1 is constitutively released from shsy5y cellsto exclude a tagging artifact another epitope taghemagglutinin ha was used hafaf1 was alsodetected in the cm conditioned medium in a dosedependent manner additional file fig s1c furthermore we examined the faf1 secretion with rat primaryneurons using aav1 adenoassociated virus 1mediated faf1 overexpression faf1 overexpression in ratprimary neuronal cells demonstrated consistent resultsin shsy5y cells fig 1b moreover with thatgalactosidase was not detected in the cm of galactosidasetransfected cells demonstrating that therelease of faf1 is not a transfection artifact fig 1c next a pulsechase experiment using chx to inhibit de novo protein synthesis was performed consistently faf1 was secreted and accumulated over timefurther confirming that faf1 is constitutively secretedfrom shsy5y cells fig 1d next we examinedwhether faf1 is also secreted by other cells faf1 wasdetected in the extracellular space of mefs and hek293cells furthermore faf1 was present in the cm of eachof a number of various cancer cell types mcf7 helapanc1 and mia paca2 cells this shows that faf1secretion is notadditional file fig s2afspecific to shsy5y cellsbecause faf1 has been implicated in pd pathogenesisfaf1transfected shsy5y cells were treated with thepdassociated stressors such as mpp h2o2 bafilomycin a1 and αsynuclein overexpression at sublethal dosesadditional file fig s3 there were no significantchanges of faf1 expression in clslysatescelldependent on various pdassociated stressortypeshowever faf1 secretion increased in cms upon allstressors used in this study implying that pdassociatedstressors positively regulate faf1 secretion fig 1eto elucidate the mechanism by which faf1 is secreted two sets of experiments were performed as follows first we examined whether faf1 is released viaexocytosis or passive diffusion as exocytosis is affectedby temperature faf1transfected shsy5y cellswere incubated at either °c or °c faf1 secretionat °c was significantly reduced compared to that at °cindicating that faf1 is released via exocytosisfig 1f second we examined whether faf1 is secretedvia the classical ergolgidependent secretory pathwayusing bfa which generates ros and disassembles golgithrough ergolgi pathway inhibition bfa did not affectfaf1 release fig 1g additional file fig s1d furthermore a signal peptide was not found in faf1when its sequence was analyzed using singalp41 furtherexcluding the possibility of ergolgimediated secretionof faf1 additionalfile fig s4 taken togetherthese data demonstrate that faf1 is released via nonclassical exocytosisfaf1 is secreted via exosomal and nonvesicular pathwaysto determine the mechanism by which faf1 is secreted exosomes were isolated from cm using a differential ultracentrifugation procedure as previouslydescribed and exoquicktc following the manufacturer™s protocol western blot analysis of exosomesisolated by ultracentrifugation revealed the presenceofthe exosome markers alix cd63 hsc70 andhsp90 but not calregulin an er resident protein a negative exosome control fig 2a exosomes isolated from shsy5y cells by exoquicktc showed anexosome marker profile consistent with that of exosomes purified by ultracentrifugation additional file fig s5a moreover nanop tracking analysisand tem data further confirmed that our purifiedexosomes exhibited a typical size distribution with adiameter ranging from to nm and a typicalmorphology of exosomes fig 2b and cendogenous as well as overexpressed faf1 proteinswere detected in the exosomal fractions isolated by bothmethods indicating that faf1 is secreted as a genuineexosomal cargo fig 2a similarly faf1 was also foundin exosomes isolated from the various indicated cell linesby exoquicktc additional file fig s5bh next weinvestigated whether faf1 is present on the membraneor in the lumen of exosomes exosomes were treatedwith mm na2co3 ph to distinguish betweenthe exosomal membrane and the lumen both alix andflotillin1 were present in the exosomal membrane positive controls whereas faf1 was mainly present in the 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is secreted via nonclassical exocytosis a shsy5y cells were transfected with vector control vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium for the indicated times se short exposure le long exposure b ratprimary neuronal cells were transduced with aav1hfaf1 viral vectors at days after transduction the culture medium was replaced withserumfree neurobasal medium for h c cells were transfected with lacz or 3xflagfaf1 plasmid at h after transfection the culture mediumwas replaced with serumfree medium and cells were cultured for h d cells were transfected with vc or 3xflagfaf1 plasmid at h aftertransfection the culture medium was replaced with serumfree medium containing chx μgml for the indicated times e left panel cellswere transfected with vc or 3xflagfaf1 plus αsyn plasmid at h after transfection the culture medium was replaced with serumfreemedium containing dmso vehicle mpp mm h2o2 μm or baf a1 nm for h right panel the graph shows the densitometricanalysis of immunoblotting of faf1 in conditioned medium cm shown in the left panel n f upper panel cells were transfected with3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium and the cells were cultured at °cor °c for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting in cm shown in the upper paneln g upper panel cells were transfected with 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with serumfree medium containing bfa μgml for h lower panel the graph shows the result of densitometric analysis of faf1 immunoblotting incm shown in the upper panel n cell lysate cl and concentrated cm were analyzed by western blotting with the indicated antibodies alllanes were loaded with the same amount of total protein the data are expressed as the mean ± sd of three independent experimentsstatistical comparisons were performed using using anova followed by tukey™s hsd post hoc analysis e and student™s ttest f and g p p p and ns not significanttheexosomeabolishedstructureslumen of the exosomes fig 2d the topology of exosomal faf1 was further examined using pk a nonspecificprotease to digest proteins outside of the exosomesexosomal faf1 but not cytosolic faf1 was protectedfrom pk treatment in the absence of triton x100 txfig 2e ˆ’tx in contrast pk treatment with tx disintegratingtheexosomemediated protection of faf1 fig 2e txour data demonstrated the presence of faf1 in thelumen of exosomes furthermore the presence of faf1in theconfirmed with theimmunogoldlabeled faf1 under anvisualization ofelectron microscope as shown in fig 2f immunogoldlabeled cd63 was mainly present outside exosomeswhereas immunogoldlabeled faf1 was present in thelumen of exosomes collectively these results consistently showed that faf1 is located in the lumen ofexosomeslumen wasexosomalbecause certain exosomal cargo proteins are alsosecreted via the nonvesicular secretory pathway [“] the possibility that faf1 is also secreted viaa nonvesicular route was investigated to this endthe cm was fractionated into exosomal pellet andnonexosomal supernatant fractions by ultracentrifugation faf1 from the cm was present in nonexosomal as well as exosomalfractions fig 2gimplying the presence of a soluble form of faf1 tofurther investigate this cm from faf1transfectedshsy5y cells was treated with antifaf1 antibodyone microgram of antifaf1 antibody almost eliminated faf1 from the cmindicating that faf1 ispredominantly secreted in a soluble form fig 2htaken together these results demonstrate that faf1is concurrently released as a bona fide cargo of exosomes and in a soluble form this new finding addsfaf1 to the list of proteins secreted by nonvesicularas well as exosomal routesrespectivelytransfection offaf1 positively regulates exosome numberthe exosomal cargos such as hsp20 hsp90 andstat3 increase exosome number [“] here weexamined whether faf1 also participates in regulating exosome number the exosome markers hsc70alix and cd63 were increased by ± 009fold ± 013fold and ± 022foldinthe cm of faf1transfected shsy5y cells compared with control cells fig 3a additional file fig s6a next we further studied exosome numberchanges using sirnamediated depletion of parkina e3ubiquitin ligase of faf1 siparkin treatment elevated secretion as well as expression of faf1 inshsy5y cells moreoversirnaresistant parkin construct reverted the increased secretion and expression of faf1 by siparkin in shsy5y cells the expression levels of alix and cd63in the cm of shsy5y cells in which parkin hadbeen depleted were elevated by ± and ± 040fold respectively fig 3b collectivelythese data imply that faf1 positively controls exosome number for the direct quantification of exosome number nanop tracking analysis wasapplied the vesicle size distribution profile showinga diverse range of sizes showed that exosomes werepresent predominantly fig 3c the exosome numbers were normalized by cell number additionalfile fig s6b the exosomes of faf1transfectedcells were increased by 25fold compared to thoseof control cells hence these data robustly demonstrate that faf1 augments exosome numberinaddition gw4869 an exosome release inhibitor interfered with the ability of faf1 to increase exosome number while monensin an exosome releasepromoter enhanced this ability implying that faf1functions upstream of the exosome release processfig 3a [ ] 0cpark cell communication and signaling page of fig see legend on next page 0cpark cell communication and signaling page of see figure on previous pagefig faf1 is released to the extracellular space via both exosomal and nonvesicular pathways a shsy5y cells plated on mm dishes weretransfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replaced with exosomedepleted medium andthe cells were cultured for h then exosomes were isolated from the cm by ultracentrifugation cl and isolated exosomes exos wereanalyzed by western blotting with the indicated antibodies calr calregulin b purified exosomes were characterized using nanop trackinganalysis c representative tem images of exosomes are shown scale bar nm left or nm right d the purified exosomes were treatedwith na2co3 after centrifugation at ¨¯g the integral membrane proteins were pelleted memb and nonintegral and luminal proteinsremained in the supernatant sol these fractions were analyzed by western blotting with the indicated antibodies e the purified exosomeswere incubated with pk μgml in the absence or presence of triton x100 tx tx with tx ˆ’tx without tx f immunogold labelingof purified exosomes with anticd63 antibodies left and antifaf1 antibodies from vctransfected middle or 3xflagfaf1transfected rightcells scale bar nm g cells were transfected with vc or 3xflagfaf1 plasmid at h after transfection the culture medium was replacedwith serumfree medium and the cells were cultured for h after the cm was isolated by ultracentrifugation the supernatant sup and pelletexo were analyzed by western blot with the indicated antibodies h after immunoprecipitation of faf1 from cm with antifaf1 monoclonalantibody the immunodepleted cm was concentrated and analyzed by western blotting with the indicated antibodies all lanes were loaded withthe same amount of total proteinsecreted faf1 is transmitted to adjacent cellswe wondered whether extracellular faf1 is internalizedby neighboring cells donor cells were transfected withgfp or gfpfaf1 plasmid for h subsequently thecm which contained gfp or gfpfaf1 was concentrated and nontransfected recipient cells were treatedwith the concentrated cm for h confocal microscopic images showed the presence of gfpfaf1 in thecytoplasm of recipient cells indicating that gfpfaf1had moved from donor cells to recipient cells in contrast gfp from the cm of donor cells was not detectedin recipient cells excluding the effect of tagging ontransmission fig 4a similarly donor cells were transfected with 3xflagfaf1 plasmid as described abovedonor cellderived faf1 also moved into recipient cellsas shown by western blotting fig 4b corroborating theimmunofluorescence results in fig 4a in these data consistently demonstrate that secreted faf1can be internalized by neighboring cellsto further validate the celltocell transfer of faf1 anin vitro coculture system in which donor cells expressinggfpfaf1 were incubated in upper transwellinsertswhile nontransfected recipient cells were incubated inlower compartments was used consistent with theabove data confocal microscopy of recipient cells revealed the presence of gfpfaf1 indicating that gfpfaf1 moved from cells in the upper transwell inserts tocells in the lower compartments fig 4c consequentlythese results show that extracellular faf1 was transferred to neighboring cells without celltocell contactwe investigated the type of extracellular faf1 thatmoves into recipient cells to this end donor cells weretreated with gw4869 an exosome release inhibitorafter which recipient cells were analyzed by confocal microscopy gw4869 failed to inhibit faf1 transmissionto recipient cells to eliminate vesiclefree faf1 cm ofdonor cells was immunodepleted wit

"tea is the second most popular beverage consumed in theworld next to water green tea is a kind of nonfermentedtea produced from the plant camellia sinensis it is favoredby people for its fresh flavor and health benefits and consumed worldwide especially in east asian countriesgreen tea contains caï¬eine and polyphenolic compoundsknown as catechins catechins are the primary bioactivesubstances and present significant biological propertiestea leaves™ drycatechins constitute up to ofweight among that egcg is the main and the most abundant catechin [ ] egcg has been traditionally regardedas beneficial mainly ascribed to its antioxidant action the antioxidant eï¬ects of egcg are manifested in scavenging free radicals in the body and inhibiting the formation ofros the results of earlier studies suggested that egcgcould decrease the risk of several human disorders associatedwith oxidative stress on the other hand egcg also displays significantprooxidant eï¬ects usually under highdose conditions theprooxidant actions of egcg play a dual role being both beneficial and harmful while achieving desired outcomes inchronic disease prevention and treatment reports about thetoxicity of egcg are also emerging a growing body ofevidence continues to demonstrate a variety of harmfuleï¬ects from excessive consumption of green tea or oraladministration of highdose egcg supplement highdoses of egcg not only cause cytotoxicity in vitro but alsoresult in living body hepatotoxicity nephrotoxicity andgastrointestinal disorders vomiting and diarrhea the oral bioavailability of egcg is not so profound inhealthy humans as it was only of the total ingestion most of the ingested egcg is absorbed in the smallintestine and substantially degraded in the large intestine bymicrobiota action the eï¬ective dosage of egcg mightbe close to or higher than the toxic dosage in practical applications considering its low bioavailability therefore it is 0coxidative medicine and cellular longevitynecessary to understand the potential toxicity doses andusage of egcg in this review the prooxidant eï¬ects ofegcg in health benefits and adverse eï¬ects were discussedespecially concerning their underlying mechanisms involvedand doses used this review is aimed at harnessing the prooxidant eï¬ects of egcg for human health maintenance whileavoiding toxicity thereby better guiding the safety consumption of green tea and egcg chemical structure andautooxidation of egcgbasic catechins contain two or more aromatic ringshydroxyl group on carbon three position andor the higherdegree of hydroxylation of the b ring would be primarilyresponsible for the potent antioxidant activities of catechinsfigure 1a previous structureactivity relationshipstudies of catechins have demonstrated the importance ofthe number and location of the phenolic hydroxyl groupson antioxidative capacity egcg has the remarkablepotential to scavenge radicals and chelate metal ion theseabilities could be ascribed to the presence of dihydroxyand trihydroxy groups in a ring b ring and d ringfigure 1b the catechol structure of egcg makes it susceptible todegradation via autooxidation figure under normal°physiological conditions ph c egcg is autooxidized and converted to oquinone through nonenzymaticaldehydrogenation of phenolic hydroxyl groups at b ring when the cell culture medium is exposed in the airegcg could be oxidized by oxygen and yields superoxide andanion radicals o2‹…egcg are essential intermediate products in egcg autoox and oxygen could function as oxidants for furidation o2ther oxidation of egcg finally resulting in the formationof oquinone accompanying the generation of hydrogen could also form substantial amountsperoxide h2o2 o2of h2o2 via disproportionation reaction one egcgmolecule could produce more than two h2o2 molecules inphosphate buï¬er at neutral ph and egcg radicals ‹…egcg o2autooxidation of egcg generates substantial ros theros comprises singlet oxygen hydroxyl radicals superoxideperoxides and h2o2 h2o2 is in a dominant position andusually is regarded as a toxic agent when the ros levelexceeds cellular antioxidant capacity oxidative stress willoccur in other words this is the result of an imbalancebetween prooxidant and antioxidant eï¬ects inclusion ofantioxidant defense enzymes such as catalase cat andsuperoxide dismutase sod could minimize h2o2 levelwhich is essential to maintain the redox balancethe concentration of egcg in the cell environmentseems to be a primary factor in explaining its prooxidanteï¬ects for example egcg treatment alone diminisheddna strand breakage of human blood lymphocytes at lowconcentrations μm while it induced dna strandbreakage at higher concentration μm thusegcg acts as an eï¬ective antioxidant at low doses withinthe range of high nanomolar and low micromolar levelswhile egcg represents a prooxidant at high doses howeverthis blurred boundary might vary depending on the type ofradical stimulants cellular environment and duration ofexposure to egcg health benefitsuntil now egcg has been a major research subject withinthe field of healthpromoting eï¬ects the potential role ofthe prooxidant eï¬ects of egcg in cancer and obesity prevention and treatment as well as the antibacterial actionsachieved demonstrable results in previous studies prooxidant eï¬ects and anticancer activity of egcgcancer is one of the most common and lifethreateningdiseases occurring among humankind egcg as a naturalproduct has drawn a great deal of attention from boththe scientific community and the general public indeedegcg has shown both prophylactic and therapeutic efficacy in multiple human cancers several mechanisms havebeen proposed to accountfor the inhibitory action ofegcg against cancer formation and growth the prooxidant eï¬ects of egcg were thought to be potential mechanisms for anticancer action the anticancer mechanismsvaried depending on the cell type dose andor time oftreatment table [“]apoptosis is the bestdescribed form of programmed celldeath the induction of apoptosis represents a universal andideal therapeutic strategy for cancer control cell apoptosiscould be triggered by either the intrinsic mitochondrial pathway or the external death receptor pathway the mitochondrial pathway could be induced by intracellularstresses such as oxidative stressthe apoptosistriggering eï¬ects of ros have beennoted in vitro table egcg inhibited cell growth ina dosedependent manner and the decrease in the numberof viable cells was mainly due to apoptosis caused by theegcginduced intracellular ros as early as the last century scientists found that egcg induced h2o2 formationin human lung cancer celllines h661 and 21bes andexogenously added cat completely prevented egcginduced cell apoptosis which suggested that h2o2 isinvolved in the apoptosis process provoked by egcg similar actions were also found in various cancersand tumor cells table thioredoxin trx and thioredoxin reductase trxr are pivotal regulators of cellularredox homeostasis decreased trxtrxr activity mightcontribute to the increased ros level high concentrationof egcg inactivated trxtrxr via the formation of egcgtrx1 and egcgtrxr conjugates which was linked to theelevation of ros level in hela cells and further promotedcancer cell death moreover one of the biochemical hallmarks of apoptosis is genomic dna fragmentation chen performed the dna fragmentation assay in theskov3 cells indicating that egcg induced apoptosis bycausing dna damage this result was consistent withother studies in ovarian and cervical cancer cells [ ]in terms of molecular mechanisms intrinsic apoptosisleads to the release of mitochondrial cytochrome c afterbeing released into the cytoplasm cytochrome c stimulates 0coxidative medicine and cellular longevityohbohohohhoohbohohohohdohocoaohobhoocaohafigure a basic structure of catechins b chemical structure of egcgohbegcgohohautooxidationph75 °cohboautooxidation·egcgoh“o2h2o2ohboooquinonefigure superoxidemediated chain reaction the formation of oquinoneapoptosome formation followed by activation of caspasecascades egcgmediated mitochondrial ros couldpromote cytochrome c release to the cytosol the antiproliferative action of egcg on prostate cancer and breast cancer is mediated through apoptosis as evident from caspase9[ ] the cells susceptible to egcginduced apoptosisalso showed activation of caspase3 moreover theincreased ros level was observed to result in the stimulationof mitogenactivated protein kinase mapk themapk signaling pathwayincluding extracellular signalregulated kinase erk jun nterminal kinase jnks andp38 plays a vital contribution in cell proliferation diï¬erentiation apoptosis and stress response egcg induced oxidative stress via generation of ros and thereafter activatedthe jnk pathway leading to changes in mitochondrial membrane potential and release of cytochrome c in ht29 humancolon adenocarcinoma cells and mia paca2 pancreaticcancer cells [ ] together these results suggest thategcginduced apoptosis is mediated through ros generation and might subsequently activate the cell intrinsic pathway in the presence of transition metals such as copper andiron h2o2 could convert to a potent oxidant hydroxyl radical via the fenton reaction nakagawa found that egcg μm produced h2o2 and triggered fenton reactionto form highly toxic hydroxyl radicals which resulted in lymphoblastic leukemia jurkat cell death in the presence offeiii and cuii egcg μm induced dna damagein hl60 cells as 8oxo78dihydro2²deoxyguanosine oxodg content increased which was a characteristic ofoxidative dna damage nevertheless no significantincrease in 8oxodg was observed in h2o2resistant colonhp100 cells suggesting that h2o2 was involved in cellulardna damage egcg could inhibit cell proliferation andinduce apoptosis through cellular dna breakage in diï¬erentcancer cell lines such dna breakage involved the mobilization of endogenous copper ions and the generation ofros moreover the observation of site specificity of dnadamage by egcg is valuable cuiimediated dna damageby egcg occurred most frequently at t and g residues egcg was able to mobilize endogenous copper ions andgenerate hydroxyl radicals in situ hydroxyl radicalsserved as the proximal dna cleaving agentleading todna breakage in the nuclei this result was possibly due tothe interaction of egcg with chromatinbound copper ionsand then the nondiï¬usible hydroxyl radicals were formed atthe binding site hence hydroxyl radical generated nearbydna was well established as the cause of strand scissionbecause the concentration of copper is significantly very highin various malignancies egcg could induce cancer celldeath through the metal iondependent pathway thispathway was independent of mitochondriamediated programmed cell deaths such action involved in metal ionmediated dna cleavage would be an important mechanismof anticancer properties of egcgin addition to being transported into the cell egcgcould also function on the cell membrane fraction to regulatethe surface growth factor receptor earlier studies foundthat autooxidation of egcg led to epidermal growth factorreceptor egfr inactivation in human esophageal cancer 0ccell linesbladder cancernbtiibreast cancermcf7mcf7cervical cancerhelahelacolon canceroxidative medicine and cellular longevitytable role of prooxidant eï¬ects in the anticancer activity of egcg based on cell culture studiesegcgconcentrationtimebiological eï¬ectsreferences μm hinduced early apoptosis through dna damage μgml hinduced cell growth inhibition and apoptosis by downregulating survivinexpression via suppressing the akt pathway and activating caspase9 μm hinduced apoptosis at low doses via activation of jnk caspase9 and caspase3while inducing necrosis at high doses which is related to diï¬erences in rosgeneration and atp levels μm μm and h hincreased cell death through dna damageinduced cell death through generation of ros and inactivation of trxtrxrhct116 μm hinduced apoptosis through induction of ros and epigenetic modulation ofapoptosisrelated gene expressionht29 μm hendometrial carcinomaishikawa μm hinduced apoptotic cell death via activating the jnk pathway accompanyingmitochondrial transmembrane potential transition and cytochrome c releaseic50 was μminduced apoptosis via ros generation and p38 map kinase activationic50 was μmesophageal cancerkyse lung cancer μm hinactivated egfr by superoxide generated from autooxidation of egcg μm μm h h h μm hdisplayed strong growth inhibitory eï¬ects against lung tumor cell linesinhibited cell growth through induction of ros ic50 was μmic50 was μminduced apoptosis via h2o2 production and hydroxyl radical formationinduced apoptosis by modifying the redox systemh661 and h1299 μmh1299lymphoblastic leukemiajurkatmyelomaim9 rpmi8226and u266oral cancerscc25 andscc9ovarian cancer μm hreduced cell viability by inducing mitochondrialocalized ros and decreasingsirt3 expressionskov3 μgml dpancreatic cancerpanc1 μm hmia paca2 μm hinhibited cell proliferation and induced apoptosis by inhibiting cell cycle arrest andinducing dna damageinduced apoptosis through generation of ros as well as caspase3 andcaspase9 activationinduced stress signals by damaging mitochondria and rosmediatedjnk activationprimary eï¬usionlymphomabcbl1 and bcprostate cancer μgml hinduced apoptosis and autophagy through ros generationpc3 and μm hreduced cell survival and increased apoptosis caused a significant alteration incaspase9 alternative splicing 0coxidative medicine and cellular longevitycell line kyse one possible explanation is thath2o2 produced from egcg autooxidation in the cell culturemedium could attack and inactivate egfr leading to theinhibition of egfr phosphorylationand preferentialit is worth considering whether high amounts of egcgcould cause damage to normal cells egcgmediated rosproduction was particularly observed in cancer cells compared with normal cells the selectivity of egcginducedapoptosis in cancer cells might be due to the diï¬erentialinducibility of rosexpression ofapoptosisrelated genes moreover tao found thategcg induced diï¬erential mitochondrial dysfunction andoxidative stress in normal and oral cancer cells these eï¬ectswere related to the diï¬erential modulation of sirtuin sirt3 and its downstream targets including glutathionegsh and sod considering the cytotoxicity of egcgin normal cells the ic50 value in normal cells was checkedand showed to be more than μm while that for thecorresponding cancer cells was μm these resultssuggested that cancer cells are more sensitive to egcg thannormal cells and ros might be selectively toxic to cancercellsin addition to being used as preventive agents individually egcg could also be used as adjuvant therapies generally cooperative interaction of two or more agents couldtarget more signaling pathways thus eï¬ectively improvingagent chemosensitivity reducing untoward eï¬ects of treatment expanding the scope of action and showing highertherapeutic outcomes drug resistance is a dauntingchallenge in cancers prooxidant activities of egcg wereproposed to contribute to overcoming drug resistancehighlighted by the fact that h2o2 production induced byegcg increased the potency of cisplatin in ovarian cancercells by three to sixfold in contrast cisplatin alone washighly resistant to the treatment in some cancer cell linescopper transporter ctr1 is a critical determinant toincrease cisplatin uptake egcg could upregulate ctr1expression through the stimulation of ros simultaneous treatment of arsenic trioxide ato with egcgshowed oxidativemediated induction of apoptosis in leukemia cancer cells egcg acted as a prooxidant andincreased intracellular h2o2 and atoinduced hemeoxygenase1 ho1 provided ferrous iron to increase thefenton reaction in both cases cellular oxidative damageeventually occurredin general under typical cell culture conditions egcghas been known to generate i extracellular ros via autooxidative reaction in the cell culture medium ii ros in cellular mitochondria and iii intracellular ros through thefenton reaction upon cell entry figure these three pathways contribute diï¬erently to cancer cells but eventuallycause cell damage and death cancer initiation and progression are generally divided into several stages when egcgacts as an antioxidant it might more eï¬ectively enhance antioxidant capacity at the cancer prevention stage whereaswhen egcg acts as a prooxidant it might be more criticalat suppressing tumor growth stage one possible suppositionis that tumor cells may be more susceptible to oxidativestress because their increased growth rate and metabolismcause a heightened basal ros level the degree of cell proliferation and diï¬erentiation seems to be one factor aï¬ectingthe ros production ability of egcg future research willbe required to determine if egcg is a much more potentros inducer in diï¬erentiated than in undiï¬erentiated cancercells although a limited amount of data has shown that theseprooxidant eï¬ects can occur in vivo it is essential to understand when and to what extent the antioxidant or prooxidanteï¬ects of egcg are working in diï¬erent stages of cancers inanimal models prooxidant and antiobesity eï¬ects of egcg obesity is ametabolic disease characterized by abnormal or excessive fataccumulation it is generally associated with an increased riskof chronic diseases including diabetes hypertension anddyslipidemia a large and growing body of studies hasinvestigated the antiobesity eï¬ects of egcg in cellular andanimal experiments and the underlying mechanismsthe clinical manifestations of obesity are adipocytehyperplasia and hypertrophy in vitro studies have well demonstrated that egcg could inhibit adipocyte growth andinduce adipocyte death through its prooxidant eï¬ects hung reported that high concentrations of egcg μm reduced the cell viability of preadipocytes by induced the appearance of dna fragmentation andincreased the activity of the apoptotic enzyme caspase3 egcg was demonstrated to raise ros level anddescend gsh level in preadipocytes and adipocytes whichinduced oxidative stress thus resulting in decreased cell number ²ampregulated protein kinase ampk represents ametabolitesensing protein kinase hwang foundthat the release of ros by egcg stimulation could furtheractivate ampk rapidly in 3t3l1 adipocytes a recent studyalso proved that ampk was activated by exogenous h2o2and this activation was not through direct redox signalingto ampk but was a secondary consequence of redox eï¬ectson other processes egcg activates ampk via the generation of ros subsequently blocks anabolic pathways and promotes the catabolicpathway and suppresses gluconeogenesis and adipogenesisconsequently leading to body weight reduction and metabolic syndrome alleviation figure the activation ofampk modulates the expression of genes and proteinsinvolved in lipid metabolism downregulates the expressionof fat synthesis proteins and upregulates lipid catabolismproteins it was shown that egcg inhibited the expressions of glucose 6phosphatase g6pase for gluconeogenesis phosphoenolpyruvate carboxykinaseforgluconeogenesis fatty acid synthase fas for fatty acid synthesis acetylcoa carboxylase acc for fatty acid synthesis hydroxymethylglutarylcoa reductase hmgrforregulatory elementbinding proteinscholesterolsrebpsfor sterol synthesis peroxisome proliferatoractivated receptor gamma pparγ for lipid synthesis andstorage and ccaatenhancerbinding protein alphacebpα for adipogenesis as well as enhanced the expression of acylcoa oxidase aco for fatty acid oxidationperoxisome proliferatoractivated receptor alpha pparαpepcksterol 0coxidative medicine and cellular longevityautooxidationrosegcgcellrosfe2cu2fentonreaction·ohegfrcytochrome ccell damagecell deathcaspase9caspase3cell culture mediumfigure prooxidant eï¬ects of egcg in cell cultureegcggeneraterosactivateampkmodulateg6pase pepck fasacc hmgr srebpsppar𝛾 cebp𝛼aco ppar𝛼 cpt1acad pgc1𝛼ucps atglfat synthesislipid catabolismantiobesityfigure eï¬ects of egcg on lipid metabolism via ros and ampkfor fatty acid oxidation carnitine palmitoyltransferase1cpt1 for fatty acid oxidation acylcoa dehydrogenaseacad for fatty acid oxidation peroxisome proliferatoractivated receptor gamma coactivator1α pgc1α for fattyacid oxidation uncoupling proteins ucps for thermogenesis and adipose triglyceride lipase atgl for triglyceridehydrolysis [“]accordingly the prooxidant eï¬ects of egcg play avital role in preventing the initiation and progression ofobesity egcg could cause oxidative stress thus damagingadipocyte directly and activating ampk and then aï¬ectingrelative genes and protein expression and signal transduction in various tissues indirectly however the increase ofoxidative stress in fat accumulation might be an importantpathogenic mechanism of obesityrelated metabolic syndrome such as diabetes firm conclusions as to whetherprooxidant eï¬ects of egcg could perform on body weightbody fat and adipose weight in humans will require morethorough clinical studies prooxidant and antibacterial eï¬ects of egcg egcgexhibits a broad spectrum of bactericidal activity against various bacteria its bactericidal eï¬ects include damage to thebacterial cell membrane and inhibition of fatty acid synthesisand enzymatic activity h2o2 which is generated byegcg appears to play an indispensable role in the bactericidal actions of egcg the bactericidal action of egcgwas related to h2o2 level as bactericidal action was inhibitedby the increase of cat concentration egcg was foundto have bactericidal activity at higher concentrations in thesalmonella assay highly correlated with h2o2 production egcg showed a dosedependent “ μm inhibition on escherichia coli e coli op50 strain growth this inhibitory action was associated with a profoundincrease in intracellular oxidative stress caused by egcghence the use of egcg as a prooxidant is well supportedby these studiesegcg was shown to have broad antibacterial spectrumeï¬ects on both grampositive and gramnegative bacterianevertheless egcg might function through diï¬erent mechanisms against grampositive and gramnegative bacteriaintracellular ros level was determined by flow cytometrythe results indicated that damage on gramnegative e colicell walls was induced by egcg depending on h2o2 release 0coxidative medicine and cellular longevity in contrast the damages on grampositive staphylococcus aureus resulted from a combination between egcg andpeptidoglycan layer because the outer membrane ofgramnegative bacteria was mainly composed of negativelycharged lipopolysaccharides which could resist the destruction of egcg they are less susceptible to egcg thangrampositive bacteria bacterial cell membrane damage not only prevents thebinding of bacteria to host cells but also inhibits the abilityof the bacteria to combine with each other and form biofilms egcg was known to attack the lipid bilayer of bacterialcell membranes leading to physical disruption of the membrane as for the cell walls results from atomic forcemicroscopy suggested that the subminimum inhibitory concentrations of egcg treatment mgl to e colio157h7 strains could lead to temporary changes in the cellwalls cui such changes were due to the damagecaused by h2o2 generated from egcg moreover egcgcaused cell membrane damage via increased intracellularros level and led to potassium leakage these are potentiallyconducive to the antibiofilm efficacy of egcg against vibriomimicus which is a foodborne pathogen in seafood andwater in addition egcg also regulates the expression of oxidative stressrelated genes oxyr and soxrs systems are activated upon exposure to oxidative stress oxyr induces katgand soxrs induce soda strongly when cells are stressed byexogenous h2o2 egcg treatment upregulated katgand soda expression in e coli these results verified the roleof ros in egcgmediated bacterial inhibition the cpxsystem is thought to control protein homeostasis in the cellenvelope when e coli was exposed to egcg apoptosis happened because of ros formation by the cpx system rpos is a general stress regulator in response to oxidativestress egcg could cause a reduction in the expression forrpos indicating that egcg induced oxidative stress in bacterium models the potential prooxidant properties of egcg could beattributedin part to its suppressive eï¬ects on bacteriamore broadly research is also needed to determine relativesignaling pathways and proteomic factors egcg is superexcellent natural products it could increase the efficacy ofbactericidal eï¬ect when it aids other fungicides morerecent attention has been focused on the impact of greentea and green tea polyphenols on the intestinal microflorawhether egcg intervention would change the diversity ofmicrobiota and lead to microbiota death is also in need offurther investigation adverse effectsin recent years egcg has become one of the most aggressively promoted food supplement products in daily lifeegcg entered the market and its safety has raised queriesthe prooxidant eï¬ect of egcg is not necessarily advantageous they might have implications regarding potential toxicity hence it is necessary to systematically explore theharmful eï¬ects of egcg and the mechanisms prooxidant and hepatotoxicity eï¬ects of egcg a considerable amount of literature has been published on hepatotoxicity of green teaderived products it is noteworthythat the hepatotoxicity of green tea and its derived productswas initially found in some diet products in after beingthe cause of liver injury in subjects france and spain governments have suspended the marketing of exolise whichwas a weightloss phytotherapeutical drug in the pasttwo decades reports on liver disorders caused by green teaingestion with overdose of egcg content have graduallyemerged the liver is a major drug metabolic organ in the bodythe bioavailability of egcg in rats was determined after min of oral administration mgkg by detecting theconcentration of egcg in plasma and diï¬erent tissuesincluding the liver the results showed that the concentrationof egcg in the liver μmolkg was four times higherthan in that in the blood plasma μmolkg moreover utilizing anatomy egcg could trigger liver damagewhereas no visible abnormalities were found in other tissuesand organs [ ] hence it could be preliminarily concluded that the liver is the toxic target organ of egcgat the cellular level egcg demonstrated cytotoxic eï¬ectin cultured rat hepatocytes it was shown that μm egcgtreatment on freshly isolated rat hepatocytes caused timedependent cytotoxicity the hepatocyte was incubatedwith egcg for h resulting in liver cell function reduceddose dependently the decrease of lactate dehydrogenase ldh a marker of cell membrane damage wasobserved in rat hepatocytes egcg also caused damageto the outer mitochondrial membrane by the fact that mitochondrial membrane potential collapsed in animal experiments table the severity of egcginduced toxicity is relevant with dose route of administration and period of treatment [ “] biochemicaland histopathological analysis showed that liver samples ofmice displayed diï¬erent degrees of liver injury liver functionindexes of plasma alanine aminotransferase alt andaspartate aminotransferase ast activity increased in adosedependent mannermalondialdehyde mda and 4hydroxynonenal hne are final products of lipid peroxidation present biochemical markers of oxidative stress metallothionein mtand γhistone 2ax γh2ax are molecular markers of oxidative stress oral high dose of egcg mgkgd to cf mice for two days significantly enhanced the formation ofmda in the liver and elevated the expression of hepaticmt and γh2ax protein and increased positive staining for4hne in liver samples intraperitoneal administrationof egcg or mgkgd for five days raised serum hne level and western blot analysis showed that hepaticγh2ax was markedly increased all these biomarkersillustrated that egcgtriggered hepatotoxicity in vivo wasinduced by oxidative stressprevious pharmacological studies have shown that undernormal physiological conditions egcg is metabolizedthrough methylation sulfation and glucuronidation andthen excreted in urine subsequently whereas at toxicdoses these pathways might be saturated and the excessive 0canimal typefemale swissalbino micemalekunmingmiceegcgdosagemgkgd andmalekunmingmice and and male nd4micemale cf1micewistar rats ofboth sexesmale cd1micemicefemaleswisswebster miceoxidative medicine and cellular longevitytable hepatotoxicity of egcg based on animal modelsroute ofadministrationdurationresultsreferenceip and po dip treatment increased serum bilirubin markers po treatment didnot show any dosedependent changes except alt marker dtolerable dose of egcg was mgkg for ip and mgkg foripipigigpo d dserum alt ast 4hne il2 il6 and il10 and hepatic γh2axwere raised hepatic nrf2target gene expression was increasedthe fatality rate was single doseserum alt ast 4hne il6 and il10 and hepatic γh2ax wereraised hepatic nuclear and cytosolic nrf2 proteins were suppressed d dmouse growth was not aï¬ected the dosage was considered asmaximum tolerable dosehepatotoxicity occurred major hepatic antioxidant enzymes weresuppressed nrf2mediated rescue response was inducedsingle dosemice died in a dosedependent manner andthe nrf2 pathway was not activated nrf2 and its target genes were h dsuppressedalt was slightly increased histopathology of the liver showedcongestion of sinusoids and central and portal veinssingle dosealt was markedly increased histopathology of the liver showeddegenerative hepatocytes and a small number of vacuoles d dmouse survival was reduced by mouse survival was reduced by hepatic mda mt and γh2axwere increasedsingle dosealt was increased by 108fold mouse survival was reduced by egcg2²cysteine and egcg2³cysteine were detected in theurineposingle dosemice were lethargic and their respiration was labored and andipipipsingle doseplasma alt was increased mice died within h h degcg thiol conjugates egcg2²cysteinyl and egcg2³cysteinyl were detected in the urine of mice died plasma alt activity was elevated severe hepaticnecrosis occurredamount of egcg would be oxidized to form oquinonewhich could react with gsh to form egcg thiol conjugates therefore it could be inferred that high dose of egcgresults in the accumulation of oquinones and the metabolites of oquinones are biomarkers of oxidative stress twoegcg thiol conjugates egcg2²cysteinyl and egcg2²²cysteinyl were detected in the pooled h urine of micetreated with a dose of or mgkg intraperitonealip injection of egcg however egcg thiol conjugateswere not found when the dose was or mgkg bwip when cf1 mice were treated with a single doseof mgkg intragastric ig administration of egcgboth egcg2²cysteinecysteine weredetected in the pooled h urine gsh conjugate ofand egcg2²²egcg was also detected in hepatocytes incubated withegcg these findings indicated that the formation ofdetectable amounts of egcg thiol conjugates appears toresult from the administration of toxic doses of egcgnuclear factor erythroidrelated factor nrf2 an essential antioxidant transcription factor regulates the expressionof many antioxidant and phase ii detoxifying enzyme genessuch as ho1 and nadphquinone oxidoreductase1nqo1 through antioxidant response element are undernormal metabolic and physiologic states nrf2 is repressed inthe cytoplasm by kelchlike echassociated protein1keap1 while under oxidative stress conditions nrf2 dissociates from keap1 and translocates to the nucleus to bind toare the activation of the nrf2are signaling pathway 0coxidative medicine and cellular longevityrepresenting a major cellular defense against oxidative stresscould stimulate the expression of downstream antioxidantenzymes a previous study revealed that toxic doses ofegcg and mgkg ip inhibited hepatic antioxidantenzymes sod cat and glutathione peroxidase and exacerbated oxidative damage in hepatocytes after treatmentwith egcg the expression of nrf2 decreased in the cytosoland increased in the nucleus indicative of nrf2 activationas a result mrna expression of ho1 nqo1 and otherhepatic nrf2target genes was induced in a dosedependentmanner accordingly a conclusion could be made that themolecular mechanisms underlying highdose egcg potentialtoxicity involve activation of the nrf2are signaling pathwayand suppression o

" leukotriene receptor antagonists ltras are broadly used for the management of allergic asthmaand have recently been indicated to inhibit carcinogenesis and cancer cell growth in colorectal cancer crcchemoprevention studies the occurrence of adenoma or crc itself is generally set as the trial endpoint althoughthe occurrence rate of crc is the most confident endpoint it is inappropriate for chemoprevention studies becausecrc incidence rate is low in the general population and needed for longterm monitoring aberrant crypt fociacf defined as lesions containing crypts that are larger in diameter and darker in methylene blue staining thannormal crypts are regarded to be a fine surrogate biomarker of crc therefore this prospective study was designedto explore the chemopreventive effect of ltra on colonic acf formation and the safety of the medicine in patientsscheduled for a poly resection as a pilot trial leading the crc chemoprevention trialmethods this study is a nonrandomized openlabel controlled trial in patients with colorectal acf and polypsscheduled for a polypectomy participants meet the inclusion criteria will be recruited and the number of acf inthe rectum will be counted at the baseline colonoscopic examination next the participants will be assigned to theltra or no treatment group participants in the ltra group will continue mg of oral montelukast for weeksand those in the no treatment group will be observed without the administration of any additional drugs at theend of the 8week ltra intervention period a polypectomy will be conducted to evaluate the changes in thenumber of acf and cell proliferation in the normal colorectal epithelium will be analyzeddiscussion this will be the first study to investigate the effect of ltras on colorectal acf formation in humanstrial registration this trial has been registered in the university hospital medical information network uminclinical trials registry as umin000029926 registered november keywords colorectal cancer chemoprevention leukotriene receptor antagonist aberrant crypt foci correspondence takuma_hyokohamacuacjpdepartment of gastroenterology and hepatology yokohama city universityschool of medicine fukuura kanazawa yokohama kanagawa japan the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0chigurashi bmc cancer page of cancer is a one of the major health issues and a leadingcause of death globally the incidence prevalence andmortality rates of colorectal cancer crc continue torise all over the world [ ] the majority of crc casesare derived from adenomatous polyps and their resection has been shown to reduce the risk of the futuredevelopment of advanced adenomas and crc [ ]thereby preventing crcrelated deaths however patients with polyps adenomatous polyps andor hyperplastic polyps represent a highrisk group for theoccurrence of metachronous colorectal polyps andorcrc then a paradigm is shifting from surveillancefor the early detection of advance adenomas or crcsand resection to novel tactics for prevention is requiredto reduce the mortality rate of this disease a number oflargescale epidemiologic andor clinicaltrials haveassessed the prophylactic agentsincluding vitamin dcalcium fiber and nonsteroidal antiinflammatory drugsnsaids such as aspirin and selective cyclooxygenase cox2 inhibitors in preventing against crc development we previously reported that the nsaidsulindac suppressed the development of sporadic colorectal adenomas to this point nsaids particularlycox2 inhibitors have been proven to offer the greatestpossibility for reducing crc risk either alone or in combination with other drugs while it has been reportedan elevated risk of serious cardiovascular events relatedwith the administration of cox2 inhibitors [ ] inview of these adverse cardiovascular effects and the lackof efficacy of other drugs that initially looked promisingthe development of novel agents that meet both safetyand efficacy in preventing crc is essentialleukotriene receptor antagonists ltras such asmontelukast and zafirlukast are commonly used for thetreatment of allergic asthma [ ] and tsai mj reported that ltras reduced cancer risk in a dosedependent manner in asthma patients it was alsoreported that the cancer incidence rate was significantlylower in ltra users than in nonltra users vs per patientsyear this means that apart fromits role in asthma ltra has also been associated withcarcinogenesis and tumormediated immunosuppression for example the overexpression of cysteinyl leukotriene receptor cyslt1r has been observed in crcand montelukast leads the apoptosis of crc cancer cells[ ] previous in vivo studies have shown the chemopreventive effect of ltras [ ] but the chemopreventive effects of ltra have not been studied in clinicalpractice therefore we designed this study to ivestigatethe chemopreventive effect of ltras in clinical practicein crc chemoprevention trials the occurrence of adenomas or crc itself is generally set as the trial endpointthough the occurrence of crc is the most confidentendpoint it is not recommended for chemopreventionstudies because crc incidence rate is low in the generalpopulation and needed for longterm monitoring furthermore there are ethical concerns about conductinglongterm trials to determine whether a test agent is effective or notaberrant crypt foci acf defined as lesions containing crypts that are larger in diameter and darker inmethylene blue staining than normal crypts [“] areregarded to be a fine surrogate biomarker of crc our group has previously reported that acf is usefulbiomarker for crc [ ] and study endpoint for achemoprevention study [ ] the advantages of chemoprevention studies with the number of colorectalacf as the trial endpoint are that longterm observationis not needed to investigate the agent efficacy and thenumber of acf can be quantitatively estimated therefore we set the acf count as a good endpoint for thisstudy to the knowledge of us this is the first clinicalstudy investigating the use of ltras as chemopreventive agents against colorectal acf in humansmethodsstudy design and settingthis study was designed as a nonrandomized openlabel controlled trial to be conducted in patients withcolorectal acf it will be performed at the departmentof gastroenterology and hepatology yokohama cityuniversity ycu hospital japan the coordinating office will be at the ycu hospital and patient registrationwill be conducted at the ycu center for novel and exploratory clinical trials ynext and data collectionwill be done using electronic data caputureethical considerations and trial registrationthe trial protocol complies with the declaration ofhelsinki and the ethical guidelines for clinical research of the ministry of health labour and welfarejapan ethical approval of this trial was obtainedfrom the ethics committee of ycu hospital on may the study protocol and informed consent documents were approved by the ycu hospital ethics committee this trial has been approved in the clinical trialact in japan and registered in the japan registry of clinical trials jrct as jrcts031180094 and the universityhospital medical information network umin clinicaltrials registry as umin000029926 all study participants will submit a written study participation informedconsent formparticipation criteriawe will recruit the patients with colorectal acf and resectable polyps for this trial the inclusion criteria are asfollows patients with resectable polyps [adenoma 0chigurashi bmc cancer page of hyperplastic polyp and sessile serrated adenomapolypssap] patients with more than five rectal acfand submit written study participation informed consent formthe exclusion criteria are as follows patients withlesions suitable for early removal a history of ltrause within months before study participation a history of malignant disease within years before studyparticipation a history of heart renal liver failure orliver cirrhosis a history offamilial adenomatouspolyposis hereditary nonpolyposis crc or inflammatory bowel disease pregnancy or the possibility ofpregnancy prohibitions of montelukast allergiesto montelukast regular use of nsaids metforminand pioglitazone and participants considered as unsuitable for the study by the researchersinterventionall eligible participants will be assigned to the ltra orno treatment group because this is an openlabel trialpatients will be assigned to the no treatment group afterthe inclusion of patients in the ltra group participantsin the ltra group will receive mg of oral montelukast for weeks and those in the no treatment groupwill be observed without the administration of any additional drugs at the end of the 8week ltra treatmentperiod a polypectomy will be performed to evaluate thechanges in the number of acf and cell proliferation inthe normal colorectal epithelium will be analyzedoutcome measurementsthe primary endpoint will be the change in the numberof colorectal acf after weeks of treatment a magnifying colonoscope pcfq290azi hz290 olympus cotokyo japan will be used in all cases procedure preparation for the colonoscopy will begin day before theprocedure each participant will be informed to take alowresidue diet and mg oral sodium picosulfate onthe evening before the procedure on the day of the procedure each participant will be given ml polyethylene glycol peg if the stools are not clear enough anadditional ml peg will be given to ensure adequatebowel cleansing in most cases conscious sedation withmidazolam “ mg and pentazocine “ mg willbe use at the start of the colonoscopy subcutaneousscopolamine or glucagon will be administered for colonic movements reduction at the time of the first colonoscopy the endoscopists will insert into the cecumand the observe entire colorectum as the endoscope ispulled back one colonic mucosal sample will be collected the number of rectal acf will be counted usinga magnifying endoscope at the end of the 8week ltratreatment period the same endoscopists will performthe polypectomies and countthe number of acfendoscopists will record all procedures on a hard diskdrive and take photograph all acf the number of acfin each participant will first be counted by the endoscopists during the procedure to provide additional validation the number of acf will be recounted by threeblinded expert endoscopists aj ht and ak by observing the recorded hard disk drive cases that these evaluators deem colonoscopy to be inappropriate will beexcluded from the final analysisthe secondary outcomes will be as follows drugsafety adverse events aes will be graded according tothe national cancer institute common toxicity criteriafor adverse events ncictcae version all trialparticipants will be provided with a trial record for thedaily dose of the trial agent and aes participants whodevelop serious ae of grade or higher will be discontinued at that time in the study the effects ofltras on cell proliferation in the rectal mucosa onecolonic mucosal sample will be collected from the samestudy patient by performing a biopsy at the time of thebaseline colonoscopy and polypectomy a biopsy will beobtained from all participants cell proliferation will beevaluated by ki67 staining briefly we will randomly select six crypts and count the number of ki67positivecells per crypt in total cells will be counted at amagnification of × using a brightfield microscopethe results will be presented as the percentage of ki67positive cells all participants will receive laboratory testsand a physical examination at the point of the baselinecolonoscopy and polypectomydrug supplymontelukast capsules will be purchased from kyorincorporation ltd tokyo japan participants will be informed to take one tablet of the study drug every nightbefore bed medication adherence will be monitored bycounting the empty medication sheets returned by theparticipants at the time of their polypectomy the participants will also be interviewed and monitored to confirm thatthey have not used any prohibited drugsaspirin metformin andor other nsaids aes will bemonitored by the investigator and graded according tothe ncictcae version if serious aes or less than drug adherence are confirmed in a participant thisparticipant will be withdrawn from the trialsample size estimation and allocationwe previously reported the administration of mgmetformin per a day for weeks reduced the number ofacf in that trial the mean number of acf per patientdecreased significantly from ± at baseline to ± at weeks p although this study is exploratory research and the accurate chemopreventive efficacy of montelukastis unknown we assume that 0chigurashi bmc cancer page of montelukast may have an effect that is equivalent to of that observed for metformin on the reduction of acfnubmer therefore we estimate that the acf numberwill change by about ˆ’ to ˆ’ on average we determined that a sample size of “ individuals in theltra group was needed to detect a significant reduction in the number of acfs in the ltra group using apaired ttest with a twosided significance level of and power assuming some dropouts we propose to recruit participants in the ltra group to confirmthat the number of acf does not change during thestudy period we propose to recruit patients in the notreatment group after consecutively accumulating patients in the ltra group therefore we propose to recruit patientsstatistical analysisthe change in the number of acf which is the primaryendpoint will be compared before and after the 8weekstudy period between the ltra and no treatment groupsby the paired ttest drug safety will be assessed by thechisquare test and the remaining results will be compared by the ttest or mann“whitney u test between thetwo groups p will be defined as statistically significant all statistical analyses will be conducted using jmp® software sas institute inc cary nc usatrial steering committee and data monitoring committeethe trial steering committee and data monitoringthe ynext thecommittee will be located atmanagement team will perform central monitoring ofthe trial status and data collection every monthstudy flowa study flow is shown in fig discussionto the knowledge of us this is the first clinical trial proposed to investigate the efficacy of ltras on colorectalacf formation ltras are broadly used for the treatment of allergic asthma and rhinitis [ ] and ltrasare reported to decrease the risk of cancer in asthma patients in a dosedependent manner previous basicresearch has reported that cyslt1r is overexpressed incrc and that montelukast induces the apoptosis ofcrc cells [ ] cyslts have recently been focusedon as significant regulators of gut homeostasis with endogenous cyslt production mediating the proliferationand survival of gut mucosal cells recent evidence focuses on the effect of leukotrienec4 ltc4 in accelerating oxidative dna damage if notadequately repaired can contribute to increase mutationrates and genomic instability dna damage andgenomic instability are major drivers of carcinogenesis cyslts also acts as leukocyte chemoattractants inaddition cyslt1 mediates th17 cell migration the storage of which associates with the progression ofinflammationassociated cancers chronic inflammation is a risk factor for cancer initiation and progression as observed in patients with inflammatory bowelfig study flow chart 0chigurashi bmc cancer page of disease furthermore leukotriene d4 ltd4 antagonists suppress chronic inflammation in a rodent modelof acute enteritis and this may be effective in preventinginflammationassociated crc ltras are leukotriene pathway inhibitors and thus they may have potentialas chemotherapy andor chemoprevention agents to reduce the effect of leukotrienes previous in vivo studieshave elucidated the chemopreventive effect of leukotriene pathway inhibitors [ ] and showed the potentialuse of ltras for chemoprevention therefore we willconduct this trial to explore the chemopreventive effectof ltras in clinical settingthis trial may have some limitations as follow firstacf are believed to be a fine surrogate biomarker ofcrc though its biological significance in humans is stillcontroversial in crc chemoprevention studies typically set the occurrence of adenomas or the crc itselfas endpoint of the study though the occurrence of crcis the most appropriate endpoint it is inappropriate forchemoprevention studies because crc incidence rate islow in the general population and needed for longtermmonitoring our group has previously reported thatacf is useful biomarker for crc and conducted a chemoprevention study for colorectal acf [ ] therefore we designed this study using the number of acf asthe primary endpoint to investigate the chemopreventiveeffect of ltras second an intervention duration of weeks may be too short to reliably detect differences between two groups since our group reported in a previous trial that oral administration of metformin for weeks reduced the number of colorectal acf in humans an intervention period of weeks should beenough to assess the changes in the number of acf ifltras have a chemopreventive effectour group previously conducted a shortterm chemoprevention study of metformin for colorectal acfand reported the preventive effect of the agent on theformation of acf then we conducted a oneyearmetformin chemoprevention studycolorectalpolyps we propose to repeat the same steps as inour metformin study for the chemoprevention studyusing montelukastforif ltras were proved to have efficacy for crc prevention the impact would be significant therefore webelieve it will be very interesting to assess whetherltras inhibit the formation of human colorectal acfabbreviationscrc colorectal cancer nsaids nonsteroidal antiinflammatory drugs cox cyclooxygenase2 ltras leukotriene receptor antagonistscyslt1r cysteinyl leukotriene receptor acf aberrant crypt fociycu yokohama city university umin university hospital medicalinformation network ssap sessile serrated adenomapolyp ncictcae national cancer institute common toxicity criteria for adverseevents peg polyethylene glycol aes adverse events ltc4 leukotriene c4ltd4 leukotriene d4acknowledgmentsthe authors would like to thank the staff for their support in recruitingeligible patients and the patients who participated in this study and theirfamily we thank cathel kerr bsc phd and melissa crawford phd fromedanz group httpsenauthorservicesedanzgroupcom for editing a draftof this manuscriptauthors™ contributionsth ja and an conceived the study th and ja equally contributed to thismanuscript ja th ka nm ty tm af and ho will perform the baselinecolonoscopies and polypectomies ja th and ka will conduct the secondcount of acf using a hard disk drive recording to ensure validity tt nm tyaf and ho will recruit participants and followup with them at an outpatientclinic the analysis and interpretation of data will be conducted by ja thand ka all authors have read the final manuscript and approved its submission for publicationfundinga grant for this research from the kanagawa institute of industrial scienceand technology kistec was awarded to th we declare that the fundingbody has no role in the design data collection analysis interpretation andwriting of the studyavailability of data and materialsthe datasets used andor analyzed during the current study will be availablefrom the corresponding author on reasonable requestethics approval and consent to participateethical approval of this trial was obtained from the ethics committee of ycuhospital on may the study protocol and informed consentdocuments were approved by the ycu hospital ethics committee this trialhas been registered in the university hospital medical information networkumin clinical trials registry as umin000029926 all study participants willsubmit a written study participation informed consent formconsent for publicationnot applicablecompeting intereststhe authors declare that they have no conflicts of interestsreceived may accepted august referencestorre la bray f siegel rl global cancer statistics ca cancer jclin “anderson wf umar a brawley ow colorectal carcinoma in black andwhite race cancer metastasis rev “vogelstein b fearon er hamilton sr genetic alterations duringcolorectaltumor development n engl j med “ winawer sj zauber ag ho mn o™brien mj gottlieb ls sternberg ss wayejd schapiro m bond jh panish jf prevention of colorectal cancer bycolonoscopic polypectomy the national polyp study workgroup n engl jmed “citarda f tomaselli g capocaccia r barcherini s crespi m italianmulticentre study group efficacy in standard clinical practice ofcolonoscopic polypectomy in reducing colorectal cancer incidence gut“zauber ag winawer sj o™brien mj lansdorpvogelaar i van ballegooijenm hankey bf shi w bond jh schapiro m panish jf stewart et waye jdcolonoscopic polypectomy and longterm prevention of colorectalcancerdeaths n engl j med “ maisonneuve p botteri e lowenfels ab fiveyear risk of colorectalneoplasia after negative colonoscopy n engl j med “author reply das d arber n jankowski ja chemoprevention of colorectal cancerdigestion “ epub oct review matsuhashi n nakajima a fukushima y yazaki y oka t effects of sulindacon sporadic colorectal adenomatous polyps gut “ 0chigurashi bmc cancer page of drazen jm cox2 inhibitorsa lesson in unexpected problems n engl jmed “ epub feb meyskens fl jr mclaren ce pelot d fujikawabrooks s carpenter pmhawk e kelloff g lawson mj kidao j mccracken j albers cg ahnen djturgeon dk goldschmid s lance p hagedorn ch gillen dl gerner ewdifluoromethylornithine plus sulindac for the prevention of sporadiccolorectal adenoma a randomized placebocontrolled doubleblind trialcancer prev res phila “scott jp petersgolden m antileukotriene agents for the treatment of lungdisease am j respir crit care med “shen z genomic instability and cancer an introduction j mol cell biol“kim hs lee g the cysteinyl leukotriene receptor cysltr1 mediates th17cell migration j immunol bernstein cn blanchard jf kliewer e wajda a cancer risk in patients withinflammatory bowel disease a populationbased study cancer “ nishikawa m hikasa y hori k tanida n shimoyama t effect of leukotrienec4d4 antagonist on colonic damage induced by intracolonic administrationof trinitrobenzene sulfonic acid in rats j gastroenterol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations dahlen se dahlen b drazen jm asthma treatment guidelines meet the realworld n engl j med “tsai mj wu ph sheu cc hsu yl chang wa hung jy corrigendumcysteinyl leukotriene receptor antagonists decrease cancer risk in asthmapatients sci rep wang d dubois rn eicosanoids and cancer nat rev cancer “ nielsen ck the leukotriene receptor cyslt1 and 5lipoxygenase areupregulated in colon cancer adv exp med biol “ burke l butler ct murphy a moran b gallagher wm o™sullivan j kennedybn evaluation of cysteinyl leukotriene signaling as a therapeutic target forcolorectal cancer front cell dev biol savari s liu m zhang y sime w sjolander a cyslt1r antagonists inhibittumor growth in a xenograft model of colon cancer plos one e73466 bellamkonda k satapathy sr douglas d chandrashekar n selvanesan bcliu m savari s jonsson g sjolander a montelukast a cyslt1 receptorantagonist reduces colon cancer stemness and tumor burden in a mousexenograft model of human colon cancer cancer lett “ roncucci l stamp d medline a cullen jb bruce wr identification andquantification of aberrant crypt foci and microadenomas in the humancolon hum pathol “ roncucci l medline a bruce wr classification of aberrant crypt foci andmicroadenomas in human colon cancer epidemiol biomark prev “ pretlow tp barrow bj ashton ws aberrant crypts putativepreneoplastic foci in human colonic mucosa cancer res “ pretlow tp o™riordan ma pretlow tg stellato ta aberrant crypts in humancolonic mucosa putative preneoplastic lesions j cell biochem suppl “takayama t katsuki s takahashi y ohi m nojiri s sakamaki s kato jkogawa k miyake h niitsu y aberrant crtpt 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" The blue nodes represent biomarkers identified in this work. The yellow nodes represent six genes which are not related with NSCLC on the NSCLC and normal specimens. The red nodes represent subtypes i.e. AC and SCC. Key gene pairs inferred by gene-subtype higher logic relationships We grouped together the gene-subtype higher logic relationships with the same logic function. Because the two logic functions AND (Type 1) and XOR (Type 8) have more intuitive biological interpretations than other logic functions we restricted our analysis to these two logic functions. The key gene pairs were defined as the gene pairs involved in the gene-subtype higher logic relationships with logic function AND or XOR. We obtained key gene pairs in total where and gene pairs were related with AC/SCC through the logic functions AND and XOR respectively (Table S6). This result may be explained by the strict parameters we chose. Gene Ontology analysis The Gene Ontology (GO) is a structured and controlled vocabularies and classifications about the annotations of genes gene products and sequences [40]. GO includes three categories of terms: biological processes molecular functions and cell components. We were focused on the biological processes enriching the genes involved in lower logic relationships. So in what follows when we say GO terms it means the GO terms in the ˜biological process™ category. According to probe-AC/SCC pairwise associations and their uncertainty coefficients we obtained a gene set containing genes without overlap and each gene attached a coefficient. A total of genes were ranked in descending order by coefficients and given as input to the Gorilla. The Gorilla gave significant GO terms like ˜tissue development™ (GO: 0009888) ˜epidermis development™ (GO: 0008544) and ˜epithelial cell differentiation™ (GO: 0030855) (Part A in Appendix S1). Given that the significant GO terms were retrieved based on the subtypes of NSCLC data it has to be checked whether the significant GO terms are also significant on NSCLC and normal specimens. The same procedure was applied to the ranked genes based on the NSCLC and normal data. The test revealed significant GO terms with significant value (Part B in Appendix S1). In total seven out of GO terms on the subtypes of NSCLC data were also significant on the NSCLC and normal specimens (). It indicates that the following seven biological processes are important for tumorigenesis of NSCLC: tissue development epidermis development epithelial cell differentiation anatomical structure development developmental process cell adhesion and biological adhesion. .0094644.t002 Significant GO terms. GO terms Description P-value1 P-value2 E1 E2 GO:0009888 tissue development GO:0008544 epidermis development GO:0030855 epithelial cell differentiation GO:0048856 anatomical structure development GO:0032502 developmental process GO:0007155 cell adhesion GO:0022610 biological adhesion ˜P-value1™ and ˜P-value2™ denote the p-value scores of GO terms based on the subtypes of NSCLC data and NSCLC and normal data respectively. ˜E1™ and ˜E2™ are the enrichment values of GO terms based on the subtypes of NSCLC data and NSCLC and normal data respectively. Further we grouped the genes closely related with the subtypes of NSCLC into two groups by the types of gene-SCC lower logic relationships. We mapped the genes which were related with SCC (AC) by Type () lower logic relationships to GO terms. Gene ontology analysis revealed GO terms with the p-value scores smaller than and the enrichment scores larger than . Among significant GO terms epithelial cell differentiation (GO: 0030855) and cell adhesion (GO: 0007155) were also involved in the seven significant GO terms which may be important for tumorigenesis of NSCLC. It indicates that dysfunction of epithelial cell differentiation and cell adhesion is important for both of the tumorigenesis of AC and SCC. In addition we mapped the identified biomarkers to GO terms. The resulted significant GO terms were cell adhesion (GO: 0007155) and epidermis development (GO: 0008544) with the p-value scores smaller than and the enrichment scores larger than . It indicates that genes annotated to epidermis development and cell adhesion may be differently regulated between AC and SCC. By mapping the higher logic relationships to GO terms we obtained pairs of GO terms with different GO terms. Among all pairs of GO terms pairs of GO terms involving GO terms were significant with the p-value scores smaller than enrichment score larger than one and the number of gene pairs larger than two. These combination of biological processes may be pivotal for differentiating AC and SCC including a combination of ˜transport™ (GO: 0006979) and ˜regulation of transcription DNA-dependent™ (GO: 0006355) a combination of ˜oxidation-reduction process™ (GO: 0055114) and ˜nervous system development™ (GO: 0007399) and a combination of ˜negative regulation of cell proliferation™ (GO: 0008285) and ˜muscle contraction™ (GO: 0006936). Discussion In this paper we improved the logic analysis method to infer sufficient and necessary conditions for the presence states (presence or absence) of a phenotype. The current method omits the integration of networks and identifies not only gene-phenotype pairwise combinations (i.e. lower logic relationships) but also triplets combinations (i.e. higher logic relationships). On one hand it avoids the incompleteness of data sources and the noise from the integration of data; on the other hand the triplets combinations reflect the combination effect of gene pairs on phenotypes other than an individual effect. Some examples of lower and higher logic relationships demonstrated the biological relevance of our results. However the accuracy of all discovered logic relationships cannot be verified because of the current limited knowledge of the relationships between genes and phenotypes. The statistics analysis strengthened the reliability of discovered logic relationships. In addition the current method was compared with the two earlier methods (the NMF method and the RA method). The current method was superior to the two earlier methods because of its ability of mining gene pairs which are closely related with phenotypes. Moreover the current method gained the higher recall rate and classification accuracy than the two earlier methods. Our results display the advantage of the current method in mining genes closely related with phenotypes. The discovered gene-subtypes logic relationships in this paper are equivalent relationships between the expression patterns (expression or no-expression) of genes and the presence states (presence or absence) of phenotypes. That is both a expression pattern of a gene and a presence state of a phenotype must be either simultaneously true or simultaneously false. For example DSC3 is expressed if and only if the specimen is SCC as DSC3 is related with SCC by the first type of lower logic relationship. If a gene is related with a phenotype by a logic relationship then either the expression pattern of a gene or the presence state of a phenotype may be determined by the underlying logic relationship. Concretely given a phenotype the expression pattern of genes in a phenotype could be determined by the logic relationship. For example the expression pattern of DSC3 in SCC depends on the type of DSC3-SCC lower logic relationship. Conversely given a expression pattern of a gene the presence state of a phenotype could also be determined by the underlying logic relationships. The type of a discovered gene-AC lower logic relationship was totally different from that of the gene-SCC lower logic relationship where the genes involved in two relationships are the same. It indicates that the totally different types of lower logic relationships between genes and phenotypes may be the intrinsic reason for the different expression patterns of genes in distinct phenotypes. A total of genes identified in our work were regarded as the biomarkers for distinguishing AC from SCC as well as novel molecular targets for targeted therapeutic agents. Besides the genes identified in the literature (DST CLCA2 KRT5 DSG3 GJB5 SERPINB13 TRIM29 PKP1 KRT6B DSC3 NKX2-1 TP63 and NTRK2) most of the rest genes (BNC1 FAT2 LASS3 and PVRL1) are likely to be the novel biomarkers to distinguish AC from SCC. The BNC1 gene is thought to play a regulatory role in ˜keratinocyte proliferation™ and the LASS3 gene is participated in ˜keratinocyte differentiation™. Both of the biological process ˜keratinocyte proliferation™ and ˜keratinocyte differentiation™ are children of ˜keratinization process™. Because the genes involved in ˜keratinization process™ are higher expressed in SCC as compared with AC [26] BNC1 and PVRL1 which are either a upstream regulatory factor or a member of these high expressed genes may be able to differentiate AC and SCC. FAT2 functions as a cell adhesion molecular and it controls cell proliferation. As ˜cell adhesion™ is one of the significantly important biological processes for tumorigenesis of NSCLC the cell adhesion molecular (FAT2) is deserved to be a biomarker to distinguish AC from SCC. Until recently the function of LOC642587 and GOLT1A has been unknown. Further experimental validation is needed to confirm the differentiating ability of these two genes. In addition the NKX2-1 gene has been considered as a novel oncogene [35] and it opens new windows for novel targeted therapies [41]. "

"We concluded that the combination of a selective MEK inhibitor and a PI3K/mTOR inhibitor was effective in suppressing the growth of gefitinib-resistant tumors caused by EGFR T790M mutation MET amplification and KRAS/PIK3CA mutation. These findings represent a promising strategy for the treatment of gefitinib-resistant NSCLC and provide a strong basis for the design of clinical trials for this purpose. Competing interests The authors declare that they have no competing interests. Authors™ contributions YQQ conceived and designed the experiments. XXW YHY YY and HL performed the experiments. XXW YHY and DDM analyzed the data. XXW YHY and HL wrote the paper. YQQ supervised the whole experimental work and revised the manuscript. All authors read and approved the manuscript. She J Yang P Hong Q Bai C Lung cancer in China: challenges and interventions Chest 2013 143 1117 1126 23546484 Weir HK Thun MJ Hankey BF Ries LA Howe HL Wingo PA Jemal A Ward E Anderson RN Edwards BK Annual report to the nation on the status of cancer "

Activation by NaturalPhytochemicals An OverviewConcetta Iside Marika Scafuro Angela Nebbioso  and Lucia Altucci Department of Precision Medicine University of Campania œLuigi Vanvitelli Naples ItalySirtuins are class III histone deacetylases whose enzymatic activity is dependent on NADas a cofactor Sirtuins are reported to modulate numerous activities by controlling geneexpression DNA repair metabolism oxidative stress response mitochondrial functionand biogenesis Deregulation of their expression andor action may lead to tissuespecificdegenerative events involved in the development of several human pathologies includingcancer neurodegeneration and cardiovascular disease The most studied member of thisclass of enzymes is sirtuin SIRT1 whose expression is associated with increasinginsulin sensitivity SIRT1 has been implicated in both tumorigenic and anticancerprocesses and is reported to regulate essential metabolic pathways suggesting thatits activation might be beneficial against disorders of the metabolism Via regulation of p53deacetylation and modulation of autophagy SIRT1 is implicated in cellular response tocaloric restriction and lifespan extension In recent years scientific interest focusing on theidentification of SIRT1 modulators has led to the discovery of novel small moleculestargeting SIRT1 activity This review will examine compounds of natural origin recentlyfound to upregulate SIRT1 activity such as polyphenolic products in fruits vegetablesand plants including resveratrol fisetin quercetin and curcumin We will also discuss thepotential therapeutic effects of these natural compounds in the prevention and treatmentof human disorders with particular emphasis on their metabolic impactKeywords sirtuin natural compounds oxidative stress human disorders polyphenolsEdited byCecilia BattistelliSapienza University of Rome ItalyReviewed byNarasimham L ParinandiThe Ohio State UniversityUnited StatesCarmen JeronimoPortuguese Oncology InstitutePortugalCorrespondenceAngela NebbiosoangelanebbiosounicampaniaitLucia Altucciluciaaltucciunicampaniait These authors share last authorshipINTRODUCTIONSpecialty sectionThis was submitted toTranslational Pharmacologya section of the journalFrontiers in PharmacologyReceived April Accepted July Published August CitationIside C Scafuro M Nebbioso A andAltucci L SIRT1 Activation byNatural Phytochemicals An OverviewFront Pharmacol 103389fphar202001225Epigenetic modifications are associated with genome stability gene transcription and metabolicregulation Acetylation is one of the most characterized histone modifications Histoneacetyltransferase HAT and histone deacetylase HDAC enzymes control the levels of histoneacetylation modulating gene expression Cavalli and Heard Sirtuins SIRT “ are enzymes classified as class III HDACs They exhibit different subcellularlocalizations SIRT1 SIRT6 and SIRT7 are nuclear although SIRT1 isoforms were also identified inAbbreviations SIRT1 Sirtuin HATs Histone acetyl transferases HDACs Histone deacetylases ROS Reactive oxygenspecies PPAR Receptor peroxisome proliferatoractivated receptor NRF Nuclear respiratory factor TFAM Mitochondrialtranscription factor A SOD Superoxide dismutase TNFa Tumor necrosis factor a IAP Apoptosis protein inhibitor Bcl2Bcell lymphoma2 family MnSOD Manganese superoxide dismutase RSV Resveratrol Que Quercetin oxLDL OxidizedLDL BBR Berberine Cur Curcumin COX Cytochrome c oxidase T2D Type II diabetes NAFLD Nonalcoholic fatty liverdisease CRM Caloric restriction mimeticFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsthe cytoplasm SIRT2 is mainly cytosolic SIRT3 SIRT4 andSIRT5 are mitochondrial and can shuttle to the nucleus Changand Guarente The enzymatic activity of SIRTs is dependent on NAD as acofactor and plays an important role in controlling geneexpression DNA repair metabolism oxidative stress responsemitochondrial function and biogenesis Deregulation of theiractivity may lead to tissuespecific degenerative events thatunderlie several human pathologiesincluding cancerdiabetes and cardiovascular diseases Haigis and Sinclair O™Callaghan and Vassilopoulos Waldman The most studied member of this enzymatic class isSIRT1 SIRT1 regulates metabolic pathways cell survivalcellular senescence and ‚ammation and acts in thepathogenesis of chronic conditions such as diabetes as well aspulmonary neurodegenerative and cardiovascular diseasesIndeed SIRT1 has been reported to play a key role intumorigenesis as an oncogene or tumor suppressor dependingon the context specificity BiasonLauber It is able tocontrol these processes via deacetylation of lysine groups ofhistone and nonhistone proteins including known transcriptionfactors FOXO MyoD p53 PGC1a Kupis Chronic ‚ammation caused by oxidative damage increasesthe risk of many chronic disordersincluding heartcardiovascular and neurodegenerative diseases obesity insulinresistance and type diabetes T2D Geto Oxidative stress plays a key role in the pathogenesis of theseconditions The overproduction of reactive oxygen speciesROS including free radicals and reactive nitrogen speciesRNS can lead to damage of cellular components such aslipids proteins and DNA The imbalance between oxidantsand antioxidants can result in cellular dysfunction apoptosisand necrosis Liguori SIRT1 guards against oxidative stress by activating genetranscription of PGC1a via deacetylation and by regulatingtranscription of factors such as the nuclear receptor peroxisomeproliferatoractivated receptor PPAR nuclear respiratory factorNRF and mitochondrial transcription factor A TFAMinvolved in modulation of biogenesis and mitochondrialfunction Ren and metabolism of glucose and lipidsRodgers SIRT1 is also able to regulate the expressionof superoxide dismutase SOD and glutathione peroxidase Sun In addition since mitochondrial dysfunction leads tothe activation of apoptosis SIRT1 can directly regulate theapoptotic process by modulating acetylation of PGC1a Zhang SIRT1 also regulates ‚ammatory responseKauppinen By modulating the acetylation level ofNFkB p65 SIRT1 is able to control transcription of genes such asIL interleukin1 tumor necrosis factor a TNFa IL8 IL6and other ‚ammatory factors Rodgers Ren Yeung Through NFkB SIRT1 also regulatesthe expression of genes such as inhibitor of apoptosis proteinIAP and Bcell lymphoma2 Bcl2 and tumor necrosis factorreceptor TNFR Ren SIRT1 protects against oxidative stress via regulation ofFOXO protein acetylation which is involved in antioxidantprocesses apoptosis and cell proliferation Wong andWoodcock By activating FOXOMsSOD pathwaySIRT1 increases the expression of manganese superoxidedismutase MnSOD and catalase counteracting oxidativestress and promoting damage repair Gu SIRT1also increases the expression of MnSOD by deacetylating p53thus enhancing cellular antioxidant capacity Brunet Zhang Ren Over the past few years the evergrowing awareness that goodhealth goes hand in hand with a healthy and balanced diet hasencouraged people to eat more fruit and vegetables and to takesupplements to make up for any deficiency D™Angelo Bioactive compounds in the diet can act as antioxidantand anti‚ammatory agents thereby reducing the negativeeffects of oxidative stress and the incidence of chronicdiseases such as obesity diabetes and cardiovascular disordersWang Several moleculesincluding naturalphytochemical compounds can modulate SIRT1 activityMiceli Numerous studies have provided evidenceof the protective effects of natural polyphenolic substances suchas resveratrol quercetin curcumin and fisetin and ofnatural nonpolyphenolic substances such as berberineMcCubrey Natural polyphenols are the largestgroup of phytonutrients and are considered potential agents forthe prevention and treatment of stressrelated oxidative diseasesThey are found in many plants and foods such as fruitsvegetables tea cereals and wine and longterm intake isassociated with health benefits Mediterranean diets are in factlinked to a reduced risk of chronic diseases due to theconsumption of olive oil and red wine which contain highamounts of polyphenols Romagnolo and Selmin Most of the evidence supporting the beneficial effects ofphytochemical compounds comes from in vitro or animalstudies while human studies evaluating the longterm impact ofphytomolecules are particularly few or inconsistent Interventionalstudies are in fact limited by issues of bioavailability andmetabolism However in vitro studies aimed at identifyingcellular targets linked to the beneficial actions of phytonutrientrich foods at concentrations ranging from nM to µM challenge thetranslatability of data After ingestion these compounds are in factdetected as phase II metabolites and their blood level does notexceed concentrations in the nM range Substantial amounts of thecompounds and their metabolites are degraded in the colon byintestinal microbiota giving rise to small phenolic acids andaromatic catabolises which are absorbed by the circulatorysystem Del Rio Interesting studies showed thatthese natural polyphenol and nonpolyphenol substances couldaffect SIRT1 expressionactivity Table de Boer Themain mechanisms of action common to polyphenol and nonpolyphenol molecules that lead to antioxidant and anti‚ammatory effects via SIRT1 activation are reported in Figure Here we focus on the natural molecules resveratrolquercetin fisetin curcumin and berberine and elucidate theireffect on SIRT1 activation and their potential to treat andorprevent several human pathologies mainly associated withmetabolic disorders Figure Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Classification of nutraceuticals based on their action and food sourceNaturalSIRT1activatorsEffectSourceReferencesResveratrol Positive effect on bloodlipid profile antioxidantDark grapesraisins peanutsQuercetinBerberineCurcuminFisetinAnticancer positiveeffect on blood lipidprofile antioxidant anti‚ammatoryAntioxidant anti‚ammatoryAnticancer antioxidantanti‚ammatoryAnticancercardiovascularpreventive anti‚ammatoryantioxidantFruits vegetablesnutsNatural componentof traditionalChinese herbCoptidis rhizomaActive componentin Curcuma longaApplespersimmonsgrapes onionskiwi kalestrawberriesD™Angelo Zordoky Hung Nabavi Nabavi Wu Hung Zendedel Kim Chen NATURAL COMPOUNDS ENHANCINGSIRT1 EXPRESSION AND ACTIVITYResveratrolResveratrol RSV a nonflavonoid polyphenol found in grapesand grape products such as red wine exerts an antioxidant actionwith reported cancer preventive properties KrisEtherton RSV also has anti‚ammatory anticancer andantineurodegenerative effects Piotrowska The roleof RSV as an immune response modulator was demonstrated inboth in vitro and in vivo studies where it reversed immunesenescence in older rats reduced ‚ammatory responses inrodents and improved immunological activity against cancercells Malaguarnera RSV was shown to be involved in theactivation of macrophages T cells and natural killer cells as wellas in the suppression processes of CD4 CD25 regulatory T cellsYang Svajger and Jeras All these effects aredue to its ability to remove ROSinhibit cyclooxygenaseCOX and trigger anti‚ammatory pathways via SIRT1activation Miceli Malaguarnera ActivatedSIRT1 interrupts TLR4NFkBSTAT axis reduces cytokineproduction by inactivated immune cells and inhibits pro‚ammatory factors derived from macrophagesmast cellssuch as plateletactivating factor and TNFa Capiralla RSVSIRT1 interaction modifies SIRT1 structure andpromotes binding activity with its substrates including p65RelA Yeung a component of the NFkB complexwhich regulates activation of leukocytes and ‚ammatorycytokines SIRT1 activated by RSV inhibits acetylation of RelAby reducing the expression of ‚ammatory factors such as TNFa IL1b IL6 metalloprotease MMP1 MMP3 and NFkBmediated Cox2 Malaguarnera AMP activatedprotein kinase AMPK is also a target of RSV as it controlsSIRT1 activity via regulation of cellular levels of NAD thusacting as an energy sensor Price Cyclicadenosine monophosphate cAMP levels activate proteinkinase A resulting in phosphorylation and activation of SIRT1FIGURE Basic mechanisms and effects of SIRT1 activation by polyphenol and nonpolyphenol moleculesFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsFIGURE Nutraceutical action on SIRT1 expression Natural substances have beneficial effects on human health by regulating SIRT1 action in different cellularprocesses wwwpubchemncbinlmnihgovWan Activated SIRT1 catalyzes the deacetylationand activation of PGC1a thereby promoting beneficial effectsin the metabolism Ren In different anisms S cerevisiae C elegans and Dmelanogaster expressing SIRT1 or its homologous genesRSV treatment is able to extend life span In mammaliansRSV administration can improve SIRT1dependent cellularprocesses such as axonal protection Araki fatmobilization Chaplin and inhibition of NFkBdependent transcription Yeung these effects areabolished in SIRT1 knockdown models Numerous studiesinvestigated the beneficial effects of RSV in cardiovasculardiseases including hypertension Theodotou cardiac ischemia Fourny and atherosclerosisChassot RSV has an effect on blood vesselsreduces ‚ammation and prevents thrombus formation andplatelet oxidation Zordoky It can also reducecardiac dysfunction oxidative stress fibrosis and apoptosis inthe heart Gupta Yamagata In addition RSVwas found to improve heart and kidney damage in rats Li et al2020a The protective effect of RSV is associated with anincrease in SIRT1 activity which deacetylates FOXO1 andactivates MnSOD downstream RSVinduced MnSOD alsoreduces oxidative stress Li 2020a A recent in vitrostudy showed that RSV reduces hypoxiainduced apoptosis inH9C2 cells through activation of SIRT1miR30d5pNFkB axisHan RSV treatment decreased cortical andhippocampal malondialdehyde levels while increasing SODactivity and SIRT1 expression in a diabetic rat model Ma RSV was shown to activate SIRT1 and improve endothelialfunction in obese mice via upregulation of PPARd expressionactivity in PPARd mutant mice Cheang It hadpreviously been observed that Akt activation together withPPARd is involved in vascularization of dbdb mice Tian RSV was subsequently reported to increasephosphorylation of Akt and transcriptional activity of PPARdin the aorta of wildtype mice thus supporting the hypothesis ofSIRT1PPARd interaction and to strongly decrease LPCinduced mitochondrial ROS in the aortic endothelium ofC57BL6 mice Cheang Taken together thesefindings highlight the beneficial effects of RSV against oxidativestress which is involved in major pathologies such as heart andmetabolic disorders Although RSV is beneficialin manycontexts its pleiotropic actions need to be better studied inorder to understand which of its described activities are directlydue to SIRT1 modulation and whether this effect is always directBecause of the pleiotropic actions of RSV clinical trials arecurrently testing its therapeutic potential in a wide range ofhuman diseases However of all the mechanisms described in invitro and in vivo studies only a few have been confirmed inhumans such as gene and protein regulation in blood or musclecells and Akt signaling pathways Ghanim Brasnyo Many clinical studies conducted in healthy patientsand volunteers using both high and low doses of RSV highlightits potential cardioprotective benefit through improvement ofendothelial function‚ammatory markers and glucosemetabolism Nevertheless the mechanisms of action are notyet well defined Despite clinical evidence of its effects thepoor bioavailability and rapid metabolism of RSV severelylimit the potential use of this molecule in the clinic Futurescientific research should focus on identifying actual metabolitesor mediators of these observed effectsTo date clinical trials have tested the efficacy safety andpharmacokinetics of RSV in the prevention and treatment of different pathological conditions wwwclinicaltrialsgovFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsRestricting the search to interventional phase studiescompleted and terminated clinical trials addressed theability of RSV to improve the pathological conditions ofpatients affected by several diseases Most of these studiestested RSVmediated effects in central nervous systemdisorders Friedreich ataxia Alzheimer™s disease Parkinson™sdisease metabolic disorders [T2Dinsulin resistancedyslipidemia hypercholesterolemia metabolic syndrome Xnonalcoholic fatty liver disease NAFLD] A phase clinicaltrial NCT01640197 tested the effects of chronic resveratrolsupplementation mg daily for days in healthy humansand found considerable improvements in cognitive performancecerebral blood flow subjective sleep mood health and bloodpressure A list of completed and terminated clinical trials inwhich RSV was tested for metabolic disorders is reported inTable Focusing on completed clinical trials with availableresults NCT02114892 evaluated the effect of RSV on metabolicsyndrome demonstrating that when administered three timesper day mgdie before meals RSV was able to treat andprotect from obesity and diabetes with beneficial effects onglucose and lipid metabolism blood pressure and bodyweight Another phase study NCT02095873 evaluated theeffects of a formulation composed of RSV and hesperetin inobese subjects and found that these molecules are dietaryinducers of glyxalase improving metabolic and vascularhealth of obese subjects Xue QuercetinThe flavonoid polyphenol quercetin Que ²²pentahydroxyflavone is a natural safe dietary supplementfound in a glycoside form in fruits vegetables and nuts whichhas antioxidant and anti‚ammatory properties Nabavi Wu In recent years the scientific community has focused on thepotential antiproliferative chemopreventive and anticarcinogenicactivities of Que as well as on its role as a modulator of geneexpression However Que was also found to have potentially toxiceffects including mutagenicity prooxidant activity mitochondrialtoxicity and inhibition of enzymes involved in hormonalmetabolism Li Due to its poor solubility short halflife and low bioavailability its medical use is limited Konrad andNieman In humans Que bioavailability is very low and absorption varies from to in subjects receiving mgdie Costa Que may reduce infection Li hepatic lipemicoxidative damage Cui Zhang et al2016b and antioxidant risk Xu In addition Que isknown to exert a modulating action on immunity Galleggiante As regards its mechanism of action in some cell linesQue was able to inhibit the production of TNF in macrophagesTang IL8 in A549 lung cells induced bylipopolysaccharide LPS Geraets and TNFa andIL1a mRNA levels in glial cells causing a decrease in neuronal celldeath induced by microglial activation Li MainlyTABLE Resveratrol in clinical trials for metabolic disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effects of Resveratrol in Patients With TypeType Diabetes DiabetesTerminated Effect of Administration of Resveratrol onType Diabetes Mellitus mg to a maximum dose of g daily mg times dailyPhase NCT01677611Phase NCT02549924Glycemic Variability in Individuals With Type Diabetes MellitusCompleted Effect of Resveratrol on Agerelated InsulinResistance and Inflammation in HumansCompleted Regulation of Intestinal and HepaticLipoprotein Secretion by ResveratrolType Diabetes Mellitus InsulinResistanceDyslipidaemia Insulin ResistanceCompleted Effects of Dietary Antioxidants to PreventHypercholesterolemia HealthyCardiovascular DiseaseHealthy Aging Through Functional FoodCompletedwith resultsCompleted Effects of Resveratrol on Inflammation inType Diabetic PatientsCompletedwith resultsEffect of Resveratrol Administration onMetabolic Syndrome Insulin Sensitivity andInsulin SecretionCompleted Resveratrol for the Treatment of NonAlcoholic Fatty Liver Disease and InsulinResistance in Overweight AdolescentsGlucose Intolerance Aortic Stiffness VasodilationType Diabetes Mellitus Inflammation Insulin Resistance Other Disorders ofBone Density and StructureMetabolic Syndrome XNAFLD Type Diabetes MetabolicSyndromeCompleted A Study of Resveratrol as Treatment forFriedreich AtaxiaFriedreich AtaxiaCompleted Effect of Banaba Lagerstroemia Speciosaon Metabolic Syndrome Insulin Secretionand Insulin SensitivityMetabolic Syndrome X mg twice daily for daysPhase NCT01354977 mg for week followed by g for weekDietary Supplement red wine for monthDietary Supplementresveratrol for monthTransresveratrol mg hesperetin mg combination months mg daily then months mg daily mg times daily beforemeals with a total dose of mg daily mg twice daily for a total dailydose of mg for days g daily mg twice daily for weeks then g daily gtwice daily for weeksBanaba capsules mg times daily before meals for daysPhase NCT01451918Phase NCT02409537Phase NCT02095873Phase NCT02244879Phase NCT02114892Phase NCT02216552Phase NCT01339884Phase NCT02767869Frontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 Activatorsin immunity and ‚ammation Que acts on leukocytes and targetsmany intracellular signaling kinases and phosphatases as well asenzymes and membrane proteins Li Theimmunostimulating effect of Que is due to induction of theexpression of interferong IFNg derived from Th1 andinhibition of IL4 derived from Th2 in normal peripheral bloodmononuclear cells Nair In addition Que reduces T cellproliferation by blocking IL12induced tyrosine phosphorylationof JAK2 TYK2 STAT3 and STAT4 Muthian and Bright Nabavi In ‚ammation Que inhibits the enzymesCOX and lipoxygenase Lee and Min Savikin Inthe RAW cell line Que was also shown to counteract LPSinduced ‚ammation by phosphorylation of tyrosinephosphatidylinositol3kinase PI3Kp85 and complexformation of tolllike receptor TLR4MyD88PI3K Endale Oxidative stress occurs following an imbalance of the body™santioxidant defence mechanisms and excessive generation of freeradicals and is involved in various pathologies such as diabetesatherosclerosis hypertension neurodegenerative diseases‚ammation and cancer Oboh Que is apowerful ROS scavenger and its antioxidant action is due tothe presence of two pharmacophores within the molecularstructure which confer a favorable configuration for freeradical elimination Costa Generally Que reducesthe effects of free radicals by transferring the hydrogen atom andstabilizing the radicals a feature that has a structurefunctionrelationship Oboh Que can also act as both an antioxidant and prooxidant agentAt low concentrations “ µM Que displayed a protective effectagainst oxidative DNA damage in vitro in human lymphocytes Li At concentrations between µM and µM Que wasable to directly eliminate ROS in vitro Costa Howeverits effect in vivo is very likely not direct but due to its ability tomodulate the cell™s antioxidant defense mechanisms moderateoxidative stress can in fact increase the cell™s antioxidant defensesresulting in general cytoprotection Halliwell Recentresearch showed that oxidized LDL oxLDL induces oxidativestress LaraGuzman Oxidative injuries promote ROSgeneration in human endothelial cells and SIRT1 regulatesendothelial function Therefore enhancement of SIRT1 activityand SIRT1AMPK axis upregulation inhibits oxidative injuryinducing endothelial dysfunction Chen Shentu Que may reduce oxLDLinduced oxidative damageby upregulating SIRT1 and AMPK Hung thereforepotentially preventing oxLDLimpaired SIRT1 inhibition linked toendothelial dysfunction These findings indicate that SIRT1 canfunction as a regulator to improve AMPK activity under oxLDLstimulation Hung It was very recently shown that Que mgkg can reduceinsulin resistance and improve glucose metabolism by reducingsensitivity to T2Dinsulin resistance in obob mice via SIRT1activation Hu In this context another study showedthat in streptozotocininduced diabetic rats Que mgkginhibits oxidative damage by increasing SIRT1 expression anddecreasing levels of NFkB a SIRT1 substrate Iskender In recent years the scientific community has focused on therole of apoptosis in cardiovascular disease showing thatoxidative stress myocardial ischemia hypoxia and ischemiareperfusion injury may induce myocardial apoptosis Donniacuo Tang and colleagues evaluated the effects of Que inimproving myocardialischemiareperfusion injury MIRinduced cell apoptosis both in vitro and in vivo SIRT1 andPGC1a expression levels were decreased in rat MIR groups butwere significantly increased after treatment with Que Tang Furthermore activation of SIRT1PGC1a pathwayupregulated Bcl2 expression and downregulated Bax exertingantiapoptotic effects The authors hypothesized that Que mightimprove MIRinduced myocardial damage via regulation ofSIRT1PGC1a and Bcl2Bax pathways Tang Que is also reported to regulate ROS generation and mitigatemitochondrial dysfunction by promoting their biogenesisSpecifically in a study to develop a therapeutic strategy forosteoarthritis Que was shown to increase expression levels ofSIRT1 PGC1a NRF1 and NFR2 TFAM and phosphoAMPKa in osteoarthritis rats confirming the hypothesis that Quemight act via the AMPKSIRT1 signaling pathway Qiu Overall these findings suggest that Que may counteractcardiovascular disease and oxidative damageThe growing body of evidence supporting the beneficial effects ofQue has led to its clinical use as demonstrated by the number ofclinical trials studies on ClinicalTrialsgov A list of completedstudies using Que in different metabolic and ‚ammatoryconditions is reported in Table Specifically a phase clinicaltrial NCT01839344 measured the effect of Que on glucosetolerance and postprandial endothelial function in subjects withT2D compared to the effect of an alphaglusidase inhibitor acarboseThe administration of g of Que led to a decrease in postprandialblood glucose NCT01839344 Given its antioxidative and anti‚ammatory capacities this flavonoid was considered a goodcandidate for antioxidant therapy in mucositis NCT01732393hepatitis C NCT01438320 idiopathic pulmonary fibrosisNCT02874989 osteoporosis NCT00330096 uric acidmetabolism NCT01881919 cytokine release NCT01106170and chronic obstructive pulmonary disease NCT01708278 Inthe latter study Que supplementation was safely tolerated bypatients with mildtosevere chronic obstructive pulmonarydisease opening the way towards the potential use of Que as atherapeutic agent for this conditionHowever as for RSV and nutraceuticals in general the resultsof molecular studies on Que obtained from in vitro investigationsand animal models are often inconsistent with data from clinicaltrials Concentration factor dose and timing of administrationand bioavailability are the two main issues that require furtherclarification Additional studies are needed to identify theoptimal concentration of Que for it to exert a beneficial effectfor example on insulin sensitivityBerberineBerberine BBR is an isoquinoline alkaloid reported to haveanalgesic anticancer anti‚ammatory and myocardialprotective properties Cicero and Baggioni It was foundFrontiers in Pharmacology wwwfrontiersinAugust Volume 0cIside et alNatural SIRT1 ActivatorsTABLE Quercetin in clinical trials for metabolic and ‚ammatory disordersStatusStudy TitleConditionsInterventionPhaseNCT NumberCompleted Effect of Quercetin in Prevention andTreatment of Oral MucositisBeneficial Effects of Quercetin in ChronicObstructive Pulmonary Disease COPDCompletedwith resultsCompleted QTrial in Patients With Hepatitis CCompleted Effects of Quercetin on Blood Sugar andChemotherapy Induced OralMucositisChronic ObstructivePulmonary DiseaseChronic Hepatitis CDiabetes Mellitus Type mg daily for weeksPhase NCT01732393 to mg daily for weekPhase NCT01708278 days mg oral single dose of mgPhase Phase NCT01438320NCT01839344Blood Vessel Function in Type DiabetesCompleted Effect of Quercetin Supplements onCompletedHealthy Males a 4Week RandomizedCrossOver TrialTargeting ProInflammatory Cells inIdiopathic Pulmonary Fibrosis a HumanTrialCompleted Efficacy of Provex CV Supplement toReduce Inflammation Cytokines andBlood PressureHyperuricemia Gout KidneyCalculi DiabetesCardiovascular DiseaseIdiopathic Pulmonary FibrosisIPFBlood Pressure mg tablet for days with meal breakfastpreferredEarly PhaseNCT01881919 doses administered over consecutive days in consecutive weeks oral administration ofquercetin mg daily mg of Provex CV supplement by mouth perday for weeksPhase NCT02874989Phase NCT01106170Completed Effects of Hesperidin on Bone MineralOsteoporosis OsteopeniaPhase NCT00330096Density and Bone Metabolism ofPostmenopausal Womento exert protective antioxidative effects in different physiologicand pathologic conditions Huang Li 2020bHowever the mechanisms underlying these effects remainunclear BBR was described as a potential antitumor agent thatinduces cell cycle arrest in G0G1 phase increases Cipp21 andKipp27 protein expression decreases expression of cyclin D1D2 and DE and the cyclindependent kinases Cdk2 Cdk4 andCdk6 promoting apoptosis in HL60 human leukemia cellsLi BBR can also deregulate telomerase activityand promote mitochondriadependent apoptosis in HepG2human hepatocarcinoma cells through caspase and caspase activation PARP cleavage induction increased expression ofthe proapoptotic protein Bax through activation of FOXOtranscription factors and inhibition of Bcl2 and Bclx antiapoptotic protein expression Hwang BBR wasobserved to exert an apoptotic effect by inducing ROSproduction and increasing MAPK and JNK activity of p38 inSW620 human colon carcinoma cells and by increasing Ca andcytochrome C release in HSC3 squamous cells Song In addition BBR inhibits the proliferation of cancer cellsthrough an anti‚ammatory pathway In oral carcinoma celllines and in SCC4 cells BBR inhibits expression of COX2 andAP1 bond decreases prostaglandin E2 PGE2 production andsuppresses NFkB IKK ERK and JNK activities FurthermoreBBR can inhibit colon cancer cell growth by activating retinoid Xreceptor a RXRa which binds RXRa and promoting bcatenin degradation Ruan However some studieshighlighted the potential ability of BBR to prevent oxidativestressinduced senescence by activating AMPK and restoringNAD levels Song Initial research revealed a significant role of SIRT1 signalingin mediating the antioxidant effect of BBR in diabetes Pang and in lipid metabolism Hasanein Thelipidlowering activity mediated by cotreatment with BBR andRSV was investigated in mice exposed to a high fat diet Zhu In vivo data showed that BBR combined with RSVlowered total cholesterol triglyceride and LDL cholesterol levelsin mice These findings were also confirmed in vitro with 3T3L1adipocytes treated with BBR or RSV alone Specifically BBR andRSV cotreatment was able to reduce lipid accumulation morerobustly than single treatments BBR in combination with RSVdisplayed hypolipidemic effects likely mediated by SIRT1expression regulation Moreover BBR pretreatment seemed tocounteract SIRT1 downregulation Zhu The antioxidant and anti‚ammatory effects of BBR werealso investigated in heart Yu BBRmediated SIRT1activation reduced MIR injury by affecting oxidative damageand ‚ammation signaling Specifically BBR exerted anantioxidant effect by decreasing the generation of cardiacsuperoxide and gp91phox expression and by increasing SODlevels Yu A previous study had also shown thatSIRT1 activation promotes antioxidant molecule production anddecreases proapoptotic proteins through FOXO1 activationthus protecting against MIR lesions Hsu As well as activating SIRT1 BBR is also able to decreaseFOXO1 acetylation triggering antiapoptotic signaling pathwaysvia Bcl2 expression and Bax and caspase3 downregulation Yu A very recent report described the protective effect of BBRagainst doxorubicininduced cardiovascular damage Wu This effect is

Breast cancer is a common malignancy in women Among breast cancer types triplenegative breast cancer TNBC tends to affect younger women is prone to axillary lymph node lung and bone metastases and has a high recurrence rate Due to a lack of classic biomarkers the currently available treatments are surgery and chemotherapy no targeted standard treatment options are available Therefore it is urgent to find a novel and effective therapeutic target As alteration of ion channels and transporters in normal mammary cells may affect cell growth resulting in the development and progression of TNBC ion channels and transporters may be promising new therapeutic targets for TNBC This review summarizes ion channels and transporters related to TNBC and may provide new tumor biomarkers and help in the development of novel targeted therapiesKeywords Triplenegative breast cancer Ion channels Ion transporters Pathological roles Targeted therapyBackgroundBreast cancer BC is the common malignancy in women its incidence is increased each year [] and it has become a significant threat to women™s health [] BC is a heterogeneous disease that can be divided into multiple molecular subtypes based on estrogen receptor ER progesterone receptor PR and human epidermal growth factor receptor HER2 expression providing important prognostic and predictive information [] There are four BC subtypes depending on receptor status luminal A luminal B HER2overexpressing and triplenegative breast cancer TNBC Among them TNBC is defined as ER PR and HER2 negative and it tends to affect younger women a0years of age it is Correspondence onlyoneliuxuemei163com 0078029sinacom Chengli Lu and Zhiyuan Ma contributed equally and share first authorship Department of Thyroid and Breast Surgery Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province China Department of Gastroenterology Affiliated Hospital of Zunyi Medical University Zunyi Guizhou Province ChinaFull list of author information is available at the end of the prone to axillary lymph node lung bone metastases and has a high recurrence rate [ ] Lehmann et a0al classified TNBC into six subtypes based on gene cluster sequence expression basallike basallike immunomodulatory mesenchymal mesenchymal stemlike and luminal androgen receptor subtypes [] After analyzing the RNA and DNA profile of TNBC tumors Matthew et a0 al classified TNBC into four subtypes including luminal androgen receptor mesenchymal basallike immunesuppressed and basallike immuneactivated subtypes [] The two classification methods have similarities and both provide theoretical bases for exploring targeted therapies for TNBCAlthough TNBC is the BC subtype that responds best to chemotherapy its recurrence and metastasis rates are higher than those of other BC subtypes [] Furthermore due to the lack of classic biomarkers TNBC lacks standard treatments guided by tumor biology and only surgery and chemotherapy are currently available as treatments [] Previous studies have shown that ion channels and transporters play important regulatory roles in mammary physiology and the initiation and progression of BC The Authors This is licensed under a Creative Commons Attribution International License which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate if changes were made The images or other third party material in this are included in the ™s Creative Commons licence unless indicated otherwise in a credit line to the material If material is not included in the ™s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder To view a copy of this licence visit httpcreat iveco mmons licen sesby40 The Creative Commons Public Domain Dedication waiver httpcreat iveco mmons publi cdoma inzero10 applies to the data made available in this unless otherwise stated in a credit line to the data 0cLu a0et a0al Cancer Cell Int Page of [] However the detailed functional role of ion channels and transporters in TNBC has not been clarified and summarized In recent studies upregulation of NaH exchanger has been shown to promote the proliferation migration and invasion of the TNBC cell line MDAMB231 [ ] In addition Ca2 channels such as mitochondrial calcium uniporter MCU can promote TNBC cell migration invasion and lung metastasis [] and Alvarez et a0al [] reported that the twopore domain potassium channel KCNK5 is associated with a poor prognosis in TNBC Therefore ion channels and transporters play important regulatory roles in the pathophysiology of TNBC but there is currently no relevant review on this topic Here we review the pathological roles of ion channels and transporters including AQPs Clˆ’ channels Ca2 channels K channels and acidbase transporters in the initiation and progression of TNBCAQP channelsAQPs which compose a family of transmembrane water channel proteins modulate the movement of water and small solutes into and out of cells and maintain suitable concentrations of water and solutes for cell survival [] At least AQP subtypes AQP012 have been identified in mammals and are divided into two families based on transfer specificity namely the classic watertransporting AQP family and the solute water and glyceroltransporting glycoprotein family [] AQP02 AQP4 to AQP68 are mainly waterselective AQP3 AQP7 AQP9 AQP10 and AQP12 also transport glycerol and possibly other small solutes AQPs also play roles in the transport of ammonia urea carbon dioxide metalloids nitric oxide and certain ions [] Expression of AQP1 AQP35 and AQP1012 has been detected in normal human mammary tissue and is closely related to milk secretion [ ] In addition deletion of œCCAATenhancer binding protein a family of transcription factors isoforms results in changes in mammary ductal morphogenesis and changes in expression of transport proteins such as AQP5 suggesting that AQP5 may be involved in mammary development [] Recent studies have shown that AQPs play carcinogenic roles by promoting angiogenesis enhancing invasive and metastatic potential and enhancing the transport of reactive oxygen species ROS [ ] In femalespecific cancers such as BC AQP1 and are the most important AQPs and they are been reported to be upregulated []AQP1 the membrane protein was the first reported mammalian AQP and plays a significant role in tumor cell migration proliferation and angiogenesis [] Clinical studies have shown that patients with TNBC have higher levels of AQP1 expression and that upregulation correlates with a poor prognosis [ ] AQP1 expression is induced by hypoxia through the EBoxChoRE transcription element which is affected by increased glucose consumption and metabolism [] AQP1 expression has been detected only in a subgroup of CK14positive basallike breast cancer BLBC cases [] CK14 has been used as a marker of basal mammary epithelial cells with in vivo regenerative ability in studies on mammary gland progenitor and stem cells [] Therefore it is speculated that expression of AQP1 is related to the stem cell characteristics of BLBC cells Hu et a0al demonstrated that AQP1 upregulation promotes extravasation and increases migration in a0vivo and in a0vitro in the mouse TNBC cell line 4T1 suggesting that this aquaporin enhances the rate of cell migration by promoting water permeability in cell protrusions [] Thus upregulation of AQP1 can promote the proliferation migration and invasion in TNBC cells Moreover in a0 vivo experiments have shown that AQP1 deficiency can reduce tumor mass volume vessel density and lung metastases in MMTVPyVT mouse mammary tumor virusdriven polyoma virus middle T oncogene mice and inhibition of AQP1 function andor expression is predicted to attenuate angiogenesis via reduced migration and invasion of endothelial cells [] Recently Irene AbreuRodriguez et a0al [] revealed that AQP1 expression is also responsive to hypoxiainducible factor HIF which may play a role in the VEGFindependent signaling mechanism inducing angiogenesis in a hypoxic environment Helen et a0al [] also reported that the triterpenoid saponins bacopaside I and bacopaside II can synergistically reduce the transcriptional expression of AQP1 and inhibit proliferation migration and invasion in MDAMB231 cells Similarly ginsenoside Rg3 a compound with anticancer activity isolated from ginseng inhibits AQP1 to attenuate cell proliferation through a mechanism that involves downregulation of AQP1 to induce cell cycle arrest in G0G1 phase by inhibiting cyclin D and E and inhibition of chemoattractantinduced cell migration and invasion by blocking AQP1mediated water flux in MDAMB231 cells [] These findings indicate that AQP1 plays an important role in the development and progression of TNBCOverexpression of AQP3 has been detected in the membranes and cytoplasm of TNBC tumor cells and is significantly associated with poor prognosis [] XuChen Cao et a0 al [] found that the presence of fibroblast growth factor2 FGF2 induced cell migration and metastasis in MDAMB231 cells by increasing AQP3 expression Moreover FGF receptor kinase FGFRK inhibitors PI3K inhibitors and MEK12 inhibitors all inhibit AQP3 expression suggesting that FGF receptor kinases increase AQP3 expression and promote FGF2induced cell migration by initiating downstream PI3K and ERK pathways In addition CuSO4 a water transport 0cLu a0et a0al Cancer Cell Int Page of inhibitor of AQP3 inhibits migration in MDAMB231 cells AQP3 downregulation reduces the proliferation invasion and migration of MDAMB231 cells while increasing sensitivity to 5fluorouracil chemotherapy The mechanism may be related to a decrease in glycerol permeability caused by AQP3 downregulation [] Overall these findings demonstrate that AQP3 plays a pivotal role in the initiation and progression of TNBC and specific inhibitors of AQP3 in clinical applications may improve the therapeutic effect of TNBC patientsSimilarly overexpression of AQP5 in the membrane and cytoplasm of TNBC cells has been detected and is significantly associated with poor prognosis [] Moreover patients with higher Ki67 expression are more likely to have abnormal AQP5 protein expression than patients with lower Ki67 expression [] Ki67 is a widely accepted proliferation marker [ ] and it is speculated that upregulation of AQP5 may promote proliferation in TNBC cellsIn summary AQP1 AQP3 and AQP5 are significantly upregulated in TNBC this upregulation is related to a poor prognosis and can promote the proliferation migration and invasion of TNBC cells These AQPs are promising new targets for the diagnosis and treatment of TNBCClˆ’ channelsCFTRCFTR is a member of the ATPbinding cassette transporter family that localizes at the apical membranes of normal epithelial cells CFTR is mainly responsible for conducting HCO3ˆ’ and Clˆ’ and promoting HCO3ˆ’ secretion in many tissues including the airway intestines and pancreas [] However when the extracellular concentration of Clˆ’ is higher than a0mmolL the permeability of CFTR to Clˆ’ is much greater than that of CFTR to HCO3ˆ’ thus CFTR mainly conducts Clˆ’ under physiological conditions [] CFTR can also transport two other anions glutathione and thiocyanate which are involved in airway inflammation and oxidative stress [ ] Interestingly Pierre et a0al [] reported that CFTR is required for the tightly connected functions of normal epithelial tissues loss of CFTR reduces epithelial resistance and epithelial integrity and this effect is not related to the anion channel function of CFTRCFTR has been reported to be associated with several cancers such as cervical cancer [] colorectal cancer [] prostate cancer [] and BC [] Significant downregulation of CFTR expression is observed in BC tissue compared to normal mammary tissue [] Zhang et a0al demonstrated that overexpression of CFTR inhibits EMT invasion and migration in MDAMB231 cells via a mechanism that involves CFTR inhibition of NFκB targeting of urokinasetype plasminogen activator [] In addition CFTR overexpression inhibits the EMT and the invasiveness of MDAMB231 cells and reduces lung metastasis in xenograft models Increasing evidence reveals that downregulation of CFTR occurs after treatment with EMTinducing factors such as TGF suggesting that as a downstream effector CFTR plays important roles in mediating various EMT effects [ ] Moreover hypermethylation of the cancer genome leads to activation of oncogenes or suppression of tumorsuppressor genes thereby resulting in tumorigenesis [] It has also been observed that the methylation level of CFTR in BC tissues is much higher than that in normal tissues and treatment with DNA methylation inhibitors in TNBC cell lines MDAMB231 and MDAMB435 can rescue CFTR mRNA indicating that CFTR methylation plays an important role in TNBC [] ΔF508 is the most common mutation in CFTR causing the protein to be retained and degraded in the endoplasmic reticulum due to misfolding [] It is worth noting that although there is no difference in the incidence of BC between ΔF508 carriers and noncarriers patients with ΔF508 CFTR mutations all have grade III cancer indicating that CFTR defects are associated with BC progression [] Therefore CFTR methylation or mutation need to be further investigated in the future which may provide novel therapeutic intervention for TNBCChloride channel The chloride channel ClC family also plays an indispensable role in the transport of Clˆ’ [] There are nine family members in humans which are divided into two categories based on their distribution and physiological function Clˆ’ channel proteins ClC1 ClC2 ClCKa and ClCKb which mainly exist in the plasma membrane and play roles in stabilizing membrane electric potential or mediating epithelial transport and Clˆ’H reverse transporter proteins ClC37 which mainly exist in the vascular intima of the endosomelysosomal pathway and are localized at the plasma membrane only to a limited extent due to protein degradation and hydrolase activity [ ] In recent years it has been discovered that ClC3 can transport one hydrogen ion in exchange for two chloride ions [] with important roles in cancers such as nasopharyngeal carcinoma [] and BCClC3 overexpression is observed in tissues and the TNBC cell line MDAMB231 [ ] Studies by Zhou et a0 al revealed that knockdown of ClC3 downregulates expression of cyclin D1 and cyclin E and increases levels of p21 indicating that knockdown of ClC3 can block the cell cycle of MDAMB231 cells at G0G1 phase inhibiting cell proliferation Moreover knockdown of ClC3 suppresses tumor growth in xenograft models and significantly reduces levels of pERK12 in MDAMB231 cells 0cLu a0et a0al Cancer Cell Int Page of This indicates that ClC3 can promote the progression of TNBC by acting on the ERK12 signaling pathway [] Nevertheless relative research on ClC3 in TNBC is still very limited and extensive work is needed in the furtherCa2 channelsCa2 is a key nutrient in milk that plays a vital role in the mineralization of bones and teeth and as a second messenger ionized Ca2 is a key regulator of proliferation migration cell cycle progression and apoptosis [] The level of Ca2 is very low in the cytoplasm ˆ’ a0molL whereas it is somewhat higher in anelles ˆ’ a0 molL and highest in the extracellular level milieu ˆ’ a0 molL Hence a small amount of Ca2 can significantly change intracellular levels to activate downstream signaling molecules including calmodulin nuclear factor of activated Tcells NFAT NFκB calmodulindependent protein kinase II calpain and others [ ] In nonexcitatory mammary cells calcium channels play important roles in lactation and the maintenance of normal physiological functions [ ]Continuous increases in intracellular Ca2 levels will drive expression of oncogenes resulting in tumor growth and development especially the metastatic behavior of cancer cells and conferring tumor cells with resistance to apoptosis [] Abnormal expression of several Ca2 transporters and ion channels such as calcium releaseactivated calcium modulator Orai1 has been observed in TNBC and may lead to oncogenic Ca2 signaling [] Interestingly specific changes in the expression and function of Ca2 channels are related to hormone receptor status and differ significantly among BC subtypes []Calcium modulator Ca2 influx mainly depends on storeoperated calcium channels SOCCs When the Ca2 concentration in the endoplasmic reticulum declines to a certain level the STIM stromal interaction molecule which is located on the endoplasmic reticulum membrane moves to a position close to the highly selective calcium channel protein Orai on the cell membrane Subsequently Orai is activated to cause Ca2 influx and storeoperated calcium entry SOCE is initiated thereby replenishing the calcium store Some researchers have proposed that the canonical transient receptor potential TRPC also participates in the above process though the mechanism remains controversial There are two different claims that both Orai and TRPC form independent channels activated by the STIM protein and that Orai and TRPC subunits form heterochannels triggered by STIM [] There are three Orai1 isomers Orai1 to Orai3 and two STIM homologs STIM1 and STIM2 SOCE has been found to be primarily mediated by Orai1 and STIM1 in TNBC [] Compared with that in nonmalignant breast epithelial cells expression of Orai1 and STIM1 is significantly higher in TNBC cell lines and is associated with a poor prognosis [ ] Liu et a0 al [] reported that hypoxia can induce expression of Orai1 Notch1 and Jagged1 and Orai1 is significantly downregulated after blockade of Notch signaling suggesting that hypoxia can increase Orai1 expression in TNBC by activating Notch signaling Notch1Orai1SOCENFAT4 axis Similarly Mognol et a0al [] found that Orai1 promotes the invasion and angiogenesis of TNBC cell lines and activates NFAT4 which can regulate genes involved in the cell cycle apoptosis angiogenesis and metastasis In addition Yang et a0 al [] demonstrated that Orai1 and STIM1 promote the migration and invasion of MDAMB231 cells both in a0vivo and in a0vitro and the authors proposed that these proteins may at least partially control cell migration by regulating focal adhesion turnover Furthermore treatment with TGF can reduce expression of STIM1 whereas blockade of SOCE can impair TGFinduced G0G1 cell cycle arrest and inhibit the proliferation of MDAMB231 cells [] Based on the above research Orai1 and STIM1 may be new therapeutic targets for TNBC Indeed some selective SOCE inhibitors have shown encouraging inhibitory effects on TNBC but they are still only in the preclinical trial stage For example phemindole a diindole derivative reduces SOCE by downregulating STIM1 expression significantly inhibits the proliferation and migration of MDAMB231 cells reduces the growth of solid tumors in mouse models and produces a targeted antitumor effect in TNBC [] In addition Miroslava Didiasova et a0al [] revealed that elevated cell surfaceassociated enolase1 ENO1 expression correlates with augmented MDAMB231 cell migratory and invasive properties Pharmacological blockade a selective SOCC inhibitor NS1643 or knockdown of STIM1 or Orai1 reduces ENO1dependent migration of MDAMB231 cells These results demonstrate the pivotal role of SOCE in the regulation of ENO1 exteriorization and thus in the modulation of TNBC cell migratory and invasive properties indicated that Orai1 and STIM1 might be promising threptic targets for TNBCSecretory pathway Ca2‘ATPaseThe secretory pathway Ca2ATPase SPCA can direct Ca2 and Mn2 from the cytoplasm to the Golgi and postGolgi vesicles Two isotypes SPCA1 and SPCA2 are known and the distribution and function of the two differ SPCA1 is commonly expressed in mammalian tissues expression of SPCA2 is limited to highly absorptive and secretory epithelial cells including mammary 0cLu a0et a0al Cancer Cell Int Page of and salivary gland cells [] SPCA1 is highly expressed in TNBC cell lines and SPCA2 is highly expressed in cell lines of other subtypes [] Interestingly based on clinical samples Desma et a0al reported SPCA1 levels to be significantly elevated in the basal subtype of BC compared with all other subtypes and it is worth noting that changes in its expression affect posttranslational modification and transport of certain proteins important for tumor progression without significantly changing cytosolic calcium signaling SPCA1 inhibition also decreased MDAMB231 cell proliferation [] Moreover SPCA1 is a key regulator of insulinlike growth factor receptor IGF1R processing in TNBC cells and promotes the production of functional IGF1R IGF1R activity is associated with poor prognosis suggesting that targeting SPCA1 is an alternative IGF1Rinhibiting strategy [ ] Overall upregulation of SPCA1 may promote the initiation and progression of TNBC The main mechanism reported to date involves SPCA1mediated increase in functional IGF1R expressionMitochondrial calcium uniporterUpregulation of MCU expression on the mitochondrial membrane is closely related to a poor prognosis in BC [] miR340 correlates negatively with the metastatic potential of TNBC cells [] it may directly inhibit MCU expression to reduce glycolysis and exercise capacity and knockdown or inhibition of MCU inhibits the growth invasion and metastasis of MDAMB231 cells [] Interestingly Anna et a0 al [] demonstrated that mitochondrial Ca2 uptake is required for TNBC progression in a0vivo and that absorption of Ca2 by mitochondria promotes the production of sustained mitochondrial reactive oxygen species activating the HIF1α signaling pathway and promoting tumor growth and metastasis In addition inhibiting or silencing MCU also block seruminduced migration of MDAMB231 cells and reduce serum or thapsigargininduced SOCE suggesting that MCU promotes TNBC cell migration by regulating SOCE [] The above results indicate that overexpression of MCU may play an important oncogenic role in the growth invasion and metastasis of TNBC cells However the precise mechanism is unclearOther promising calcium channel targets in TNBC include TRPV6 [] Overall calcium channels are promising targets for TNBC treatment but most compounds targeting these channels are only in the preclinical trial stage Thus further research is neededK channelsThrough the action of NaKATPase two K molecules are transported into a cell in exchange for three sodium molecules which increases the intracellular K concentration K channels on the cell membrane are numerous and in humans more than genes encode major K channel subunits [] K channels play key roles in maintaining acidbase balance by functioning in concert with the NaH exchanger and NaKATPase [] controlling electrical excitability of nerves and muscles and participating in energy metabolism and other physiological processes In addition K channels can help regulate cell proliferation and cell cycle progression and are involved in tumorigenesis [] Many studies have reported dysregulated K channel expression in human cancers including BC astrocytictype brain cancer and prostate cancer [ ] Tumorrelated K channels can be divided into four main categories according to their domain structures and activation mechanisms voltagegated potassium channels which are controlled by changes in membrane potential calciumactivated potassium channels which are activated by intracellular calcium inwardly rectifying potassium channels and twoporedomain potassium channels K2P KCNK [] However the carcinogenic mechanism of K channels remains rather clear Nuria et a0 al [] proposed that K channels may participate in and regulate tumor progression through permeationrelated mechanisms including changes in membrane potential Ca2 driving forces and cell volume regulation and nonconductive mechanisms dependent on proteinprotein interactionsThe Kv111 channel also known as human etheragogorelated gene hERG1 is not expressed in normal breast cells but is expressed in BC with a relationship with subtype Indeed TNBC exhibits lower expression of Kv111 compared with other subtypes [] Olivia Crociani et a0al [] showed that the mRNA levels of Kv111 change throughout the cell cycle peaking in G0G1 phase Moreover Lansu et a0al [] reported that stimulation of Kv111 led to inhibition of proliferation in MDAMB231 cells and that an agonist the diphenylurea derivative NS1643 caused a significant inhibition of cell proliferation This phenomenon can be linked to a rapid decrease in the cyclin E2 protein level which causes accumulation of cells in G0G1 phase and an increase in tumor suppressor proteins and markers for cellular senescence including p21 p16INK4a and galactosidase activity Therefore Kv111 inhibits TNBC cell proliferation by activating a cellular senescence program [] Breuer et a0 al confirmed that NS1643 reprograms the EMT by attenuating the Wntcatenin signaling pathway inhibits cancer cell stemness and significantly reduces the metastatic spread of breast tumors in a MDAMB231 mouse model [] Regardless cardiotoxicity is an important limiting factor for potential therapeutic molecules acting on Kv111 Although the activator is well tolerated 0cLu a0et a0al Cancer Cell Int Page of in BC potential effects include tachycardia [] Overall the potential benefits of Kv111 activators as anticancer drugs outweigh their side effectsIn addition many other channels are altered in TNBC For example some K2P channels with differential expression may serve as novel molecular markers associated with TNBC RNASeq analysis of K2P channels has shown that overexpression of KCNK5 KCNK9 and KCNK12 and low expression of KCNK6 and KCNK15 are related to TNBC [] The above findings indicate that K channels play an important role in TNBC and are expected to be diagnosis markersAcid‘base transportersThe pH of milk is significantly lower than that of plasma indicating that there may be some acidbase transporters in the mammary gland that regulate the pH between the extracellular fluid and milk [] A uniform feature among solid tumors with high metabolic and proliferative rates is a significantly different pH from that of normal tissue [] Cancer cells can maintain a weakly acidic intracellular pH that is even more alkaline than that of normal cells suggesting that tumor cells have a powerful pH regulation system a0[]The NaH exchanger NHE a membrane transporter mainly catalyzes the exchange of intracellular H for extracellular Na in mammals thereby maintaining the pH balance inside and outside the cell [ ] There are subtypes of NHE with tissue and membranespecific expression patterns NHE15 are located on the plasma membrane and NHE69 are on intracellular anelle membranes NHE10 is only expressed in osteoclasts [] In addition NHE plays indispensable roles in maintaining normal mammary structure and physiological functions [] NHE1 SLC9A1 is universally expressed in epithelial cells and upregulated in BC tissues compared to normal tissues [] Studies have shown that hypoxia various growth factors and hormones among others can activate NHE1 and enhanced NHE1 activity can reduce extracellular pH and promote metastasis of MDAMB231 cells [] Furthermore it has been proposed that NHE1 promotes metastasis and remodeling of the extracellular matrix by acidifying the extracellular microenvironment [] In addition NHE1 knockdown reduces the migration invasion and growth of xenograft tumors of MDAMB231 cells increasing the susceptibility of these cells to paclitaxel [ ] Moreover knockdown of NHE1 or NBCn1 SLC4A7 in the MDAMB231 cell line significantly reduced the steadystate intracellular pH value after acid load the ability to restore pHi and the primary tumor growth of xenografts in a0 vivo but NBCn1 knockdown prolonged tumorfree survival and reduced cell proliferation [ ] It has been confirmed that NHE1 and NBCn1 promote the development of TNBC through different mechanisms There are two main NHE1 inhibitors amiloride and cariporide which are more effective than amiloride and highly selective [] Amiloride is a potassiumsparing diuretic and has blocking effects on a variety of ion channels such as NHE and the NaCa2 exchanger Cariporide is a highly specific and powerful NHE1 inhibitor that is relatively well tolerated in humans with heart disease [] Moreover a study has suggested that KR33028 a novel small molecule inhibitor of NHE1 produces a cellular phenotype comparable to that of NHE1 knockout cells and significantly decreases rates of migration invasion and colony growth in TNBC cell lines MDAMB231 MDAMB468 and Hs578T [] The above findings suggest that NHE1 may play an important role in the progression TNBCAdditionally other acidbase transporters are also altered in TNBC and are expected to emerge as new targets for TNBC treatment For instance NBCe1 SLC4A4 knockdown reduces cell proliferation invasion and migration in TNBC cells expressing high levels of NBCe1 [] The above findings all suggest that the acidbase transporters have essential functions in the occurrence and development of TNBC but further research is neededConclusionsDysfunction of ion channels and transporters in the mammary resulted in development and progression of TNBC Despite extensive work has been performed to investigate the expression pattern functional diversity regulatory mechanism and pathophysiology of different ion transporters in TNBC the systematic review is rare in this field Therefore this review focuses on different pathological function of multiple families in the development and progression of TBNC including the AQPs Clˆ’ channels Ca2 channels K channels and acidbase transporters Fig a0 Table a0 We hope that we can provide a basic systemics and summarised knowledge to this field advocating researchers play more attention on the pathophysiological role of ion channels and transporters in the development and progression of TNBC which may provide novel targets for the clinical diagnosis and treatment of TNBC 0cLu a0et a0al Cancer Cell Int Page of Fig Pathological roles of ion channels and transporters in triplenegative breast cancer cells Alteration and dysfunction of AQPs Clˆ’ channels Ca2 channels K channels NaHCO3ˆ’ transporter and NaH exchanger results in abnormality of ion transport and disorder of multiple signaling pathway including WNT PI3K TGF Notch and VEGF etc eventually promoting TNBC cell proliferation migration and invasion but inhibiting apoptosis

Construction of a novel prognosticpredicting modelcorrelated to ovarian cancerWeichun Tang12 Jie Li3 Xinxia Chang12 Lizhou Jia1 Qi Tang12 Ying Wang4 Yanli Zheng4 Lizhou Sun5 andZhenqing Feng121National Health Commission Key Laboratory of Antibody Technique Nanjing Medical University Nanjing People™s Republic of China 2Department of Pathology Nanjing MedicalUniversity Nanjing People™s Republic of China 3Department of Nursing The Second Affiliated Hospital of Nantong University Nantong People™s Republic of China 4Department ofGynaecology and Obstetrics The Second Affiliated Hospital of Nantong University Nantong People™s Republic of China 5Department of Obstetrics and Gynecology First AffiliatedHospital of Nanjing Medical University Nanjing People™s Republic of ChinaCorrespondence Zhenqing Feng fengzhenqingnjmueducn or Lizhou Sun lizhou sun163comBackground Ovarian cancer OC is one of the most lethal gynecological cancers worldwide The pathogenesis of the disease and outcomes prediction of OC patients remainlargely unclear The present study aimed to explore the key genes and biological pathwaysin ovarian carcinoma development as well as construct a prognostic model to predict patients™ overall survival OSResults We identified upregulated and downregulated differentially expressedgenes DEGs associated with OC Gene Ontology GO term enrichment showed DEGsmainly correlated with spindle microtubes For Kyoto Encyclopedia of Genes and GenomesKEGG pathways cell cycle was mostly enriched for the DEGs The protein“protein interaction PPI network yielded nodes and edges Top three modules and ten hub geneswere further filtered and analyzed Three candidiate drugs targeting for therapy were alsoselected Thirteen OSrelated genes were selected and an eightmRNA model was presentto stratify patients into high and lowrisk groups with significantly different survivalConclusions The identified DEGs and biological pathways may provide new perspective onthe pathogenesis and treatments of OC The identified eightmRNA signature has significantclinical implication for outcome prediction and tailored therapy guidance for OC patientsBackgroundOvarian cancer OC is the most lethal malignant disease in the female reproductive system with over new cases and deaths each year worldwide [] Epithelial OC accounts for “ ofovarian malignancies listed as the most common histological type Since the ovaries locate in the deeppelvis with mere symptoms emerging at the beginning of ovarian morbid change the early detectionfor the malignancy is truly difficult Hence when OC is detected the patient is usually at an advancedstage with invasion and metastasis accompanied [] For patients in the early stage the 5year survivalrate can reach “ whereas for advancedstage patients the number is mere ˆ¼ [] Thereforeit is imperative to explore the molecular mechanisms of malignant biological behavior of OC cells andto develop more reliable molecular markers for predicting recurrence and evaluating prognosis furtherguiding clinicians for therapyAt present various highthroughput microarrays and nextgeneration sequence genomic datasetswhich were deposited in the Gene Expression Omnibus GEO [] and The Cancer Genome Atlas TCGAdatabases have been widely analyzed for identifying differentially expressed genes DEGs which couldserve as candidate diagnostic or prognostic markers further effectively improving our understanding ofthe disease from genetic perspective Whereas since the existence of tissue or sample heterogeneity inThese authors contributedequally to this workReceived April Revised July Accepted July Accepted Manuscript online July Version of Record published August The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261each independent experiment as well as the discrepancy of different data processing methods and technology platforms the DEGs identified from a singlecohort study may generate false positives Herein the Robust Rank Aggregation RRA method which analyzes the significant probability of all elements by a probabilistic model is developedto identify statistically significant genes from multiple datasets and provide more accurate and valuable informationfor clinical use far beyond one gene list [] To date a bunch of novel prognostic markers have been discovered topotentially improve the efficacy of diagnosis and prognosis of OC [] However these identified markers were onlyeffective for partial stages or grades and were difficult to apply widely Hence a prognostic model which is basedon signature gene expression level with high discriminating power to effectively assist prognosis prediction for eachpatient is required in clinical practiceIn the present study we downloaded six primary microarray datasets from the GEO database which containeda total of samples with OC samples and normal samples The geneset and relative clinical information on ovary tissues of OC patients and healthy females from TCGA and GTEx portal were also downloadedIntegrated DEGs between cancerous and normal ovarian samples were screened using the ˜limma™ R package andRRA method Gene Ontology GO and Kyoto Encyclopedia of Genes and Genomes KEGG pathways enrichmentof DEGs were performed for nextstep functional analysis The Search Tool for the Retrieval of Interacting GenesDatabase STRING and the Connectivity Map CMap online database were then used to analyze the association ofDEGs and explore the molecular mechanisms as well as drugs involved in tumorigenesis Through survival analysisprognostic mRNAs were also selected By performing Cox regression analysis we identified an eightmRNA signature model with the ability to predict the prognosis of OC patients and independent from clinical factors Our studyprovides reliable molecular markers and prognostic models for early detection and outcome prediction as well aseffective drug targets for treating OCMethodsData collectionThrough searching on the GEO Repository with ˜ovarian cancer™ we downloaded the gene expression profiles ofGSE54388 GSE40595 GSE38666 GSE27651 GSE18520 and GSE14407 and the corresponding annotation files fromthe GPL570 [HGU133 Plus ] Affymetrix Human GenomeU133 Plus Array platform GSE54388 contains ovarian tissue samples with normal ovarian surface epithelium and tumor epithelial components from highgradeserous OC patients GSE40595 includes OCassociated stroma and epithelium samples which consist of cancerassociated stroma samples and epithelial tissues from highgrade serous OC patients along with stromal component and ovarian epitheliums from the normal ovary GSE38666 comprises stroma and matchedovarian epitheliums from healthy females as well as cancer stroma and matched cancer epitheliums from OCpatients GSE27651 incorporates normal ovarian surfaces epithelial and serous borderline ovarian tumors lowgrade serous ovarian carcinomas and highgrade serous ovarian carcinomas GSE18520 covers advancedstage highgrade primary OC specimens and normal ovarian surface epithelium tissues GSE14407 involves healthy ovarian surface epithelial samples and paired serous OC epithelium Note that all samples from these GEOdatasets are classified into the cancerous or normal part to be clear the normal stromal and surface epithelium is defined as normal ovarian tissues whereas the borderline tumors as well as cancerous stromal and epithelial tissues areconsidered as malignancies Besides we also downloaded the FPKM format gene expression data and relative clinicalinformation of OC patients™ samples and normal ovarian tissues from TCGA and GTEx portal respectivelyScreening for DEGs and integration of microarray dataWe used the ˜limma™ R package [] to integrate the expression profiles from TCGA and GTEx portal standardize the data from the integrated TCGA and GTEx expression matrix as well as six GEO datasets and furtherscreen the DEGs between ovarian cancerous and normal samples The list of DEGs obtained from six GEO microarray datasets by limma analysis was further integrated by the ˜RRA™ method to get prioritized commonly upor downregulated gene list The final overlapped DEGs for subsequent biological function analysis were the combination of prioritized jointly dysregulated genes from six GEO microarrays and the results from TCGA and GTExdatabases The cutoff criteria were set as FDR and log2fold change FC GO term and KEGG pathway enrichment analysisGO classified the known genes into three main biological progress Molecular Function MF Cellular ComponentCC and Biological Process BP [] KEGG provides researchers highlayer functions of the biological system frommolecular level information [] The Enrichr online tool amppharmmssmeduEnrichr allows for GO term The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression heatmap of the top significantly dysregulated genes in six GEO datasetsHierarchical clustering that shows the expression profiles of mRNAs from A GSE14407 B GSE18520 C GSE27651 DGSE38666 E GSE40595 F GSE54388annotation and KEGG pathway for a cluster of genes [] We explored the biological functions of overlappedDEGs and hub modules from our protein“protein interaction PPI network using Enrichr website Pvalue was considered as significant enrichment Likewise the functional biological pathways of the top ten hub genes fromPPI network were also analyzed by the FunRich tool version [] and the top five enriched pathways of up anddownregulated genes were displayed as bar charts respectively We set the Pvalue as statistically significant The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261PPI network construction and analysisPPI networks display the relationships of various proteins according to their physical or biochemical propertiesSTRING is a database that encompasses the interaction information between known proteins and potentially interacted proteins [] In order to explore the correlations between the DEGs we used the STRING database to constructa PPI network and visualize our results by Cytoscape software [] Confidence score was set as significantMolecular Complex Detection MCODE was utilized to select hub modules of PPI networks in Cytoscape [] Weset the degree cutoff node score cutoff kscore and max Depth was set as the criterion Thenthe significant modules were performed by GO and KEGG analyses Top ten genes were defined according to thehigh degree of connectivity in STRING network [] The coexpression analysis of ten hub genes was performed bySTRING eitherValidation of the hub genesWe downloaded the raw geneset of OC patients from TCGA to explore the expression differences of hub genes inlow and highgrade tumor tissues of OC and draw the survival plot using Kaplan“Meier plotter webtool [] Thegene and protein expression level of graderelated hub genes were then confirmed by Oncomine and The HumanProtein Atlas HPA database [] Meanwhile we explored the genetic alteration information of the selected tenhub genes in OC patients by plugin cBioPortal cBio Cancer Genomics Portal tool which deposits the genomicsprofiles of various cancer types and provides analysis and visualization of the genomics datasets []Identification of candidate small molecule drugsThe CMap database was able to predict potential drugs which might reverse or induce the biological state encoded incertain gene expression signatures in OC [] The DEGs from our study were used to query the CMap databaseThe enrichment scores which represent the similarity were calculated ranging fromˆ’ to The positive connectivity score means an inducing influence on the input signature whereas drugs with negative connectivity score presentreversion impact on the characteristic in human cell lines and are considered as candidate therapeutic molecules After sorting all instances the connectivity score of various instances was filtered by Pvalue Next we investigatethe structures of these candidate molecular drugs from the Pubchem database pubchemncbinlmnihgovEstablishment and evaluation of the prognostic modelThe OC patients from the TCGA project were randomly classified into the training cohort n188 and the testingcohort n186 OSrelated genes were determined by performing univariate Cox regression analysis in the trainingcohort with the ˜Survival™ R package and further selected for the nextstep screening Least Absolute Shrinkage andSelection Operator LASSO is a parameter selection algorithm which shrinks all highdimensional regression coefficients and generates the penalty regularization parameter λ via the crossvalidation routine by ˜glmnet™ R packageTo select the optimal prognostic mRNAs we adopted LASSO regression among the selected candidate genes and further perform multivariate Cox proportional hazards regression to evaluate their independent prognostic values Theriskscore model for predicting outcomes of OC patients was the sum of each optimal prognostic mRNA expressionlevel multiplying relative regression coefficient weight calculated from the multivariate Cox regression modelRisk score patient cid2Coefficient mRNAi × Expression mRNAiiAll training cohort patients were classified into high and lowrisk groups according to the median risk score TheKaplan“Meier curves of two diverse groups were plotted using ˜survfit™ function and the receiver operating characteristic ROC curve was unfolded for OS prediction to estimate the sensitivity and specificity of the prognostic modelCox multivariate analysis was also performed to examine whether the risk score was independent of the clinical characters such as age tumor stage and grade Next we used the testing group to check the efficacy of the prognostic riskmodel Each individual in the testing cohort was also categorized as high or lowrisk case by comparing the patient™srisk score with the cutoff value calculated from the training cohort Kaplan“Meier curve analysis timedependentROC analysis and Cox multivariate analysis were performed eitherSearching tumorinfiltrating immune cells associated with patients™prognostic signaturesThe TIMER webtool allows for systematical evaluations of the relationship between the six immune cell types inthe tumor microenvironment which are B cell CD4 T cell CD8 T cell neutrophil macrophage as well as dendritic The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The volcano plot of all gene expression distribution in six GEO datasetsVolcano plot of differentially expressed mRNAs of A GSE14407 B GSE18520 C GSE27651 D GSE38666 E GSE40595 FGSE54388cell and clinical impact in various cancer types via a novel statistical method [] To further explore the prognosticsignature we used the TIMER online tool to search the most significant tumorinfiltrating immune cells according tothe TCGA OC gene data To be clear the relative gene expression levels of six types of immune cells for each patientin high and lowrisk groups from training and testing cohort were measuredResultsThe DEGs among six GEO microarray datasetsThe top significantly up and downregulated genes from each microarray dataset were displayed in the heatmapsFigure 1A“F and the distribution of all gene expression was presented in volcano plots Figure 2A“F ThroughRRA analysis of expression microarrays we identified DEGs which consisted of upregulated and downregulated genes and displayed the top dysregulated genes by ˜pheatmap™ R package in Figure Next weanalyzed the expression profiles of TCGA and GTEx getting dysregulated genes Intriguingly when these DEGs were combined with the DEGs from GEO datasets we found that genes were commonly dysregulatedin these two databases with upregulated Figure 4A and downregulated genes Figure 4BGO term and KEGG pathway enrichment analysis of DEGsTo study the potential biological function of the DEGs we performed biological pathway analysis and identifiedsignificantly enriched pathways via Enrichr web tools In GO term Figure 5A for the BP group the DEGs weremostly enriched in ˜regulation of attachment of spindle microtubes to kinetochore™ ˜cellular response to laminar fluidshear stress™ and ˜microtubule cytoskeleton anization involved in mitosis™ In MF group the dysregulated geneswere highly correlated to ˜microtubulebinding™ ˜microtubule motor activity™ and ˜tubulinbinding™ As for CC group The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Heatmap showing the top upregulated genes and top downregulated genes according to PvalueEach row represents one gene and each column indicates one dataset Red indicates upregulation and blue represents downregulation The numbers in the heatmap indicate log FC in each dataset calculated by the ˜limma™ R package Abbreviation log FClogarithmic fold changethe DEGs were closely related to ˜condensed nuclear chromosome kinetochore™ and ˜mitotic spindle™ KEGG pathwayanalysis showed DEGs highly enriched in ˜cell cycle™ and ˜Alanine aspartate and glutamate metabolism™ Figure5B The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The intersection of up and downregulated DEGs of GEO and TCGA datasetsA upregulated intersected DEGs in both datasets B downregulated intersected DEGs in both GEO and TCGA dataset Theintersected DEGs were defined as the significant DEGsPPI network construction and modules analysisUsing the STRING database and Cytoscape software a total of DEGs were mapped into the PPI network whichincluded nodes and edges Figure 6A The PPI enrichment Pvalue was 10e16 The top three key modules Figure 6C“E within PPI network were then selected Module MCODE score Module MCODEscore Module MCODE score and the biological function of Module which consisted of nodesand edges was further analyzed GO analysis indicated that Module1 was mainly associated with ˜regulation ofattachment of spindle microtubules to kinetochore™ ˜condensed nuclear chromosome kinetochore™ and ˜microtubulemotor activity™ KEGG analysis showed that ˜cell cycle™ and ˜oocyte meiosis™ were the most highly enriched pathwaysSupplementary Figure S1The screening of Hub genes and their characteristicsThe top ten hub genes with the highest degree of connectivity were CDC45 CDK1 TOP2A CDC20 CCNB1CEP55 UBE2C HMMR FOXM1 and TPX2 Figure 6B The coexpression analysis results of the hub genes demonstrated that these genes actively interacted with each other Supplementary Figure S2 Besides we established theinteraction network of ten hub genes with their related genes and explored the biological role Supplementary Figure S2AC“F of the network by FunRich The gene alteration type and frequency as well as the most frequentlyaltered neighbor genes were also exhibited Figure Gene alteration frequency of ten hub genes among TCGAOC samples was beyond with most genes showed amplified and multiple altered Figure 7AB The top threemost frequently altered genes were FOXM1 CDC20 and CCNB1 with FOXM1 and CDC20 largely amplified whileCCNB1 deep deleted Through analysis of OC patients™ geneset from TCGA we found that CCNB1 UBE2C CDK1CEP55 as well as FOXM1 expressed significantly higher in highgrade tumors and predicted worse outcomes sincepatients overexpressed above genes owned lower overall survival OS and diseasefree survival DFS rates Figure The Oncomine database showed results from various studies were consistent to our finding Supplementary Figure S3 The HPA website also demonstrate that proteins translated by such five hub genes were overexpressed in OCtissues Supplementary Figure S4 HMMR and TPX2 were also negatively correlated to patients™ prognosis while noexpression difference was observed in diverse tumor grades and CDC20 was positively associated with tumor gradebut not correlated to patients™ outcomesRelated small molecule drugs screeningIn total DEGs were analyzed in CMap to screen small molecule drugs and the candidate molecules with top tenconnectivity score are listed in Table Five of these molecules showed a negative correlation and suggested potentialin clinical applications Among them Trichostatin A pyrvinium and 8azaguanine showed a significantly negativecorrelation and the stuctures of such candidate molecule drugs was found in the PubChem database and shown inSupplementary Figure S5 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure GO and KEGG functional annotation for the significant DEGsA The top ten enriched BP of the DEGs B The top ten enriched CC of the DEGs C The top ten enriched MF of the DEGs DThe top ten enriched KEGG pathways of the DEGs The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The PPI network and top hub genes as well as top three modules were constructedA The PPI network of the DEGs B The hub genes of the DEGs C“E Top three hub modules were identified by Cytoscapeplugin MCODE C module1 D module2 E module3Table The top ten OCrelated small molecules with highly significant correlations in results of CMap analysisRankCMap nametrichostatin A8azaguaninepyrviniumisoflupredonequinpirolevorinostatgenisteinantimycin AheptaminolmidodrineMeanˆ’ˆ’ˆ’ˆ’ˆ’NEnrichmentP valueˆ’ˆ’ˆ’ˆ’ˆ’ The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The gene mutation overview of ten hub genes in TCGA OC patientsA Ten hub genes are altered in of queried patients B The summary of mutation type of ten hub genes in OC patientsC The network of hub genes and the most frequently altered neighbor genesTable Univariate cox regression identified DEGs correlated to patients™ OSGene IDCCND1SYNE4CCDC80TMC4MCCFOXQ1KRTCAP3CXCR4IL4I1DEFB1CSGALNACT1KLHL14MCUR1HRAbbreviation HR hazard ratioHR95LHR95HPvalueConstruction of prognostic model and evaluation of its predictive abilityUnivariate Cox regression analysis revealed that of DEGs were significantly correlated to patients™ OS in thetraining cohort Table The OSrelated genes were listed as follows CCND1 SYNE4 CCDC80 TMC4 MCC The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical characteristics of CCNB1 CEP55 CDK1 FOXM1 and UBE2C in OC patientsA Five genes were overexpressed in high grade G1 and G2 compared with low grade G3 and G4 in OC BC The OS time Band DFS time C of five genes in OC patients The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Identification of prognosisrelated mRNAs using LASSO regression modelA LASSO coefficient profiles of the mRNAs associated with the OS of OC B Plots of the crossvalidation error rates Each dotrepresents a λ value along with error bars to give a confidence interval for the crossvalidated error rateTable Multivariate Cox regression selected eight DEGs correlated to patients™ OSGene IDTMC4KLHL14CXCR4CCDC80KRTCAP3DEFB1SYNE4FOXQ1HRHR95LHR95HPvalue845E08Abbreviation HR hazard ratioFOXQ1 KRTCAP3 CXCR4 IL4I1 DEFB1 CSGALNACT1 KLHL14 and MCUR1 Through LASSO Cox regression we narrowed the number of prognosisassociated genes to according to the minimum criteria Figure Next based on the multivariate Cox model of candidate mRNAs retained their prognostic significance and werefinally selected as independent remarkable prognostic factors which were TMC4 KLHL14 CXCR4 CCDC80 KRTCAP3 DEFB1 SYNE4 and FOXQ1 Table To predict patients™ outcomes we developed an individual™s risk scoremodel as follows risk score × expression value of TMC4 × expression value of KLHL14 ˆ’ × expression value of CXCR4 × expression value of CCDC80 ˆ’ × expression value of KRTCAP3 ˆ’ × expression value of DEFB1 × expression value of SYNE4 × expression value of FOXQ1 On the basis of the median risk score patients were divided into high orlowrisk groups Kaplan“Meier curve analysis showed that the OS time of the lowrisk group was significantly longerthan the highrisk group P1147e07 Figure 10E ROC curve analysis revealed the area under the ROC curveAUC of the prognostic model was Figure 10D Meanwhile the risk scores Figure 10A of OC patients inthe training group were ranked for displaying their distribution and the survival status Figure 10B was marked onthe dot plot The expression pattern of eight prognostic mRNAs between high and lowrisk groups was also shown inthe heatmap Figure 10C Univariate and multivariate Cox regression analyses concerning the risk score and clinicalfactors showed that the prognostic model was able to serve as an independent prognostic indicator Figure 11ABROC curve analysis also showed that the AUC value of the model was much significantly higher than the tumor stage AUC grade AUC and patients™ age AUC Figure 11C Interestingly when The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA training modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E Kaplan“Meier survival ofthe prognostic model for the TCGA OC training cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the risk model in training cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the training model and OC patients™ OS status when compared with or combined with clinical factorscombined the risk score with clinical factors the ROC curve of combination model was much higher than each aloneFigure 11DAs for the testing cohort we divided the group into highrisk and lowrisk individuals based on the trainingcohort cutoff risk score The outcomes of low and highrisk groups™ patients of the testing cohort were also measuredand the OS time of the highrisk group was significantly shorter than the lowerrisk group P1721e02 Figure12E The AUC of the prognostic model was Figure 12D The risk scores distribution Figure 12A and survivalstatus Figure 12B of OC patients as well as the eightprognostic gene expression heatmap Figure 12C in the testinggroup were also present Meanwhile the independency of the prognostic model was confirmed in testing cohort sinceunivariate and multivariate Cox regression analyses showed the model correctly predicted high or lowrisk factroups patients™ outcomes without relying on any clinical factors Figure 13AB ROC curve analysis showed that theprognostic model exhibited better sensitivity and specificity when compared with tumor stage grade and patients™age for the AUC value of the model was much higher than later Figure 13C In accordance with results from trainingcohort the combination of risk score and clinical factors showed better OS prediction capability Figure 13DThe prognostic signature correlating to immune cells infiltrationThrough TIMER webtool we analyzed the relative gene expression levels of six types of immune cells for each patientand found that genes concerning macrophage fraction were expressing significantly higher in the highrisk groupP005 compared with the lowrisk group in training cohort Figure Interestingly same result was also observed in the testing cohort Figure The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure Prognostic analysis of the TCGA testing modelA The risk score B survival status C expression heatmap D timedependent ROC curves and E Kaplan“Meier survival ofthe prognostic model for the TCGA OC testing cohort The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The clinical independency of the prognostic risk signature in testing cohortUnivariate A and multivariate B regression analyses as well as timedependent ROC curve analysis CD of the prognostic valuebetween the testing model and OC patients™ OS status when compared with or combined to clinical factorsDiscussionIn the present study we used the RRA methods to jointly analyze six GEO OC microarrays which contained OC and normal samples identifying DEGs and overlapped them with dysregulated genes of OC cohort fromTCGA and GTEx portal finally getting upregulated and downregulated genes Functional analysis showedthat DEGs were significantly enriched in the cell division cycle to be clear in the process of the mitotic spindleSpindle microtubules have been proved to play crucial role in physiological and pathological processes As for celldivision only when all chromosomes linked to spindle microtubules through kinetochores and the spindle assemblycheckpoint is satisfied this process could step to anaphase [] Suraokar et al found that the mitotic spindle assemblycheckpoint and microtubule network were significantly altered in malignant pleural mesothelioma MPM whileusing epothiloneB a nontaxane small molecule inhibitor targeting the microtubules could greatly decrease theviability of MPM cell lines [] Rogalska et al compared the antiproliferative capacity of epothilone B with paclitaxelon OC cell line SKOV3 found that this effect of Epo B was greater than latter [] The researches above wereconsistent with our study that the mitotic spindle process was dysregulated in OC progression playing importantroles in OC cell proliferation and tumor developmentPPI network construction of DEGs included nodes and edges among which we identified three keymodules Interestingly the top1 module was also highly associated with spindle microtubules and chromosome kinetochore confirming the role of cell cycle in OC pathogenesis The top ten hub genes from the PPI network were alsorecognized which were CDC45 CDK1 TOP2A CDC20 CCNB1 CEP55 UBE2C HMMR FOXM1 and TPX2 The Authors This is an access published by Portland Press Limited on behalf of the Biochemical Society and distributed under the Creative Commons AttributionLicense CC BY 0cBioscience Reports BSR20201261101042BSR20201261Figure The expression level of immune cells related genes in high and lowrisk groups of the training cohortA Bcell fraction B dendritic fraction C CD4 Tcell fraction D CD8 Tcell fract

" autism spectrum disorder asd is a developmental disorder and the effective pharmacologicaltreatments for the core autistic symptoms are currently limited increasing evidence particularly that from clinicalstudies on asd patients suggests a functional link between the gut microbiota and the development of asdhowever the mechanisms linking the gut microbiota with brain dysfunctions gutbrain axis in asd have not yet beenfull elucidated due to its genetic mutations and downregulated expression in patients with asd ephb6 which alsoplays important roles in gut homeostasis is generally considered a candidate gene for asd nonetheless the role andmechanism of ephb6 in regulating the gut microbiota and the development of asd are unclearresults here we found that the deletion of ephb6 induced autismlike behavior and disturbed the gut microbiota inmice more importantly transplantation of the fecal microbiota from ephb6deficient mice resulted in autismlikebehavior in antibiotictreated c57bl6j mice and transplantation of the fecal microbiota from wildtype miceameliorated the autismlike behavior in ephb6deficient mice at the metabolic level the disturbed gut microbiota inephb6deficient mice led to vitamin b6 and dopamine defects at the cellular level the excitationinhibition eibalance in the medial prefrontal cortex was regulated by gut microbiotamediated vitamin b6 in ephb6deficient mices our study uncovers a key role for the gut microbiota in the regulation of autismlike social behavior byvitamin b6 dopamine and the ei balance in ephb6deficient mice and these findings suggest new strategies forunderstanding and treating asdkeywords gut microbiota asd ephb6 vitamin b6 dopamine ei balance correspondence tgaosmueducn lijming3sysueducn ying li and zhengyi luo contributed equally to this work2state key laboratory of an failure research key laboratory of mentalhealth of the ministry of education guangdonghong kongmacao greaterbay area center for brain science and braininspired intelligenceguangdong province key laboratory of psychiatric disorders collaborativeinnovation center for brain science department of neurobiology school ofbasic medical sciences southern medical university guangzhou people™s republic of china1department of pathology sun yatsen memorial hospital sun yatsenuniversity guangzhou people™s republic of chinafull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cli microbiome page of autism spectrum disorder asd which affects approximately of the population around the worldis mainly characterized by impaired social interactionand communication and restricted and repetitive behavior although early behavioral and educationalinterferences have shown effective ameliorative roleson autistic symptoms of asd patients the effectivepharmacological therapies for the treatment of coreautistic symptoms remain limited [ ]thataccumulating evidence showsthe gutbrainmicrobiota axis plays a key role in regulating homeostasis of the human body gut microanisms reportedlyparticipate in many neuropsychiatric disorders suchas anxiety disorders depression and epilepsy in most asd patients changes in gut microanismsand serious gastrointestinal problems have been observed [“] interestingly several studies have foundthat the gut microbiota play important role in modulating the asdlike phenotypes of mice [“] aclinical study showed that microbiota transfer therapycan improve gastrointestinal problems and autisticsymptoms in asd patients aged to years and thisbenefit can last for years [ ] these studiessuggest that the gutbrainmicrobiota axis might havea significantimpact on the development of asdhowever the contribution of the gut microbiota tothe dysregulation of brain function has not been fullyelucidatedephb6 which belongs to the eph family of receptortyrosine kinases is located on chromosome 7q in twostage genome research on susceptibility lociinautism found transcripts mapped to the chromosome 7qregion that are associated with a predisposition to autincluding ephb6 more recent studies haveismsuggested that ephb6 is a candidate asdassociatedgene [“] and recent genomic studies have foundthat ephb6 is mutated in some asd patients [ ]most importantly transcriptome analyses have shownthat ephb6 is downregulated in asd patients [ ]although ephb6 plays an important role in regulatingeph receptor signaling networks t cellfunctionsintestinal epithelium and epithelialdevelopment ofhomeostasis [“]the role and mechanisms ofephb6 involved in regulation of the gut microbiota andasd remain unclearin our study we found that ephb6 is functionallyassociated with asd and regulates autismlike socialbehavior by gut microbiotamediated vitamin b6 anddopamine moreestablished thefunctionallink between dysregulated gut microbiotaand excitationinhibition ei imbalance in the medial prefrontal cortex mpfc a key gutbrain functional axis in ephb6deficient miceimportantly weresultsthe deletion of ephb6 led to autismlike behavior andgut microbial disturbance in micealthough ephb6 has been identified as a candidate geneassociated with asd whether and how ephb6 functions inasd remain unclear to address these unanswered questions we established ephb6knockoutko mice andfound that ephb6 was deleted in different tissues includingthe colon brain lung and spleen in these mice comparedwith ephb6 wildtype wt mice additional file figure s1cd however the brain and body weights thebody length and the daily dietary consumption were similarbetween the two groups of mice despite the deletion ofephb6 additional file figure s1ehthan the wt mice fig 1bpatients with asd often display repetitive stereotypedbehavior and social deficits interestingly we found thatthe ko mice spent more time selfgrooming than thewt mice fig 1a in the marble burying test the komice buried similar marbles as the wt mice additionalfile figure s1j and in the social partition test the komice spent less time sniffing at the partition regardlessof whether a familiar or novel mouse was placed in thecagein the threechambered social approach task both the wt and komice spent similar lengths of time in bilateral chambersduring the first 10min trial which indicated that the experimental environment was normal fig 1c howeverthe ko mice spent a similar length of time in chamberswith an unfamiliar mouse or inanimate object fig 1dand also showed less preference for the social mousestranger over the object than the wt mice fig 1f“gif a novel social partner stranger was placed in theempty wire cage the ko mice still spent a similar lengthof time in the two chambers fig 1e and showed lesspreference for the novel mouse over the familiar mousethan the wt mice fig 1h these results sufficientlyconfirmed that the ko mice exhibited abnormal socialinteraction olfactory cues have generally been consideredto be of the utmost importance in communication amongmice [ ] in the olfactory habituationdishabituationtest repeated presentation of cotton swabs saturated withthe same odor resulted in increasingly decreased lengthsof time spent sniffing at cotton swabs and the presentation of cotton swabs saturated with a new odor increasedthe time spent sniffing these findings were obtained withboth the wt and ko mice however the ko miceshowed less interest in cotton swabs saturated with socialodor than the wt mice fig 1i these results indicatedthat the ko mice exhibited communication deficits eventhough their ability to discriminate and habituate differentodors was normalasd is often accompanied by other mental diseasessuch as hyperactivity anxiety and intellectual disabilityin the open field test the ko mice showed the same 0cli microbiome page of fig see legend on next page 0cli microbiome page of see figure on previous pagefig the deletion of ephb6 led to autismlike behavior and gut microbial disturbance in mice a the 8weekold male ko mice spent more time selfgrooming than wt mice n mice for each group b in social partition test ko mice spent less time sniffing the partition than wt mice n mice respectively c“h in threechambered social approach task time spent in chambers during different 10min trials c“e trajectory diagram duringthe second 10min trial f were showed ko mice showed less preference for the social mouse over the object g and less preference for the novelsocial mouse over the familiar social mouse h than wt mice n mice respectively i in olfactory habituationdishabituation test ko mice spentless time sniffing social odors than wt mice n mice for each group j in elevated plus maze test ko mice spent less time in open arm and moretime in closed arm than wt mice n mice respectively k the intestinal permeability of 8weekold wt and ko mice was detected using fitcdextran n mice for each group l the mrna expressions of tight junction molecules were detected in colon of 8weekold wt and ko mice n mice respectively m the mrna expressions of cytokines were detected in colon of 8weekold wt and ko mice n mice respectively n“r 16srrna gene sequencing of gut microbiota of 8weekold wt and ko mice the species richness n and diversity o of gut microbiota were similarwhile the microbial composition p was different between the two groups relative abundance of different bacteria in phylum level was showed qat genus level the relative abundance of mucispirillumn was decreased in ko mice r n mice for each group data shown are mean ± sem ormedian ± iqr twotailed unpaired student™s t test a c“e g“h j“m mannwhitney test n“o q r mixed design anova with genotype asindependent factor and stimulitrials as repeatedmeasure factor b i anosim analysis p p p p wt ephb6 mice koephb6ˆ’ˆ’ mice fitc fluorescein isothiocyanate statistical values are presented in additional file table s2locomotor activities and spent almost the same time inthe center area as the wt mice additional file figures1kl in the elevatedplusmaze testthe ko micespent less time in the open arm and more time in theclosed arm than the wt mice fig 1j which impliedthat the ko mice displayed anxietylike behavior in themorris water mazethe ko mice exhibited normalspatial learning and memory similarly to the wt miceadditional file figure s1mo collectively the resultsshowed that the deletion of ephb6 in mice resulted inautismlike behavior including stereotyped behavior andsocial deficits accompanied by anxietylike behavior butdid not result in any evidence of intellectual disabilityephephrin signaling reportedly modulates gut epithelial development and homeostasis and it is also generallyaccepted that many asd patients present gastrointestinal gi symptoms [ ] and a changed gut microbiota composition we then questioned whether komice would suffer from gi problems measurement ofthe intestinal permeability by fluorescein isothiocyanatefitcdextran revealed that the intestinal permeabilityof ko mice was significantly increased compared withthat of the wt mice fig 1k accordingly the mrnaexpression of cldn4 a tight junction molecule in thecolon of ko mice was lower than that in the colon ofwt mice fig 1l in addition we found that the colonof the ko mice presented substantially increased mrnaexpression of il1β a proinflammatory factor and decreasedexpression of il6 which exerts an antiinflammatory effectcompared with that of the wt mice fig 1m the gi problems in the ko mice were not accompanied by morphological changes in the distal ileum proximal colon liver orlung additional file figure s1pthe integrity of the intestinal mucosa is important formaintaining the balance of the ecological environmentin the animals™ gut we then detected the fecal microbialpopulations of mice by 16s rrna gene sequencing nodifferences in the microbial species richness and diversitywere found between the two groups fig 1n o notablya principal coordinates analysis of the braycurtis distanceshowed that the fecal microbiota of the ko mice clustereddifferently from that of the wt mice fig 1p which indicated that the gut microbial composition differed betweenthe two groups at the phylum level the differencesbetween the two groups were caused by a decreased abundance of deferribacteres in the fecal microbiota of the komice fig 1q at the genus level mucispirillum which isa genus belonging to the phylum deferribacteres wasdecreased in the fecal microbiota of the ko mice fig 1rin general our results indicated that the deletion of ephb6in mice resulted in increased intestinal permeability andchanges in the gut microbial compositionmany studies have indicated that gi problems and thebehavioral abnormalities associated with asd always appearin parallel in patients we thus questioned which of thesesymptoms appears first in the ko mice and found that themicrobial species richness and diversity did not differ between the 34weekold wt and ko mice additional file figure s2ab the principal coordinates analysis revealedthat the gut microbiota of 4weekold ko mice clustered differently from that of 4weekold wt mice additional file figure s2d whereas the gut microbiota of 3weekold komice clustered similarly to that of 3weekold wt miceadditional file figure s2c in addition 4weekold butnot 3weekold ko mice showed increased selfgroomingand decreased interest in social odors compared with sameaged wt mice additional file figure s2eh these resultsfurther implied a possible relationship between the abnormalbehavior and gut microbial dysbiosis in mice with deletion ofephb6transplantation of the fecal microbiota from ephb6deficient mice caused autismlike behavior in spf c57bl6j miceasd is generally considered a neurodevelopmental disorder postnatal developmental disorder can also causeautism in patients and the postnatal mutation ofnrxn1 in neurons leads to autismlike behavior in mice 0cli microbiome page of additionally the gut microbiota of asd patientscould induce autismlike behavior in mice thereforeto study the relationship between gut microbial dysbiosisand autismlike behavior in mice with deletion of ephb6we gavaged the fecal microbiota from 8weekold malewt or ko mice to 3weekold spf male c57bl6j micefor week fig 2a three weeks after the gavage offecal microbiota the gut microbial composition of spfc57bl6j mice treated with the fecal microbiota fromthe ko mice differed from that of spf c57bl6j micetreated with the fecal microbiota from the wt micefig 2b“d more interestingly c57bl6j mice that weregastrically perfused with the fecal microbiota from theko mice displayed increased selfgrooming fig 2e andpartially decreased social behavior fig 2f“i comparedwith the control mice the two groups of mice showedsimilar behaviors in the open field test and elevated plusmaze test additional file figure s3ad furthermorewe orally gavaged the suspending solution offecalmicrobiota from the wt or ko mice to antibioticpretreated spf male c57bl6j mice after pretreatmentwith antibiotics for days 3weekold spf male c57bl6j mice was gavaged orally with the fecal microbiota of8weekold male wt or ko mice for days fig 2japproximately weeks after fecal microbial colonizationwe similarly found that the gut microbiota of spf c57bl6j mice treated with the fecal microbiota from the ko miceclustered differently from that of the control mice fig 2k“m we subsequently found that c57bl6j mice that weregastrically perfused with the fecal bacteria from the komice showed increased selfgrooming fig 2n and partiallydecreased social behavior fig 2o“r additionally the twogroups of mice showed similar behaviors in the openfield test and elevatedplusmaze test additional file figure s3eh moreover the fecal microbiota from weekold but not 3weekold ko mice induced increasedselfgrooming and partial social deficits in 3weekold spfc57bl6j micecompared with c57bl6j micegavaged with fecal microbiota from sameaged wtmice additional file figure s4ah collectively thefecal microbiota from ephb6deficient mice caused increased selfgrooming and partially impaired socialbehavior in c57bl6j micewe subsequently questioned whether the gut microbiota continue to play a role in autismlike behavior inadult mice first we orally gavaged a mixture of antibiotics to 6weekold male spf c57bl6j mice for weekand found that this antibiotic treatment greatly disruptedthe gut microbiota and induced decreased selfgroomingand partial social deficits in young adult c57bl6j miceadditional file figure s5ai these results indicatedthat the gut microbiota was related to autismlike behavioreven in adult mice and that different gut microbiota compositions likely contributed to different behaviors such asselfgrooming and social behavior we then gavaged thefecal microbiota from 8weekold male wt or ko micedirectly to 6weekold spf male c57bl6j mice for week and found that the fecal microbiota from ko micealso induced a disturbed gut microbiota increased selfgrooming and partial social deficits in adult c57bl6jmice fig 3a“i unexpectedly we also found that metabolites of the gut microbiota from the ko mice inducedpartial social deficits in c57bl6j mice fig 3j“m thegut microbiota without metabolites from the ko mice stillcaused partial social deficits in c57bl6j mice additionalfile figure s5jmoverall our results indicated that the gut microbiotaplays an important role in autismlike behavior even inadult micetransplantation of the fecal microbiota from wildtypemice ameliorated autismlike behavior in adult ephb6deficient miceno previous study has focused on the effectiveness ofmicrobiota transplantation in adult asd patients wesubsequently orally gavaged the fecal microbiota from weekold male wt mice to 8weekold ko mice for week a week later we found that the gut microbiota ofthe ko mice gavaged with the fecal microbiota of thewt mice clustered differently from that of the ko micegavaged with sterile pbs fig 4b the phylumlevelanalysis revealed that the relative abundance of deferribacteres was increased in the ko mice gavaged with thefecal microbiota of the wt mice fig 4c at the genuslevel mucispirillum which is a genus belonging to thephylum deferribacteres was also increased in the komice treated with the fecal microbiota of the wt micefig 4da functional analysis revealed that the ko mice exhibited increased social behavior fig 4f“i after gavage withthe fecal microbiota from the wt mice and a decreasedtendency of selfgrooming fig 4e these results indicated that gut microbial dysbiosis was responsible forautismlike behavior in mice with deletion of ephb6gut microbiotamediated vitamin b6 homeostasisregulated social behavior in ephb6deficient micebecause the abnormal behaviors were likely due tobrainrelated problems we attempted to determine howthe gut microbiota affected the brain and subsequentlycaused autismlike behavior in ephb6deficient micefirst we attempted to identify the key region of thebrain affected by the dysregulated gut microbiota inmice with deletion of ephb6 and that was responsiblefor the resulting autismlike behavior studies on asdpatients or mouse models have shown that the hippocampus cerebellum and mpfc are implicated in asd[ ] after processed with a threechambered social 0cli microbiome page of fig see legend on next page 0cli microbiome page of see figure on previous pagefig transplantation of the fecal microbiota from ephb6deficient mice caused autismlike behavior in 3weekold spf c57bl6j mice a“ischematic of the fecal microbiota transplantation a the 3weekold spf male c57bl6j mice were orally gavaged with the fecal microbiotafrom 8weekold male wt or ko mice each contained eight healthy mice from at least three cages for week after weeks the fecalmicrobiota of the treated c57bl6j mice were sequenced b“d eight treated c57bl6j mice of each group were selected randomly from at leastthree cages and selfgrooming test e olfactory habituationdishabituation test f threechambered social approach task g“i open field testand elevated plus maze test were conducted with an interval of at least days e“i n mice respectively j“r schematic of the fecalmicrobiota transplantation j the 3weekold spf male c57bl6j mice were orally gavaged with antibiotics ampicillin vancomycin neomycinmetronidazole for days and then orally gavaged with the fecal microbiota from 8weekold male wt or ko mice each contained eight healthymice from at least three cages for another days after days the fecal microbiota of the treated c57bl6j mice were sequenced k“m sixtreated c57bl6j mice of each group were selected randomly from at least two cages and selfgrooming test n olfactory habituationdishabituation test o threechambered social approach task p“r open field test and elevated plus maze test were conducted with an intervalof at least days n“r n mice respectively data shown are mean ± sem or median ± iqr twotailed unpaired student™s t test e h i nq r mannwhitney test b c k l mixed design anova with genotype as independent factor and stimulitrials as repeatedmeasure factor f oanosim analysis d m p wt col or ko col colonized with the fecal microbiota from ephb6 or ephb6ˆ’ˆ’ mice abx pretreated withantibiotics ampicillin vancomycin neomycin metronidazole statistical values are presented in additional file table s2approach task the protein expression of cfos in thempfc of the ko mice was significantly higher than thatin the mpfc of the wt mice additional file figures6ac asd is generally considered to be caused by anincreased ratio of synaptic excitation and inhibition andasd children exhibit elevations in the restingstateneuronal activity therefore whether the mpfc ismodulated by the gut microbiota of the ko mice needsto be further investigated because the mpfc tissue wastoo small for some experiments we used pfc tissuefrom mice in our subsequent studythe first question we asked was whether the bacteriacould directly modulate the mpfc undoubtedly we didnot detect any bacterial dna or colonies in the pfctissues of the wt or ko mice additional file figures6de because metabolites of the gut microbiota fromthe ko mice also induced social deficits in c57bl6jmice we hypothesized that some substances that hadbeen affected by gut microbial dysbiosis caused socialdeficits in the ko miceto identify the significantly changed metabolites wedetected the metabolites in the target tissue that is thepfc of the ko mice using nontargeted metabolomicsstrategies surprisinglythe metabolites in the pfcshowed significant differences between the two groupsof mice as demonstrated by orthogonal partialleastsquares discriminant analysis fig 5a a kegg pathwayanalysis identified four pathways that were significantlyenriched in the differentially changed metabolites andthese included the vitamin b6 metabolism pathway dueto the decreased relative abundances of pyridoxaminepm and pyridoxal ²phosphate plp in the pfc ofthe ko mice fig 5b“dvitamin b6 in the body is mainly derived from dietand gut bacteria synthesis and is then absorbed in theintestine we then detected the levels of vitamin b6 inthe feces blood and pfc of mice and found that theephb6deficient mice presented increased fecal levels ofpyridoxine pn decreased plasma levels of pm andplp and decreased levels of plp in the pfc fig 5e“kone week after gavage the ko mice gavaged with thefecal microbiota from the wt mice exhibited decreasedlevels of pn in feces and tended to show increased levelsof pm in plasma and increased levels of plp in plasmaand the pfc compared with the ko mice gavaged withsterile pbs fig 5e“k these results indicated that thegut microbiota regulated the level of vitamin b6 in thefeces blood and pfc of mice probably by regulatingthe absorption of vitamin b6 in intestinewe subsequently supplied vitamin b6 to the ko mice toclarify its effect on autismlike behavior however intragastric supplementation with vitamin b6 did not amelioratethe social deficits in the ko mice additional file figures7ac one hour after the intraperitoneal injection of mgplp the ko mice presented higher levels of plp in plasmafig 6b and increased social behavior fig 6d“f comparedwith the control mice no changes in selfgroomingfig 6c and social novelty fig 6g were detected inthe ko mice after the injection of plp additionallythe intraperitoneal injection of or mg of plp exerted noeffect on the social behavior of c57bl6j mice fig 6h“jmoreover after being fed without vitamin b6 for weeksc57bl6j mice presented lower plasma plp levels anddecreased social behavior fig 6k“n conclusively ourresults proved the existence of a relationship between gutmicrobiotamediated defects of vitamin b6 and socialdeficits in ephb6deficient micegut microbiotamediated vitamin b6 homeostasisregulated dopamine in the pfc of ephb6deficient micevitamin b6 as a cofactor has been implicated in morethan biochemical reactions in cellsincluding thebiosynthesis and catabolism of amino acids and neurotransmitters as the most important active substances in the brain we first detected neurotransmittersin the pfc of mice by highperformance liquid chromatography hplc and found similar levels of glutamategammaaminobutyric acid gaba glycine aspartic 0cli microbiome page of fig fecal microbiota transplantation from ephb6deficient mice partially induced social deficits in 6weekold spf c57bl6j mice a“ischematic of the fecal microbiota transplantation a the 6weekold spf male c57bl6j mice were orally gavaged with fecal microbiota from weekold male wt or ko mice for week after week the fecal microbiota of the treated c57bl6j mice were sequenced b“d n mice foreach group and selfgrooming test e olfactory habituationdishabituation test f threechambered social approach test g“i were conductedwith an interval of at least days e“i n mice respectively j“m fecal metabolites from 8weekold male wt and ko mice were orallygavaged to 6weekold spf male c57bl6j mice for week j after week olfactory habituationdishabituation test k and threechamberedsocial approach task l m were conducted with an interval of at least days n mice respectively data shown are mean ± sem ormedian ± iqr twotailed unpaired student™s t test e h i m mannwhitney test b c mixed design anova with genotype as independentfactor and stimulitrials as repeatedmeasure factor f k anosim analysis d p p wt col or ko col colonized with fecalmicrobiota or fecal metabolites from ephb6 mice or ephb6ˆ’ˆ’ mice statistical values are presented in additional file table s2acid serine and glutamine in the wt and ko micegavaged with sterile pbs or the fecal microbiota fromthe wt mice fig 7a b interestingly the pfc of theko mice exhibited decreased dopamine levels and increased 5hydroxytryptamine 5ht levels than that ofthe wt mice fig 7c treatment with the fecal microbiota from the wt mice increased the level of dopaminebut did not affect the level of 5ht in the pfc of theko mice compared with the levels found in the komice gavaged with sterile pbs the levels of noradrenaline epinephrine and dihydroxyphenyl acetic aciddopac did not differ among the three groups of micefig 7c more excitingly the level of dopamine in thepfc of spf c57bl6j mice gavaged with the fecalmicrobiota from the ko mice was lower than that in thepfc of c57bl6j mice gavaged with the fecal microbiotafrom the wt mice additionalfile figure s8aeadditionally the intraperitonealinjection of plp increased the level of dopamine in the pfc of the komice fig 7d and vitamin b6 deficiency decreased thelevel of dopamine in the pfc of spf c57bl6j micefig 7e brieflyindicated that gutthese results 0cli microbiome page of fig transplantation of the fecal microbiota from wildtype mice ameliorated autismlike behavior in adult ephb6deficient mice a schematic ofthe the fecal microbiota transplantation the 8weekold male wt and ko mice were orally gavaged with the fecal microbiota from 8weekoldmale wt mice eight healthy mice from at least three cages or sterile pbs for week after week the fecal microbiota of the treated wt andko mice were sequenced b“d and behavioral tests were conducted with an interval of at least days e“i b“d 16s rrna gene sequencing ofthe fecal microbiota from mice principal coordinates analysis of braycurtis distance b the relative abundance of deferribacteres c at phylumlevel and the relative abundance of mucispirillum d at genus level were showed at phylum level the range of “ on x axis was used for therelative abundance of p_bacteroidetes p_firmicutes and p_proteobacteria and the range of “ on x axis was used for other bacteria n mice respectively e“i selfgrooming test e olfactory habituationdishabituation test f and threechambered social approach task g“iwere performed n mice respectively data shown are mean ± sem or median ± iqr oneway anova e h i kruskalwallis testc d mixed design anova with genotype as independent factor and stimulitrials as repeatedmeasure factor f anosim analysis b p p p wt ephb6 mice ko ephb6ˆ’ˆ’ mice fmt fecal microbiota transplantation pbs phosphatebuffered saline statisticalvalues are presented in additional file table s2microbiotamediated vitamin b6 homeostasisaffect the level of dopamine in the pfc of micecouldto determine whether the decrease in dopamine contributed to the autismlike behavior of ephb6deficientmice and considering the fast metabolism of dopaminein the brain we injected agonists of dopamine receptorsinto the mpfc of mice the deletion of ephb6 had noeffect on the mrna expression of dopamine receptorsor tyrosine hydroxylase th in the mpfc or ventraltegmental area vta fig 7f we then injected anagonist of dopamine d1 receptor d1r skf38393or dopamine d2 receptor d2r quinpirole into thempfc of mice the results showed that the ko miceexhibited increased social behavior fig 7g“j afterinjection with skf38393 compared with the ko miceinjected with artificial cerebrospinalfluid acsfhowever no differences were found between c57bl6j mice injected with acsf and c57bl6j mice 0cli microbiome page of fig gut microbiota regulated vitamin b6 in ephb6deficient mice a“d in nontargeted metabolomics analysis the metabolites in pfc of weekold male wt and ko mice were differently clustered by orthogonal partial least squares discriminant analysis a the enriched keggpathways associated with differential metabolites b the relative abundance of pyridoxamine pm c and pyridoxal ²phosphate plp d wereshowed n mice respectively e“k the fecal microbiota from 8weekold wt mice or pbs were gavaged to 8weekold wt or ko mice for week e one week later the level of pm f plp g and pyridoxine pn h in feces of mice were detected n mice respectively thelevel of pm and plp in plasma i j n mice respectively and level of plp in pfc k n mice respectively of mice were alsodetected data shown are mean ± sem r b“d oneway anova f“k p p p wt ephb6 mice ko ephb6ˆ’ˆ’mice fmt fecal microbiota transplantation pbs phosphatebuffered saline pfc prefrontal cortex pm pyridoxamine plp pyridoxal ²phosphate pnpyridoxine statistical values are presented in additional file table s2injected with skf38393 fig 7k“min contrastquinpirole did not increase social behavior in the komice additionalfile figure s8fh and the d1rantagonistinc57bl6j mice fig 7n“q in short these results indicated that dysregulated gut microbiota and vitaminb6 defect led to autismlike behavior via the d1rmediated pathway in ephb6deficient miceinduced decreased social behaviut microbiota regulated the ei balance in the mpfc ofephb6deficient miceit is generally thought that d1rs modulate ga

" child maltreatment leads to enormous adverse short and longterm health outcomes the aim ofthis study is to estimate the burden of disease and the cost of illness attributable to child maltreatment in japanmethods an incidencebased topdown cost of illness analysis was conducted to estimate medical costs andburden of disease attributable to child maltreatment based on a societal perspective the assessment includedshortterm and longterm medical costs and burden of disease measured by disabilityadjusted life years dalysthat generates mortality and morbidities based on several national surveys and systematic review we consideredthe main types of child maltreatment as exposure for which the incidence was obtained from literature reviewbased on population attributable fractions pafs burden of disease of physical and mental health consequencesattributable to child maltreatment were estimated then dalys were converted into monetary value the lifetimeeconomic burden was finally estimated by combining with medical costs and subject to sensitivity analysisresults the lifetime disease burden expressed in dalys was estimated at dalys ci dalys for the cohort victims in based on the incidence according to literature review the overall lifetimeeconomic burden was billion usd equivalent to million times of gross domestic product gdp per capitaamong the total economic burden costs of suffering and pain based on dalys were accounting for theseestimates were “ times of conservative estimates which used incidence data from official reported casess this study found that the national lifetime cost was huge and equivalent to million gdp percapita and its burden of disease was approximately equal to that of colon and rectum cancers or stomach cancerour findings particularly in terms of revealed the considerable burden of disease in long term and potential effectsof the strengthened maternal and child care as the preventive strategykeywords child maltreatment burden of disease study lifelong health consequences disabilityadjusted life yeardaly costofillness correspondence gairuoyanipssgojp1department of health policy national center for child health anddevelopment tokyo japan3department of empirical social security research national institute ofpopulation and social security research uchisaiwaicho chiyodakutokyo japanfull list of author information is available at the end of the the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cmo bmc public health page of child maltreatment is a raising concern in public healthand social welfare in japan the reported number ofsuspected cases of child maltreatment is increasing from in to in according to theministry of health labour and welfare mhlw ofjapan child maltreatment is categorised into four essential types physical sexual or psychological includingwitnessing domestic violence wdv abuse and neglect exposure to multiple types and repeated episodes ofmaltreatment during childhood is associated with highrisks to enormous adverse health outcomes causing asignificant social and economic burden on individualsfamilies and societies those adverse outcomes duringchildhood include child death injuries and disabilitiesdevelopmental and behavioural problems moreover therelated physical and mental health conditions persistinto adulthood leading to the onset of chronic diseasesdepression drug alcohol misuse and risk sexual behaviour suicide ideation [ ]the number ofthe related analysis ofthe government has introduced a couple of protectivemeasures with increasing public budget [“] assessment of costs and burden of disease helps developmentof resource allocation and priority setting in public sector paralleling with growing concerns on child maltreatmenttheprevalence health consequences and economic burdenis increasing so far for the economic burden there aretwo typical research frameworks one is a comprehensively costs evaluation from healthcare social educa[“]tional areas and loss in productivity another one is to measure related economic and diseaseburden [ ] wada reported the socialcosts of child abuse in japan included direct costs ofdealing with abuse and the indirect costs related to longterm damage from abuse during the fiscal year onthe other hand the first framework is likely to underestimate longterm deleterious effects of child maltreatmenton which evidence derived from longitudinal studies isless available compared to that on the shortterm counterpart by integrating previous evidence our costofillness study aimed to assess lifetime economic anddisease burden of mortality and morbidities attributed tochild maltreatment based on the later frameworkinorder to address the evidence gap we extended cost calculations for monetary values converted from disabilityadjusted life years dalys covering related mortalityand morbidities methodsan incidencebased victims estimated by incidencetopdown approach or attributable risk approach measuring the proportion of a disease that is due to exposureto risk factor was applied in this study from a societalperspective we employed the following steps to estimate the total economic burden constituted by directand indirect costs population attributable fraction paf wasgenerated to estimate longterm impactscosts attributed to child maltreatment shortterm and longterm direct medical costs wereassessed by using national expenditure databasesindirect costs measured include productivity losscaused by abusive head trauma and economicburden deriving from dalys finally sensitivity analyses were performed for theplausible range of the discount rate and theincidence prevalenceestimating pafin the topdown approach paf for each disease i measured that how health outcomes and their associatedcosts may be attributed to child abuse using the following formula [ ]pafi ¼ p rri ˆ’ °p rri ˆ’ ¾¾ ¾ °p prevalence of child abuse rr the relative risk of theoutcome i in those who experienced child abuse compared with those who did notrisk ratio rr or odds ratio orseveral previous related systematic reviews and metaanalyses summarised the relevant health consequences[ ] as adverse childhood experiences acesoften intertwine with child maltreatment cluster in children™s lives and cumulatively lead to poor health outcomes we pooled the ors from a recent systematicreview and metaanalysis for the effect of multiple aceson health rather than that for each category of childmaltreatmentin japan thethe pooled prevalencea literature review was performed to synthesize the evidence on epidemiological characteristicsthe consequencesreview focused on thosepublished between december and march onmedline pubmed web of science scopus and ciniis japanese literature details of the search strategy search terms used and inclusion and exclusion criteria are provided in the additional file we combinedour review results with those studies in japan includedin an existing systematic review and calculated thesimple sizeweighted mean incidenceprevalenceinthe median value was also calculated toaddition 0cmo bmc public health page of examine the robustness supplementary table theannual incidence rate was obtained by the formula incidence rate ¼prevalenceaverage durationdue to the lack of local data on the average duration we adopted that published in australia theaverage years for physical abuse and years forsexual abuse based on this findingthe weightedaverage of years was used for other categories ofabusedirect medical costsshortterm medical costsfor abusive head trauma aht is the leading causeof death due to child abuse among children youngerthan years old we estimated its hospitalizationcosts as shortterm medical costs by multiplying theincidence of aht under years old the agespecific population in and admission medical fee per case there were two reported incidences one is the œpossibleincidence considering countable possibility ofaht cases at most and another one is the œpresumptiveincidence representing victims had intracranial injuriesor intentionalinjuries with certain icd10 code weused the œpossible incidence for the general calculationand the latter one in sensitivity analysis the total possible aht cases aged under years was about times ofthe presumptive counterparts longterm medical costsfor longterm medical costs we used national healthcare expenditures and patient survey tosimulate disease burden ofrelevant health consequences by sex and age group “ “ “above and then multiplied with pafs to calculatethe attributable costs in the victim cohort of [ ] on the other hand we did not include selfharm and collective violence because of the limitationto distinguish the two in the reported overallinjurycasesindirect costsin this study we considered differential and loss of earning as a result of human capital depreciation is causedby mortality and morbidities it was presented as a monetary value of dalys and gdp per capita [ ]dalys and its monetary valuethe disease burden indicator daly aggregates yearsof life lost for premature death and years lost due todisability for morbidities related data wereobtained from the who global burden of diseasegbd using the pooled ors as described bykaren we matched each relatedhealth outcome with the cause of disease burdenin the who gbd categories though it was difficultto match some outcomes with the cause of gbdsupplementary table then monetary value was converted from daly attributable to child maltreatment by multiplying dalyand gdp per capita with adjustment of purchasingpower parity in productivity losses due to aht fatal casesproductivity loss due to fatal cases of child maltreatment was calculated based on the reported fatal caseswhich figure was obtained from official data andthe average lifetime income subject to discounting in there were abuserelated deaths reported injapan not including family suicide with the averageonset age of years the discounted lifetime income from to years old was calculated by assuming the longterm growth in labour productivityto be per year dalys losses of survival ahtfor disease burden due to survival aht we considered sequelae such as vision loss brain damage andreduced life span and longterm health consequences as developing diseases in adulthood we calculated the disease burden of aht in bymultiplying average cases and the estimated meanlifetime daly loss per case at different severity mildmoderate and severe longterm dalys losses of other diseasesthen the longterm health consequences were calculatedusing the following formuladaly losses ¼ σ pafi 03original dalyi°i different child abuse ˆ’ related health outcome°¾¾sensitivity analysesa discount rate of is generally performed which wasrecommended in the domestic guideline for costeffectiveness analysis whereas especially in the usa discount rate of has been selected and applied inthe cost estimate reports of centers for disease controland a best practices for the social return on investmentanalysis recommended by experts and guidelines assuch the parameter potentially affects the finally resultswe adopted a plausible range of to for sensitivityanalysis 0cmo bmc public health page of in addition we also calculated costs and diseaseburden using the incidenceprevalence data based onofficially reported child abuse cases to calculate theconservative incidence of child abuse by categorieswe obtained the official data of victim cases reportedby child consultation facilities in and thendivided them by the total population number in corresponding age data by sex were not availablecocurrentinformation was not available and theoverlapped cases were not considered supplementarytable the initial victim age is assumed to be years old according to an ageweighted incidence calculation based on official reported cases we assumed the probable abuserelated death cases to be times of the reported cases based on the ratio of thepresumptive and the possible incidence of aht casesamong children aged under years resultsthe main results showed in tables were discounted at and conservative estimates were given for sensitivityanalysesthe pooled incidence prevalence and disease burdenthe estimations on different types of child maltreatment incidence draw from literature reviews variedregarding differences between sex except physicalabuse girls suffered more than boys in sexual abuseand witnessing domestic violence table the estimated lifetime disease burden associated with childmaltreatment onset in was considerable dalys with a ci of dalys to dalys table the top causes of totaldisease burden due to child abuse were suicide attempts cardiovascular disease and depression cancercostofillness analysis for child maltreatmenttable demonstrates lifetime costs attributed to childabuse onset in the total direct cost was estimatedtable estimated incidenceprevalence of child abuse in japanestimates aincidence bmalefemalephysical abuse sexual abuse psychological abusewdv c other d prevalencemalefemaleneglect a sample sized weighted mean valueb incidence rate prevalence average durationc wdv witnessing domestic violenced not specified as wdv often expressed as emotionalpsychological abuseto be usd million 95ci million11 million while the total indirect cost was estimatedto be usd million 95ci million52 million accounting for of the total lifetimecosts which were almost million times gdp percapita economic loss initiated from dalys in longterm costs of suffering and pain accounted for ofthe overall estimatessensitivity analysesconservative estimates based on the reported cases incidence showed a tendency similar to that observed in thedisease burden based on the literature review amongwhich psychological abuse including wdv accountedfor the majority of reported child abuse cases however the incidence estimated from the review weremuch higher than those reported by child protectionagencies the conservative estimation leading to about“ times difference gap on child maltreat burden bydifference discount rate table discussionour results indicated that disease and economic burdenattributable to child maltreatment is substantial in particular that originated from the longterm health consequences accounts for the majoritybased on literature review the pooled incidence ofchild maltreatment in japan is much higher than officially reported which is consistent with the findingsof other studies [ ] because of difficulty toidentify the actual cases and a public attitude to consider child abuse as a private affair in the society theofficially reported cases are likely to represent the tipof an icebergthe fourpsychological abuse including wdv representedthe majority of reported cases the results of the literature review also showed a gender difference in theprevalence oftypes of child abuse sizeweighted mean values girls were found to be morelikely to experience the harmful practices comparedto boys particularly sexual abuse this tendency wasalso observed in other countries in east asia and pacific region comparing those living in othercountries in the east asia and pacific region [ ]japanese children tended to less likely to experiencephysical abuse boys vs girls vsalthough it is difficult to directly compare the results across different study settings due to the different methodologies parameters and target populationsadopted the ingredients of the lifetime economic anddisease burden considered in our studyincludingmedical costs and monetary value of disease burdenare similar to that adopted in previous studies [ 0cmo bmc public health page of table longterm daly lost attributable to child abuse in japandiseases attributed to child abuse asuicide attemptdalys confidence intervalcancercardiovascular diseasedepressionrespiratory diseaseliver or digestive diseaseanxietyproblematic drug useabusive head traumaproblematic alcohol usediabetessexually transmitted infectionsviolence victimisationviolence perpetrationtotaldalys monetary value billion usa simple size weighted mean prevalence at discounted rate] still our results showed that the disease burdenwas about “ times of the conservative estimationdue to the huge gap of incidence generated from literature and that officially reported the number isconsistent with an australian research that showed awide distribution ofthe annual prevalence rangingfrom to in the conservative lifetimecourse simulation the initial victim age is assumed tobe years old according to an ageweighted incidencecalculation based on official reported cases whichwas also consistent with previous studies our study in particular highlighted dalys in longterm attributable to child maltreatment accountingin the overallfor a relevant proportion lifetime costs the estimation of disease burden attributed to child maltreatment dalys wascomparable to the total dalys due to colon and rectum cancers dalys in or stomachcancer dalys in to our knowledge this is the first study to estimatelifetime economic burden of child maltreatmentinjapan based on an epidemiological model the idea ofthis method is to convert diseaseinduced losses ofwellbeing into economic terms by multiplying theannual number oflost life years due to disease bysubreginal per capita income so far few studies hadever taken this part of costs into account potentiallyleading to an underestimation of health and economictable lifetime costs attributable to child abuse for the first time in ciitems of the costs usd milliondirect costs medical costsshortterm ahtlongterm other diseasesindirect costsabuse death a productivity lossessurvival aht dalys blongterm loss of other diseases btotal costsaht abusive head traumaa we used “ times of base line data for range costs of child abuseb costs of suffering and pain dalys converted into monetary value by multiplying a gross domestic product per capita million gdp per capita 0cmo bmc public health page of table sensitivity analyses on incidence resource and discounted ratesensitivity analysisliterature based estimation adisease burden in dalys 95cieconomic burden usd million 95cidr dr dr conservative estimation bdr dr dr dr discounted ratea estimated based on literature review simple size weighted average prevalenceb estimated based on the number of consultation cases disposed about child abuse at child guidance centres probable estimate of abuse death was assumedabout times confirmed aht casespossible cases of the costs of conservative estimatechild maltreatmentimpacts ofin addition weadopted conservative calculation methodology in thesensitivity analyses to estimate the burden of childmaltreatment for more reliable range estimationsthere are several limitations to this study first thecooccurrence of multiple types of child abuse isprevalent resulting in difficulties to identify theadverse effects separately in order to minimize possible consequent overestimation we used the pooledors of multiple adverse childhood health experiencesinstead of each types of child maltreatment and itsseverity second we focused on the economic burdendue to the mortality and morbidity of child maltreatment but did not consider nonhealth human capitalaspectslikeother economic burden estimation studies the availability of data on the related medical costs were limited wehealthconsequences and explored their unit costs for the estimates to address the knowledge gap thirdtargeted majorneverthelessthereproductiverecently in japan a continuum ofintensive supports to mothers and childrearing families encompassingcycle has been widelyimplemented in most local authorities such an integral approach serves as an essential preventive strategy against child maltreatment and other harmfulpractices by early detection and intervention of highrisk households in pregnancy postpartum and childrearing periods thisstudy can provide decisionmakers information on the economic burden of childmaltreatment as well as an important input in futureeconomic evaluations costeffectiveness analysis oncurrently ongoing intervention and policy in additionour results hint an emphasis on preventive interventions on suicide attempts and depression which aretop causes of the attributable disease burden due tochild maltreatmentour study demonstrated that lifetime disease and economic burden due to child maltreatment in japan is substantial its disease burden was approximately equal tothe burden of colon and rectum cancers or stomach cancer in particular it is important to include the longterm disease burden in future studies related to diseaseburden and cost of illness for both technical and policyperspectivessupplementary informationsupplementary information accompanies this paper at httpsdoi101186s12889020093978additional file table a1 studies included in the quantitativesynthesis table a2 health outcomes and pooled ors used in this studyaht not included table a3 incidence rate by age and average onsetage based on the number of consultation cases disposed about childabuse at child guidance centersadditional file systematics review “2018520findpossible literature including japanese studies on risk of health outcomesattributable to child maltreatment figure a1 study selection prismaflow diagramabbreviationsdalys disabilityadjusted life years pafs population attributable fractionsgdp gross domestic product mhlw ministry of health labour and welfarewdv witnessing domestic violence aht abusive head traumaicd international classification of diseases gbd global burden of diseaserr risk ratio or odds ratio aces adverse childhood experiencesacknowledgementswe are grateful thank members of health informatics department kyotouniversity of public health school for their kind supportauthors™ contributionsmx and gr designed the study mx did the calculation and draft themanuscript gr and ty takahashi contributed to the revise ty tachibanatb and nt critically reviewed and provided important intellectual feedbackon the revise all authors have read and approved the manuscriptfundingthis study is granted by health labour sciences research grant japanagency for medical research and development and as part of an ipss 0cmo bmc public health page of project on the realization of œjapan™s plan for dynamic engagement of allcitizens the funders did not have any role in the study design datacollection and analysis interpretation of data or in writing the manuscriptavailability of data and materialsall the raw data is publicly accessible from respective official website asreference national healthcare expenditures and patient survey the datasets analysed during the current study are available from thecorresponding author on reasonable requestethics approval and consent to participatenot applicableconsent for publicationnot applicablecompeting intereststhe authors declare no conflict of interestauthor details1department of health policy national center for child health anddevelopment tokyo japan 2department of health informatics kyotouniversity school of public health kyoto japan 3department of empiricalsocial security research national institute of population and social securityresearch uchisaiwaicho chiyodaku tokyo japan4maternalchild psychiatry department of psychosocial medicine nationalcenter for child health and development tokyo japan 5faculty ofeconomics saitama university sakuraku japanreceived march accepted august referencesgilbert r widom cs browne k fergusson d webb e sjtl j burden andconsequences of child maltreatment in highincome countries lancet“number of consultation cases disposed about child abuse at child guidancecenters in japan in japanese [httpswwwestatgojpstatsearchfilespage1layoutdatalisttstat000001034573cycle8tclass1000001108815tclass2000001108820second21]definition and present condition of child abuse in japanese [httpswwwmhlwgojpseisakunitsuitebunyakodomokodomo_kosodatedvabouthtml] accessed july currie j spatz widom c longterm consequences of child abuse andneglect on adult economic wellbeing child maltreatment “hughes k bellis ma hardcastle ka sethi d butchart a mikton c jones ldunne mp the effect of multiple adverse childhood experiences on healtha systematic review and metaanalysis lancet public health 201728e356“fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai11901000koyoukintoujidoukateikyokusoumuka002_1pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile06seisakujouhou11900000koyoukintoujidoukateikyoku0000180499pdf] accessed july fy budget outline for child abuse prevention [httpswwwmhlwgojpfile05shingikai12601000seisakutoukatsukansanjikanshitsu_shakaihoshoutantou0000058633pdf] accessed july the economics of child abuse a study of san francisco [httpssafeandsoundwpcontentuploads201709economicsofabuse_report_sfcapc1pdf] accessed july fang x brown ds florence cs mercy ja the economic burden of childmaltreatment in the united states and implications for prevention childabuse negl “ habetha s bleich s weidenhammer j fegert jm a prevalencebasedapproach to societal costs occurring in consequence of child abuse andneglect child adolesc psychiatry ment health mccarthy mm taylor p norman re pezzullo l tucci j goddard c thelifetime economic and social costs of child maltreatment in australia childyouth serv rev “ wada i igarashi a the social costs of child abuse in japan child youth servrev “ miller tr steinbeigle r wicks a lawrence ba barr m barr rgjp disabilityadjusted lifeyear burden of abusive head trauma at ages “ pediatrics20141346e1545“fang x fry da brown ds mercy ja dunne mp butchart ar corso psmaynzyuk k dzhygyr y chen y the burden of child maltreatment inthe east asia and pacific region child abuse negl “ corso ps fertig ar the economic impact of child maltreatment in theunited states are the estimates credible child abuse negl “ macroeconomics and health investing in health for economicdevelopment [httpwhqlibdocwhointpublications2001924154550xpdf]accessed july segel je costofillness studies”a primer rtiunc center of excellence inhealth promotion economics jo c costofillness studies concepts scopes and methods clin molhepatol metrics population attributable fraction paf [httpwwwwhointhealthinfoglobal_burden_diseasemetrics_pafen] accessed july norman re byambaa m de r butchart a scott j vos t the longtermhealth consequences of child physical abuse emotional abuse and neglecta systematic review and metaanalysis plos med 2012911e1001349kalmakis ka chandler ge health consequences of adverse childhoodexperiences a systematic review j am assoc nurse pract “ unicef child maltreatment prevalence incidence and consequences inthe east asia and pacific region new york unicef rothman kj epidemiology an introduction oxford university press joyce t huecker mr pediatric abusive head trauma shaken babysyndrome [updated aug ] in statpearls [internet] treasure islandfl statpearls publishing available from httpswwwncbinlmnihgovbooksnbk499836 yamaoka y fujiwara t fujino y matsuda s fushimi k incidence and agedistribution of hospitalized presumptive and possible abusive head traumaof children under months old in japan j epidemiol httpsdoi102188jeaje20180094japanese population projection [httpwwwstatgojpdatajinsui2016np] accessed july summary of patient survey [httpswwwmhlwgojpenglishdatabasedbhsssps_2014html] accessed july kirigia jm mburugu gn huka gs the indirect cost of disability adjusted lifeyears lost among the elderly in kenya int arch med httpsdoi1038232483 mortality and global health estimates [httpwwwwhointghomortality_burden_diseaseen] accessed july japan gdp gross domestic product [httpscountryeconomycomgdpjapanyear2016] accessed july the results of verification of death cases caused by child abuse threport [httpswwwmhlwgojpstfseisakunitsuitebunya0000173329_00001html] accessed july miller tr steinbeigle r wicks a lawrence ba barr m barr rg disabilityadjusted lifeyear burden of abusive head trauma at ages pediatrics20141346e154550 httpsdoi101542peds20141385shiroiwa t fukuda t ikeda s takura t moriwaki k development of anofficial guideline for the economic evaluation of drugsmedical devices injapan value health “ moore se scott jg ferrari aj mills r dunne mp erskine he devries kmdegenhardt l vos t whiteford ha burden attributable to childmaltreatment in australia child abuse negl “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations 0c"

to machine learning with python a guide for data scientists o™reilly media inc california demÅ¡ar j statistical comparisons of classifiers over multiple data sets j mach learn res “ yates a the ensembl rest api ensembl data for any language bioinformatics “ kim e r chang d k colorectal cancer in inflammatory bowel disease the risk pathogenesis prevention and diagnosis world j gastroenterol diovasc dis “ schulte d small dense ldl cholesterol in human subjects with different chronic inflammatory diseases nutr metab car smedley d biomartbiological queries made easy bmc genom quinlan a r hall i m bedtools a flexible suite of utilities for comparing genomic features bioinformatics “ 101093bioin forma ticsbtq03 rom¡n j evaluation of responsive gene expression as a sensitive and specific biomarker in patients with ulcerative colitis inflamm bowel dis “ 101002ibd23020 song r identification and analysis of key genes associated with ulcerative colitis based on dna microarray data medicine baltimore e10658 101097md00000 schwegmann k detection of early murine colorectal cancer by mmp29guided fluorescence endoscopy inflamm bowel dis “ 101097mib00000 oliveira l g d positive correlation between disease activity index and matrix metalloproteinases activity in a rat model of colitis arq gastroenterol “ 101590s0004 shin js antiinflammatory effect of a standardized triterpenoidrich fraction isolated from rubus coreanus on dextran sodium sulfateinduced acute colitis in mice and lpsinduced macrophages j ethnopharmacol 158pt a “ 101016jjep201410044 owens d w lane e b keratin mutations and intestinal pathology j pathol “ 101002path1646 macfie t s duox2 and duoxa2 form the predominant enzyme system capable of producing the reactive oxygen species h2o2 in active ulcerative colitis and are modulated by 5aminosalicylic acid inflamm bowel dis “ 10109701mib00004 0e palmer n p concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease one e0222952 101371journ alpone02229 wei z large sample size wide variant spectrum and advanced machinelearning technique boost risk prediction for inflammatory bowel disease am j hum genet “ amrhein v greenland s mcshane b scientists rise up against statistical significance nature “ wasserstein r a0l schirm a a0l lazar n a0a moving to a world beyond œp  maeda y fully automated diagnostic system with artificial intelligence using endocytoscopy to identify the presence of histologic inflammation associated with ulcerative colitis with video gastrointest endosc “ 101016jgie201809024 acknowledgementsthis research was partially supported by grants from the natural sciences and engineering research council of canada nserc to hu grant number rgpin and to lpc grant number rgpin hmk was partially supported by funding from memorial university™s school of graduate studiesauthor a0contributionsconceptualization hu and lpc methodology hmk hu and lpc analysis hmk and lpc writing hmk hu and lpc experiments hmk supervision hu and lpccompeting interests the authors declare no competing interestsadditional informationcorrespondence and requests for materials should be addressed to hu a0or a0lpcreprints and permissions information is available at wwwnaturecomreprintspublisher™s note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliationsscientific reports 101038s41598020705830vol1234567890wwwnaturecomscientificreports 0copen access this is licensed under a creative commons attribution international license which permits use sharing adaptation distribution and reproduction in any medium or format as long as you give appropriate credit to the original authors and the source provide a link to the creative commons license and indicate if changes were made the images or other third party material in this are included in the ™s creative commons license unless indicated otherwise in a credit line to the material if material is not included in the ™s creative commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtain permission directly from the copyright holder to view a copy of this license visit httpcreat iveco mmons licen sesby40 the authors scientific reports 101038s41598020705830vol0123456789wwwnaturecomscientificreports 0c'

" preproof 0c highlights f0b7 propolis made by bees from bioactive plant resins has antiviral activity f0b7 propolis potentially can interfere with host cell invasion by sarscov2 f0b7 propolis blocks proinflammatory pak1 a kinase highly expressed in covid19 patients propolis a resinous material produced by honey bees from plant exudates has long been used in traditional herbal medicine and is widely consumed as a health aid and immune system booster the covid19 pandemic has renewed interest in propolis products worldwide fortunately various aspects of the sarscov2 infection mechanism are potential targets for propolis compounds sarscov2 entry into host cells is characterized by viral spike protein f0b7 propolis is a safe widely consumed functional food with medicinal properties f0b7 standardized propolis has consistent properties for lab and clinical research preprooffactor in advanced covid19 disease propolis has also shown promise as an aid in the treatment of various of the comorbidities that are particularly dangerous in covid19 patients including induced lung inflammation fibrosis and immune system suppression propolis components have inhibitory effects on the ace2 tmprss2 and pak1 signaling pathways in addition antiviral activity has been proven in vitro and in vivo in preclinical studies propolis promoted α this immunoregulation involves monocytes and macrophages as well as jak2stat3 nfkb and inflammasome pathways reducing the risk of cytokine storm syndrome a major mortality interaction with cellular angiotensinconverting enzyme ace2 and serine protease tmprss2 this mechanism involves pak1 overexpression which is a kinase that mediates coronavirusimmunoregulation of proinflammatory cytokines including reduction in il6 il1 beta and tnfrespiratory diseases hypertension diabetes and cancer standardized propolis products with consistent bioactive properties are now available given the current emergency caused by the covid19 pandemic and limited therapeutic options propolis is presented as a promising and 0crelevant therapeutic option that is safe easy to administrate orally and is readily available as a natural supplement and functional food keywords propolis sarscov2 covid19 antiviral antiinflammatory pak1 blocker introduction the covid19 pandemic is of grave concern due its impact on human health and on the deadly disease most require more robust data in clinical trials before they can be widely and safely used isolation and stayathome measures do not effectively protect essential workers economy it is much more deadly than influenza and other types of diseases that recently have had worldwide impact forcing countries to take unusual measures such as limiting travel closing schools businesses and other locations where many people can come into contact with each other various public healthcare strategies have been adopted in an attempt to reduce the impact of the disease but with limited effectiveness as the virus continues to spread often through asymptomatic patients unfortunately tests to determine if people are infectious or were previously infected are not widely available often are costly and frequently do not provide timely and accurate results various therapeutic alternatives have been proposed and tested however preproofœnonessential professions return to the workplace they become more at risk for infection in this scenario any options that could help ameliorate disease progression and its consequences even marginally would be useful the world needs safe alternatives to help reduce the impact of this especially health care personnel who have become infected and are dying at alarming rates economic and other necessities limit how well and how long these isolation measures can be maintained especially in poor countries and in poor communities such as slums and favelas [ ] as populations gradually try to get back to normalcy reducing social distancing and people in natural products which have historically been widely used to help avoid and alleviate diseases are among the options being considered as an adjuvant treatment for sarscov2 infection because they are generally inexpensive widely available and rarely have undesirable side effects some have proven antiviral activity an important advantage of using natural remedies is that people who have other health problems or who have mild flurelated 0c infection infection by sarscov2 the virus that causes covid19 is characterized by binding symptoms but do not have the means or courage to visit an already overcrowded medical facility could take simple and inexpensive measures to help reduce the impact of infection with sarscov2 considering the large number of deaths and other types of damage that the covid19 pandemic is causing there is an urgent need to find treatments that have been approved as safe and potentially able to inhibit the new coronavirus reduce its infectivity andor alleviate the symptoms of infection [ ] along this line propolis and its components emerge as potential candidate materials that could help to reduce the pathophysiological consequences of sarscovfactors increased pak1 levels also suppress the adaptive immune response facilitating viral replication [ ] sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory impairment and pulmonary failure immunologicalinflammatory between viral spike proteins and angiotensinconverting enzyme ace2 activation of the spike protein is mediated through proteases such as tmprss2 which play important roles in the viral infection after entry followed by endocytosis coronavirus infection causes pak1 upregulation a kinase that mediates lung inflammation lung fibrosis and other critical mortality preproofphenomena such as cytokine release syndrome have been shown to be important in the spectrum of sarscov2 infection these mechanisms are associated with an dysfunction more than the viral load per se along this line a retrospective observational study found higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα in patients with severe inflammatory diseases by affecting various metabolic cycles recently several studies have shown that propolis extract and some of its components act against several important targets in the pathophysiological context of the disease caused by sarscov2 such as reducing tmprss2 expression and reducing ace2 anchorage which would otherwise facilitate entry of covid19 compared to individuals with mild disease there is considerable evidence that propolis can reduce and alleviate the symptoms of the virus into the cell this is in addition to immunomodulation of monocytes macrophages reducing production of and eliminating il1 beta and il6 reduction of the transcription factors 0cviral replication and antiinflammatory action [ ] propolis and its properties are particularly relevant to sarscov2 infection such as immune system fortification reduced nfkb and jak2 stat3 and blocking pak1 which determine inflammatory activities and fibrosis caused by covid19 various comorbidities have been associated with severe covid19 symptoms and a greater chance of patients requiring intensive care these include hypertension and diabetes also mortality rates of covid19 patients are much higher in those with cardiovascular disease chronic respiratory disease and diabetes [ ] there is considerable evidence that these conditions could be alleviated by treatment with propolis this includes research in animal models of diabetes [ ] hypertension [ ] and cardiovascular disease [ ] propolis has properties that preproofthese plant products to make propolis which they use to protect the colony the production and use of propolis by honey bees evolved to the point that these social bees have considerably fewer immune genes than solitary insect species bees in colonies that produce more propolis are healthier and live longer and propolis consumption by the bees augments their immune [ ] as this variability can affect their medicinal properties standardized propolis products have been developed to help meet the need for a product that does not vary in the main bioactive components and is safe with minimal interaction with pharmaceutical drugs and proven efficacy in clinical trials in recent decades it has been shown to have antimicrobial including the composition of propolis varies according to the plant species available in each region propolis is a product derived from resins and plant exudates plants defend themselves from pathogens mainly by producing phytochemicals many of which have been extracted and used in medicine plant defense substances collected by bees include phenols and terpenoids phytochemical compounds that show promise for the inhibition of coronavirus in humans include quercetin myricetin and caffeic acid all components of propolis honey bees and many other species of social bees recognize these antimicrobial properties and selectively collect and process response to bacterial challenge antiviral antiinflammatory immunomodulatory antioxidant and anticancer properties propolis has historically been widely used to alleviate various diseases [ ] it also has been considered among other natural alternatives as an adjuvant treatment for sarscov2 infection 0ccompared to the propolis that the bees make from these plant materials because it is generally inexpensive widely available and rarely causes undesirable side effects some types of propolis that are highly valued for their medicinal properties such as brazilian green propolis are mainly produced by bees from materials they collect from specific plants in this case baccharis dracunculifolia after the botanical origin of the propolis has been identified extracts of the plant can be made to develop useful products such as a medicinal mouthwash however the medicinal properties of these plant extracts are often inferior can be safely consumed these propolis products and the raw material for their manufacture are extensively exported by brazilian companies especially to asian countries including japan south among natural medicine alternatives propolis has been widely studied and is already extensively consumed in many countries [ ] for example propolis products such as throat sprays and extracts are produced by hundreds of companies in brazil and are sold as a health aid in nearly every pharmacy throughout the country demonstrating on a practical basis that they preproofin fact propolis has a wide spectrum of pharmacological properties and is a dietary supplement that is commonly consumed by both healthy and sick people as a preventative precaution and for treatment it is also used in veterinary medicine due its antibacterial antifungal antiviral korea and china [ ] the importance of propolis in chinese japanese russian and korean medicine is reflected in the number of patents for propolis containing products registered by including about by china and “ each for japan russia and korea since about new propolisrelated patents were applied for in the us patent office it is a key ingredient in traditional chinese medicine japanese scientists have isolated and patented various brazilian propolis components for cancer treatment demonstrating their usefulness why propolis may be a good fit for dealing with covid19 antiparasitic hepatoprotective and immunomodulatory activities in the wake of the coronavirus outbreak south korea has seen a boon in the use of functional foods according to their ministry of food and drug safety œhealth functional foods are nutrients that have been proven to be beneficial to health in march of this year in response to the coronavirus pandemic the ministry eased regulations for propolis which is considered a 0ccould help fight against the covid19 pandemic active site of this enzyme is a relevant target for drug discovery functional food and allowed new oral formulations however despite considerable evidence that propolis can reduce and alleviate disease symptoms its acceptance as a healthpromoting supplement in human medicine has been limited in many countries such as the usa because of a relevant criticism that propolis products are not standardized and vary in their components and biological activity in part this is because propolis varies with the species of plants available in each region from which the bees collect resins to produce it [ ] however standardized propolis products have recently become available to help fill the need for a product that does not vary in the main bioactive components and effectiveness [ ] one such option a standardized brazilian propolis extract blend has been tested for safety and effectiveness in clinical trials for treating kidney disease and diabetes denture stomatitis and burn patients therefore propolis as a nutraceutical or functional food should be considered as a resource that preproofcaffeic acid phenethyl ester cape galangin chrysin and caffeic acid substances found in several different types of propolis around the world appeared as potential drugs against this viral target table specifically cape was predicted to interact with sarscov2 mpro in a potential targets in order to inactive the virus and reduce the damage that it causes the main protease of coronavirus sarscov2 mpro 3chymotrypsinlike cysteine enzyme is essential similar study therefore although it will be necessary to run in vitro assays to evaluate the potential antisarscov2 effects of propolis andor its constituents these in silico results are along this line hashem evaluated various natural compounds with an in silico approach molecular docking to try to find useful options for treating sarscov2 infection curiously for coronavirus processing of polyproteins and for its life cycle and therefore inhibition of the some propolis compounds can potentially interact with sarscov2 mpro the research community has examined the genetic code of coronavirus and the mechanisms underlying the damages caused by sarscov2 to help search for drugs andor well boding propolis can interact with ace and tmprss2 potentially blocking or reducing sarscov2 invasion of the host cell 0c sarscov2 strongly binds to angiotensinconverting enzyme ace2 using this enzyme as a receptor for invasion and replication in the host cell [ ] causing damage and increasing interpersonal transmission [ ] consequently ace inhibitors have been considered as useful drug alternatives however potential deleterious effects on users of angiotensinconverting enzyme ace inhibitors and angiotensin receptor blockers arbs have tool against potential cardiovascular events inhibition of ace2 enzyme is an important target for treatment against sarscov2 myricetin caffeic acid phenethyl ester hesperetin and pinocembrin rutin interacts with zinc fingers of the active sites of ace2 a metalloprotease that presents the same zinc finger in ace1 emerged as a concern for treatment of covid19 patients an observational study involving patients did not confirm this suspicion and therefore these classes of drugs remain an important infection [ ] güler et al prepared an alcoholic extract of propolis and identified some hydroxycinnamic acids caffeic acid pcoumaric acid tcinnamic acid and cape the flavanons rutin and myricetin and the flavones hesperidin chrysin and pinocembrin using molecular docking evaluations they found that rutin had the highest binding energy to ace2 followed by preproofvarious characteristics including inhibition of ace they found strong inhibition for most of the propolis types they studied with higher than ace inhibition the best results were found in addition to the in silico evidence osés et al evaluated several types of propolis for with the propolis components catechin and pcoumaric acid ace2 and tmprss2 transmembrane serine protease on the surface of host cells are used by sarscov2 via interaction with spike glycoproteins in order to proceed with invasion and replication vardhan sahoo studied several molecules commonly found in medicinal herbs using molecular docking procedures with relevant targets such as rnadependent rna polymerase rdrp ace2 and spike glycoproteins and compared the resulting scores with those of hydroxychloroquine limonin was the most active compound however quercetin and kaempferol also propolis compounds gave high docking scores kaempferol was studied in prostate cancer models and the expression of tmprss2 was reduced showing a potential mechanism of action for an antitumoral effect kaempferol could be an important propolis component for use against covid19 since it is involved in the inhibition of tmprss2 0c potentially interacting with ace2 rdrp and spike glycoprotein sgp besides its antiviral activity table propolis blocks pak1 potentially avoiding lung fibrosis and restoring a normal immune response among the possible targets for controlling covid19 damage the major œpathogenic contributes to their suppression pak1 inhibitors can both help combat the virus and restore a normal immune response kinase pak1 is key it is an essential component in malaria and viral infections but it is also involved in a wide variety of other diseases and disease conditions including cancer inflammation and immunosuppression when abnormally activated consequences of pak1 activation include lung fibrosis which is an aggravating factor in covid19 pak1 is activated by rac xu et al demonstrated that caffeic acid and its ester cape components of propolis can inactivate rac consequently inhibiting pak1 the inactivation of pak1 directly or upstream can potentially attenuate coronavirus pathogenesis bcells and tcells are lymphocytes that produce specific antibodies against viruses and other intruders and pak1 preproofimprove the immune response its components increase neutralizing antibody titers activate phagocytosis and increase ifnγ levels and the number of lymphocytes an increase in ifnγ levels was also detected by shimizu et al who evaluated the mechanisms involved in the cape caffeic acid phenethyl ester is a potent inhibitor of activation of nfkb in myelomonocytic cells anse et al demonstrated that propolis cape quercetin hesperidin and some other propolis flavonoids can inhibit the cytokine production of th1 and th2 type t cells while increasing tgfbeta an important antiinflammatory cytokine moreover cape can attenuate oxidative stress and inflammation through downregulation of jak2stat3 signaling propolis from europe and temperate asia usually made by bees from resins collected from poplar trees has predominantly flavonoid compounds while green propolis from baccharis dracunculifolia a propolis exclusively found in brazil has various kinds of flavonoids and prenylated phenylpropanoids such as artepellin c baccharin and drupanin these and all other types of propolis can inactivate pak1 artepillin c selectively inhibits pak1 table some studies have shown that propolis can act as an immunostimulant with the ability to effects of some types of propolis in a herpes simplex animal model 0c as well as having an immunomodulatory effect in which cape inhibits il6 phosphorylation and stat3 which are important for proinflammatory th17 development besides the antiinflammatory effect of cape and kaempferol paulino et al evaluated the antiinflammatory effect of artepellin c in rat paw edema and in cell cultures demonstrating that the activity was at least in part mediated by prostaglandin e2 and no inhibition through nfkb modulation artepillin c is an important biomarker of brazilian green propolis botanical source baccharis dracunculifolia immune modulation is desirable since coronavirus infection dysregulates the immune response in the initial phases of infection which facilitates viral replication however in later stages of covid19 the body develops an exaggerated inflammatory response which can greatly damage the lungs and other ans propolis different from typical immunosuppressants can help avoid immunosuppression during the initial phases of disease and in later stages reduce an exaggerated host inflammatory response inhibiting excess il6 il2 and jak signaling cape a propolis component is also known as an immunemodulating agent and should be considered as an alternative to help reduce an exaggerated inflammatory response in a mouse model propolis had immunomodulatory action in vivo on tolllike receptor expression and on preproofreplication and infectivity potentially decreasing lung inflammation due to antiinflammatory properties while promoting immune system fortification these are useful properties that could there is ample evidence for interference of propolis andor its components with viral propolis has been tested against various viral disease anisms initial successes have prompted research to determine the most useful components which may be modified to produce more active and specific pharmaceuticals viruses that were controlled by propolis in animal models with suggestion for control in humans include influenza [ ] herpes simplex virus type and hiv [ ] shimizu et al evaluated three different types of propolis in ethanol extracts using a murine model of herpes simplex virus type despite the chemical differences proinflammatory cytokine production help minimize the symptoms and deleterious effects of covid19 figure figure propolis as an antiviral substance 0cdue to the different plant origins of the resins the bees used to produce the propolis baccharis dracunculifolia baccharis eriodata and myrceugenia euosma all three propolis extracts not only had direct antihsv1 effects but also stimulated immunological activity against intradermal hsv1 infection in mice antiviral activity of propolis has been reported for dna and rna viruses poliovirus herpes simplex virus and adenovirus in an in vitro model cultured cells the best results were obtained against poliovirus and herpes virus with inhibition of the latter at a propolis concentration of ugml the propolis components chrysine and kaempferol caused a concentrationdependent reduction of intracellular replication of herpesvirus strains when host cell monolayers were infected and subsequently cultured in a drugcontaining medium quercetin another propolis component had the same effect but only at the highest concentrations tested ugml against various human herpes simplex virus strains with a intracellular replication reduction of approximately while it reduced the infectivity of bovine herpes virus human adenovirus human coronavirus and bovine coronavirus about the reduction was in the preproofimpairment and pulmonary failure inflammatory response to infection sarscov2 infection is associated with increased levels of chemokines and activated proinflammatory cytokines that lead to the development of atypical pneumonia with rapid respiratory immunologicalinflammatory phenomena such as cytokine release syndrome have been shown to be important mortality factors in sarscov2 infection higher serum levels of proinflammatory cytokines such as il6 il1 and tnfα are found in patients with severe covid compared to those of individuals with mild disease molecular mechanisms involved in this immune process are the targets of various synthetic medicines being tested in patients including ciclesonide hydroxy chloroquine ivermectin and ketorolac which are pak1 blockers pak1 raccdc42activated kinase is overexpressed in the lung in response to sarscov2 infection and is a critical mediator of the cytokine storm that frequently results in mortality the most critical cases of covid19 which require ventilatorassisted intensive care and often result in prolonged ventilator dependency and death are a result of an exaggerated case of rotavirus antiinflammatory and immunomodulatory properties of propolis 0cin hospitalized patients fortuitously propolis components are effective pak1 blockers table there is considerable evidence that propolis can reduce and alleviate the symptoms of inflammatory diseases and has immunomodulatory properties [ ] however these properties can vary according to the plant origin of the propolis as well as the extraction processsolvent used and the inflammatory protocol cell culture animal models induction by lipopolysaccharides when the propolis extracts are tested tests with animal models have shown that propolis can reduce the levels of il6 and tnfalpha which are key proinflammatory mediators and increase the levels of the regulatory cytokine il10 kaempferol a propolis component reduces il6 tnfalpha and vegf vascular endothelial growth factor via the preproofifnγ in serum fernandes et al found that propolis exerted a positive adjuvant effect on vaccines that were developed against canine coronavirus they assayed ifnγ which is an effective way to measure the cellular response induced by a vaccine in a mouse model propolis added as an adjuvant to inactivated swine herpesvirus type vaccine stimulated increased cellular propolis is considered a safe immunostimulant and a potent vaccine adjuvant propolis has been widely tested as a vaccine adjuvant because it induces an earlier immune response and provides a longer protection period it is also included as an adjuvant ingredient in traditional chinese medicine propolis flavonoids have potential as adjuvants enhancing igg il4 and propolis has potential as a vaccine adjuvant erknfkbcmycp21 pathway table tests on macrophage cell cultures also demonstrated that propolis inhibits the production of il1 beta an important component of the inflammasome inflammatory pathway in diseases such as rheumatoid arthritis lupus and other autoimmune diseases although the mechanisms of action are not well elucidated these propolis components have potential as complementary supplements in the preventive treatment of chronic inflammatory diseases and humoral responses increasing ifnγ [ ] propolis enhanced the immune response to inactivated porcine parvovirus vaccine in guinea pigs when added to a trichomonas vaginalis protein vaccine propolis increased the igg antibody response times in mice 0ccancer is considered a relevant comorbidity factor for covid19 cancer patients have a patients cancer patients compared to the protein alone propolis was also effective as an adjuvant in the immunization of cattle with bovine herpesvirus it improved the humoral and cellular responses in mice inoculated with inactivated virus vaccines propolis as an adjuvant gave a similar immune response increasing ifnγ levels to alum and freund™s adjuvant in mice vaccinated with an hiv1 polytope vaccine candidate with less risk of undesirable side effects comorbidities and evidence of how propolis can help reduce their impact in covid19 to risk a visit to a clinic or hospital to determine if they have cancer alternative therapies could help retard cancer or reduce the impact of cancer and cancer treatment in covid19 times higher risk of progressing to severe covid19 disease than patients without comorbidities also the hospital environment during the coronavirus pandemic can interfere with or delay the treatment that cancer patients should receive patients with symptoms may choose not preproofacid cape quercetin and chrysin propolis and its components normally have little impact on normal cells displaying differential cytotoxicity in liver cancer melanoma and breast cell carcinoma cell lines [ ] propolis enhances the activity of tumor various types including bladder blood brain breast colon head and neck kidney liver pancreas prostate and skin cancers propolis could help prevent cancer progression in various parts of the world it is considered an alternative therapy for cancer treatment propolis extracts have been found to inhibit tumor cell growth both in vitro and in vivo including inhibition of angiogenesis demonstrating potential for the development of new anticancer drugs various components of propolis have been shown to inhibit cancer cell growth including cinnamic propolis has potential as a complementary therapy for cancer it has shown efficacy against necrosis factor related apoptosis inducing ligand trail in cancer cells hypertension and cardiovascular disease 0c hypertension and cardiovascular disease are considered relevant comorbidities for covid19 propolis has demonstrated antihypertensive effects in rat models [ ] in cameroon it is used in popular medicine to treat various ailments including high blood pressure propolis has been widely used as a dietary supplement for its health benefits including cardiovascular protective effects [ ] in a human trial consumption of propolis improved critical blood parameters including hdl gsh and tbars levels demonstrating that obesity a natural antiobesity agent it could contribute to a reduced risk for cardiovascular disease obesity is a major comorbidity and predictor of increased mortality in covd19 patients obesity and sarscov2 both induce an inflammatory process exacerbating sarscov2 infection in the obese propolis reduced inflammation and prevented hyperlipidemia and metabolic syndromes in highly caloric diet induced obesity in mice body weight gain visceral adipose tissue liver and serum triglycerides cholesterol and nonesterified fatty acids were all reduced in the propolis fed mice [ ] caffeic acid phenethyl ester a propolis component is preproofplatelet aggregation was reduced by propolis in tests on human blood in vitro and in other in vitro tests caffeic acid phenethyl ester cape a wellstudied bioactive propolis component inhibits collagen induced platelet activation thromboembolism thrombosis and microthrombosis microthrombosis disseminated intravascular coagulation and consequent multian failure are common in severely affected covid19 patients with associated high mortality rates anticoagulants are sometimes prescribed to such patients because they can reduce mortality tang et al an elevated level of plasminogen activator inhibitor1 pai1 is a biomarker and risk factor for thrombosis and atherosclerosis [ ] various types of evidence demonstrate that propolis can reduce platelet aggregation and other thrombosisrelated parameters propolis decreased thrombotic tendencies in mice by suppressing lipopolysaccharideinduced increases in pai1 levels [ ] propolis downregulated plateletderived growth factor and platelet endothelial cell adhesion molecules in lowdensity lipoprotein knockout mice 0c old age the elderly are more often affected by chronic inflammation characterized by systemically increased levels of proinflammatory cytokines which can contribute to development of a cytokine storm a major cause of covid19 mortality propolis has antioxidant properties which could help retard o

Human transcription factor and protein kinase gene fusions in human cancerKari Salokas Rigbe G Weldatsadik Markku VarjosaloOncogenic gene fusions are estimated to account for upto of cancer morbidity Recently sequencelevel studies have established oncofusions throughout all tissue types However the functional implications of the identified oncofusions have often not been investigated In this study identified oncofusions from a fusion detection approach DEEPEST were analyzed in detail Of the oncofusions we found almost are expected to produce functional proteins with features from both parent genes Kinases and transcription factors were the main gene families of the protein producing fusions considering their role as initiators actors and termination points of cellular signaling pathways we focused our indepth analyses on them Domain architecture of the fusions and their wildtype interactors suggests that abnormal molecular context of protein domains caused by fusion events may unlock the oncogenic potential of the wild type counterparts of the fusion proteins To understand overall oncofusion effects we performed differential expression analysis using TCGA cancer project samples Results indicated oncofusionspecific alterations in gene expression levels and lower expression levels of components of key cellular pathways in particular signal transduction and transcription regulation the sum of results suggests that kinase and transcription factor oncofusions deregulate cellular signaling possibly via acquiring novel functionsAt any given moment multitudes of molecular networks are activated in cells throughout the body An important feature of these networks is highly concerted regulation of key signaling and deviation from homeostasis can result in diseases such as cancer Cancer is a complex progressive multistep disorder which stems from mutations caused by genomic instability1 The accumulation of genetic and epigenetic abnormalities ultimately leads to the transformation of normal cells into malignant derivatives Two highly enriched gene groups being mutated in the majority of cancer types are protein kinases PKs and transcription factors TFs23 PKs mediate most signal transduction events in cells by phosphorylation of specific substrates thus modifying their activity cellular localization andor association with other proteins TFs are the œtransistors of the cellular signaling circuits controlling the transcriptional outcome of activated signaling by binding to regulative elements of their corresponding target genes and driving or suppressing their expression Therefore it is easy to understand why mutational deregulation of these two gene groups can have such an impact on tumorigenesisIn addition to harboring activating or inactivating somatic point mutations PKs and TFs account for a large fraction of all human fusion genes involved in cancer COSMIC Catalogue of Somatic Mutations in Cancer cancersangeruk4 and dbCRID Database of Chromosome Rearrangements in Disease5 Chromosomal translocations creating fusion genes are among the most common mutation class of known cancer genes and they have long been identified as driver mutations in certain types of cancer6 Recently oncogenic fusion genes hereafter oncofusions OFs have been found in many hematological and solid tumors demonstrating that translocations are a common cause of malignancy78 Fusion mutations occur when two different gene regions fuse together via translocation Examples of consequences of chromosomal fusion to protein structure range from missense mutations to expressionchange inducing promotergene “combinations to fully functional fusion proteins with neomorphic properties A classic example of gained functions is the breakpoint cluster regionAbelson tyrosineprotein kinase BCRABL1 translocation in chronic myeloid leukemia9 Alternatively a protooncogene is fused to a strong promoter and thereby the oncogenic function is upregulated due to the strong promoter of the upstream fusion partner This is common in lymphomas where oncogenes are juxtaposed to the promoters of the immunoglobulin genes10 and also in prostate cancer where ETS TF ERG is fused with TMPRSS2 regulatory Systems PathologyBiology Research Group Institute of Biotechnology HiLIFE University of Helsinki Helsinki Finland email markkuvarjosalohelsinkifiScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0csequences thus obtaining androgen receptor ARresponsive expression11 The current understanding favors the aberrant gene function model rather than promoterinduced overexpressionThe frequency of recurrent OFs varies depending on the specific type of cancer12“ but identified translocations are estimated to account for up to of cancer morbidity8 Recent fusion prioritization study found that inframe transcripts were the most powerful predictor of driver fusions16 confirming the intuition that inframe transcripts are crucial to function Notably breakpoints were also observed to preferentially avoid splitting of domains Together with frameshift conservation such trends could reflect a selection on fusion proteins to maintain protein stability and evade degradation pathways17Nextgeneration sequencing NGS of genomes and transcriptomes from primary human cancer cells is constantly revealing new gene fusions that are involved in driving tumorigenesis including examples found in colorectal carcinoma bladder carcinoma breast cancer and acute lymphoblastic leukemia ALL1518“ Furthermore NGS has provided enough detailed sequence information of the fusion breakpoints allowing a0us to initiate systemslevel research on human oncofusions As a result various algorithms have been developed to mine OFs from large cancer datasets such as TCGA However the concordance among the different algorithms is very low that metacaller approaches utilizing consensus calls have been employed21 which limit novel OF discoveries Recently a new statistical method DEEPEST22 was developed to overcome these limitations In this study oncofusions that involve PKs and TFs were selected from the data produced by DEEPEST applied to the TCGA datasetIn most cases it is not possible to draw definite s about the mechanisms or extent by which individual translocations contribute to cancer Predicting protein function from a sequence has proven an extremely difficult task With gene fusions the task is even more daunting However an unexpectedly large number of PKs and TFs have been found to be mutationally activated or have increased expression due to gene amplification or translocation in cancer6 The high number of PKs and TFs with relatively low individual mutational frequency suggests either that a large number of signaling pathways can contribute to cancer or that many PKs and TFs can regulate the same pathways when activated unphysiologically Some additional support for this hypothesis comes from the interconnectivity of the PKTFoncofusionsIn this study fusions predicted to produce inframe proteins were analyzed to understand the proteinlevel implications of fusion events The fusions were analyzed from the perspective of their domain architecture to understand likely modes of action of the novel proteins Furthermore known interactomes of the participating wild type proteins were used to determine possible mechanisms of action pathways of interest and possible treatment vectors for affecting as many different fusions as possible As a result multiple cellular signaling pathways were found to intersect with major subsets of these fusions and multiple individual key interactors such as NTRK1 with over and EGFR with over interacting fusions were identified as potential targets of interestMaterials and methodsfusion selection and annotation Fusions that involve protein kinase genes23 and transcription factors24 were selected from the fusions that were identified by applying DEEPEST to the whole TCGA dataset22 Of these were determined to be unique by considering Ensembl gene IDs biotypes chromosomal breakpoints AGFusion assigned fusion effects and resulting protein sequences AGFusion was used to annotate these gene fusions to the human genome assembly GRCh38 v89 from Ensembl For analysis involving gene pairs the pair entry was used in alphabetical order eg ERGTMPRSS2 instead of TMPRSS2ERG in all cases Fusions were considered protein coding if both genes contributed over amino acids to the productclinical data Clinical data for TCGA samples was obtained from the GDC data repository The data was matched to AGFusion output data based on TCGA barcode eg TCGAWBA80K using a custom inhouse python script Stage information from the clinical data was simplified where possible eg Stage IIA was changed to Stage II Entries such as Stage Stage X and III NOS were ignored Tissue entries were simplified from detailed ICDO topographical codes to more general eg C569 †’ C56 and mapped to names accordingly Chromosomal sequence information from GRCh38 v89 was used to categorize breakpoints into chromosomal interval groupsInteractor analysis Interactors for wild type proteins of all fusion partners were obtained from IMEx consortium25 and any interactions that were not confirmed to be physical by experimental methods were discarded Interactors were added to the interactor set from each fusion while leaving out the fusion pair genes themselves Annotations for interactors were obtained using Uniprot and Reactome From Reactome mappings to all levels of pathway hierarchy were used Dijkstra™s algorithm26 implemented with a custom python script was then used to establish shortest paths to Reactome root nodes for each network node A weight of was used for all network edgesDomain annotation For the protein producing fusions sequences of the protein products were produced using the AGFusion tools Duplicate fusions based on fusion genes and protein sequence were discarded Domains were taken from AGFusion output and mapped to protein sequence in the wild type protein The intactness of domains was then determined by matching the WT domain sequence to the predicted fusion protein sequence and only full length intact domains were picked for further study A domain was classified as PK or TFspecific if ‰¥ of all its occurrences were in PK or TF proteins respectivelyData visualization Data illustrations were made with CorelDRAW Excel and inhouse python scripts using Matplotlib and Seaborn Cytoscape27 was used for creating network figuresScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cDifferential expression analysis Gene expression quantification HTSeqcounts “files were downloaded from GDC data portal Samples where OFs with intact fulllength PK or TF domains were detected were grouped together based on fusion gene pairs The groups were then analyzed with DESeq228 using other samples with protein producing nonPKTF fusions as controls For each pair group differential expression analysis against an equal number of control samples picked from samples in which other proteinproducing fusions were found Analysis was repeated times for each fusion pair For the resulting significantly differentially expressed genes qvalue basemean and expected values were averaged across all runs and a fold change value calculated based on these GO annotations were then added from ensembl annotations and Reactome pathways from first mapping ensembl gene IDs to Uniprot via Ensembl BioMart and then to Reactome lowest level pathway terms via Reactome Zscore value for pathway level overunderexpression was calculated by a method used in GOplot29 ieby deducting the number of underexpressed genes from the number of overexpressed genes and dividing the result by the square root of the number of significantly changed genes FDR corrected p ‰¤ ResultsDetection of oncofusions from TCGA dataset reveals enrichment of PK and TF fusions In this study we focused on protein producing OF genes Translocation of chromosomal regions can result in either inframe or outofframe OFs Fig a01A To characterize the proteins produced by known OFs in the TCGA dataset which currently contains data from different cancer projects we launched an analysis to understand the potential functional space of the protein producing fusions Fig a01B and especially those that involve either a PK or a TF or both PKTF fusionsThe DEEPEST dataset included fusions detected from cancer samples Of these were unique fusions Fig a01C upper panel Among the unique OFs were predicted to retain frame and also produce potentially functional proteins where both genes contributed over inframe amino acids Fig a01B Supplementary table a0S1 The limit of amino acids was the length of the shortest nonrepeat domain present in the fused proteins Examining the resulting protein producing OF set we noticed an abundance of those involving PK or TF Indeed these fusions constituted protein producing OFs Fig a01C lower panel Generally the proportion of PKTF fusions a0was under except in the PKTF”fusion prone cancers acute myeloid leukemia cholangiocarcinoma thyroid carcinoma and thymoma The number of OFs per sample varied across cancer types The types most prone to protein producing fusions were sarcoma SARC with an average of protein producing fusions per sample esophageal carcinoma ESCA fusions uterine corpus endometrial carcinoma UCEC stomach adenocarcinoma STAD breast invasive carcinoma BRCA uterine carcinosarcoma UCS and ovarian serous cystadenocarcinoma OV Due to the prevalence of PK and TF genes in the fusions we next investigated if they are enriched in particular cancers While in most cancers PKTF fusions made up around “ of all protein producing OFs the percentage reached in acute myeloid leukemia LAML samples in thymoma THYM in thyroid carcinoma THCA and and in kidney renal papillary cell carcinoma KIRP and cholangiocarcinoma CHOL respectively Fig a01C Acute myeloid leukemia is well known as an OFprone cancer30 However aside from the four fusions detected between ABL1 and BCR the high percentage was mostly TFdriven with KMT2A RUNX1 and RARA being found in and fusions respectively This is in contrast to the peak in THCA which is driven by BRAF fusions fusions of RET of NTRK1 and of NTRK3 among other protein kinasesReading frame retention is common in pK and tf oncofusions The fusions consisted of unique gene pairs and individual genes of the pairs did not have any protein producing fusions The top protein producing fusion was RPS6KB1VMP1 with unique protein producing fusions in the dataset all the others having less than Fig a02A There were fusion gene pairs that were predicted to produce protein in at least fusions in fusions in and in fusion Out of the fusion gene pairs that produced or more unique proteins were PKTF fusionsTo better understand the behavior of prolific gene pairs we next mapped tissue annotations from TCGA to fusions of each gene pair based on barcodes from samples where a fusion of the gene pair was present In contrast to RPS6KB1VMP1 and ITGB6RBMS1 which were seen in samples from different cancers pairs were seen in samples of only one cancer type Out of these cancerspecific fusions were predicted to produce or more unique proteins with ERGTMPRSS2 predicted to produce different unique proteins supplementary table a0S2 PKTF fusions featured different PK or TF genes ERG being the most common TF and ERRB2 the most common PK supplementary table a0S3 Between and percent of oncofusions in each cancer project were unique highest being sarcoma with unique gene pair combinations and thyroid carcinoma the lowest with supplementary table a0S4 Protein producing fusions followed a similar theme unique protein producing fusions making up between and of all oncofusions in each given cancer project supplementary table a0S4We next looked in more detail what cancer stages PK and TF fusions were detected in The most prominent group was stage II breast invasive carcinoma which also had the most samples in the data set Fig a02B In total of the PK and TF fusions were found in stage I samples in stage II in stage III and in stage IV On average samples had PKTF fusions per sample However in some cancers PK or TF fusions are enriched towards the more severe stages Discounting stage groups with less than samples groups had more than fusions per sample ESCA stage III samples in particular had PKTF fusions per sample while STAD and BRCA stage IV samples had and respectively and STAD stage I had Supplementary table a0S5 The distribution of protein producing OFs mirrored that of PKTF fusions quite closely supplementary figure S1A In terms of chromosomal breakpoint locations those in the PKTF fusions varied compared Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cFusion gene Fusion gene PromoterProtein coding regionetis noisuFTranslocationChr Chr Chr Chr Inframe fusionOutofframe fusionTCGA DEEPEST dataset gene fusionsAGFusionEnsemblGRCh38TCGAclinical dataUniprotKinases Manning et al TFs Lambert et al et al Gene expressionquantification dataprotein producing uniqueoncofusions Protein codingProtein kinase or Transcription factoroncofusionsPfamdomainsIMEx consortium interactor databaseCOSMICcancer genecensusReactomepathwaysClinical overviewDomain analysisInteraction analysis Pathway analysisDifferential expressionanalysisSamplesOncofusions per sampleABCselpmas fo rebmuNCCAACLBACRBCSECLOHCDAOCCBLDACSEMBGCSNHHCKICRKIPRKILMALGGLCHILDAULCSULOSEMVODAAPGPCPDARPDAERCRASMCKSDATSTCGTACHTMYHTCECUSCUMVUnietorp ni FT ro KP fo noitroporPsnoisufocno gnicudorpelpmas rep snoisufocno fo rebmuNACCBLCABRCACESCCHOLCOADDLBCESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diffuselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinoma Uterine carcinosarcomaUCSUVM Uveal melanomaScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0c—‚Figure a0 Schematic illustration of the gene fusions workflow and the number of gene fusions in human cancer A Schematic description of gene fusions formation Fusions are formed mainly via balanced and unbalanced chromosomal rearrangements such as translocations deletions inversions and insertions This usually leads to formation of a fusion gene with the ² end of Gene and ² end of Gene If the fusion occurs between two protein coding genes depending on whether the reading frame is violated and where exactly the fusion occurs a fusion protein may be transcribed with features and domains from both partners Other possible outcomes include full or truncated ² gene under the control of the promoter of the ² gene B Workflow used in this study Analysis progressed from the total set of fusions discovered by the DEEPEST method22 and moved towards more specific kinase TF containing protein producing oncofusions We started with TCGA databased fusion set from Dehghannasiri et a0al for which we generated protein sequences with AGFusion Domains were added by matching sequence to Uniprot proteins annotated with Pfam domains after which nonunique entries were dropped Fusions were classified as protein producing if both gene fragments were predicted to produce AA of protein sequence From this set the two most prominent protein groups were protein kinases and transcription factors and thus we focused further analysis on the unique protein kinase or transcription factor containing fusions using the full protein producing fusion set for comparison Known interactions for wild type fusion proteins were obtained from IMEx consortium and used for estimating maximal foreseeable effect on signaling pathways from Reactome Finally TCGA gene expression quantification data was used to probe observable effects of kinaseTF fusions using other protein producing fusions as background C Top Breakdown of samples and fusion mutations by TCGA project Largest single contributor of samples with fusions was TCGA breast invasive carcinoma project BRCA which had the highest number of samples and identified fusion mutations Bottom Proportion of protein producing fusions that include PK or TF genesto all protein producing fusion mutations but the prominent role of PKTF fusions is illustrated by overlapping hotspots supplementary figure S2Intact inframe domains are commonly retained in OFs To understand the contribution of each OF to the overall development or survival of cancerous cells the functional consequences of any given mutation and its impact on the pathways the proteins are involved in must be understood To this end we analyzed all identified unique protein producing fusions and the fulllength inframe domains of the fusion proteinsWhile AGFusion does predict protein sequence for each fusion partner and corresponding conserved or lost domains a domain is counted as conserved already if only amino acids are included in the sequence To adapt this to the study of fulllength domains we first mapped the Pfam identifiers of the domains to sequences in the wild type proteins from Uniprot The domains were then defined as conserved only if the full sequence was present in the fusion protein This resulted in conserved domains in all protein producing fusions Over of these domains are in PKTF fusions which account for of all protein producing fusions supplementary tables S1 S6 suggesting overall domain count strongly favors PK and TF genes perhaps indicating that these fusions produce more functional proteins in comparison to all protein producing fusionsThe most conserved domain was the protein tyrosine kinase domain Fig a03A supplementary table a0S6 which was conserved in fusions This was followed by the PH domain a common domain in intracellular signaling proteins and proteins of the cytoskeleton and the protein kinase domain To assess retention of nonobvious PK or TF domains we classified domains as PK or TF specific if over of the copies were found in PK or TF halves of the fusion proteins This resulted in copies of different TFspecific domains predicted to exist in the fusions compared to copies of PKspecific domains Most common TF domains were zinc finger C2H2 type KRAB and HLH DNA binding domains present in and copies respectively Many TF domains such as KRAB are involved in both transcriptional activation and repression depending on the molecular contextOn average protein producing fusions in samples of most cancer projects tended to have close to intact full length domain per protein producing OF PKTF fusions on average had more intact domains in all except for projects Fig a03B On average fusions in all projects tended to have between and intact domains while PKTF fusions featured a slightly higher average Although some cancers do appear to have particularly many domains this is mostly due to low count of fusions detected in the project Exception seems to be acute myeloid leukemia with detected protein producing fusions of which contain either a PK or a TF Most striking differences being seen in mesothelioma thyroid carcinoma rectum adenocarcinoma and uveal melanoma with and more retained domains on average in PKTF fusions compared to protein producing fusions respectivelyOn the cancer project level thyroid carcinoma had the highest percentage of PK domains of all domains identified in the samples of the project supplementary table a0S7 supplementary Figure S3 which totaled to only exceeded by breast invasive carcinoma with PK specific domains of all BRCA domains and lung adenocarcinoma LUAD with Proportion of TF domains varied less Kidney renal papillary cell carcinoma had of its intact domains in the TFspecific set followed by acute myeloid leukemia with and rectum adenocarcinoma and prostate adenocarcinoma both at Aside from prostate adenocarcinoma these projects had samples in the TCGA datasetinteractors of fusion partners can point to impact of ofs To understand what kind of implications the functional changes of lost conserved PK or TF specific domains in new combinations could have for the Scientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cNonprotein producing fusionProtein producing fusionLMEKLASMBRBGTIPMVBKSPRTXNURXNURHPMRYLKNWCREATMKLLEPXOFDNCCNPTPCDCCMUPNAKNICCATRFGFSSRPMTGREPAGPBBREARARLMPGRPTPTRCBLBAKRTNVTEBTBZASARFNZPBPSLEHYTTLDAMIUCMCCSAHDTMBMTPALDNYMZAYEDHSABRLBRSMTONCSRAYMUPSGRHPCMProtein kinaseTranscription factorProtein kinase and transcription factorAsnoisufocno fo rebmuNBIJNNAMDKCCMNBDDZDPONEPIRTSLYCHALAGTSfroCIV IIIIIIACCBLCABRCACESC CHOLCOADDLBC ESCAGBMHNSC Adrenocortical carcinomaBladder urothelial carcinomaBreast invasive carcinomaCervical squamous cell carcinomaand endocervical adenocarcinomaCholangiocarcinomaColon adenocarcinomaLymphoid neoplasm diï¬uselarge Bcell lymphomaEsophageal carcinomaGlioblastoma multiformeHead and neck squamous cell carcinoma Kidney chromophobeKidney renal clear cell carcinomaKidney renal papillary cell carcinomaKICHKIRCKIRPLAML Acute myeloid leukemiaLGGBrain lower grade gliomaLIHCLiver hepatocellular carcinomaLUADLung adenocarcinomaLUSCLung squamous cellcarcinomaMESOMesotheliomaOVOvarian serous cystadenocarcinomaPAADPancreatic adenocarcinomaPCPGPheochromocytoma and paraganglioma PRADREADSARCSKCMSTADTGCTTHCATHYMUCEC Prostate adenocarcinomaRectum adenocarcinomaSarcomaSkin cutaneous melanomaStomach adenocarcinomaTesticular germ cell tumorsThyroid carcinomaThymomaUterine corpus endometrialcarcinomaUCS Uterine carcinosarcomaUVM Uveal melanomaFigure a0 Clinical characterization of protein producing oncofusions by cancer stage A The most common protein producing gene pairs in oncofusions In total protein producing fusions were comprised of unique gene pairs predicted to produce one or more unique protein products The most common pair was RPS6KB1VMP1 with over unique proteins followed by ITGB6RBMS2 and ALKEML4 with each and LYRM9P3H4 and RUNX1RUNX1T1 at Kinase and TF fusions were common in top protein producing gene pairs illustrated by blue shading for the presence of a protein kinase in gene pair red for TF and orange for both B Sunburst diagram of project and stage distribution of PKTF oncofusions The innermost layer represents the number of fusions in each project The layers radiating out are the proportion of fusions detected in Stage I II III and IV samples in order from in to out Total numbers of fusions from each stage is marked under the stage indicatorsScientific RepoRtS 101038s41598020710408Vol1234567890wwwnaturecomscientificreports 0cProtein kinase or TF containing oncofusionsProtein producing oncofusionsAsniamod fo rebmuNniamodHS staeper niryknAniamod III epyt nitcenorbiFniamodC taeper atebG niamodDW niamod HC ygolomohninoplaC fitomnoitingocer ANRregnfiDHPniamodZDP epyt HC regnfi cniZniamod lanimret Cdevresnoc esacileHfitom ahpla eliretS niamodMAS xobBARKniamodHS niamodZOPBTBniamod HS tnairaV niamodnirehdaC niamodgnidnib lorecylglycaidsretse lobrohP niamodgnidnibANDxilehpoolxileH niamodFEGohR niamodPAGohRtaeper nirtcepSnietorp tropsnart noIniamod nilubolgonummIA ssalc niamod rotpecer LDLniamodomorBniamodAHF taeper hcir enicueL niamod tesVnilubolgonummI niamod epyt nidnopsobmorhTniamodMIL niamodWW niamod ekilFGEriap niamoddnahFEniamod lartnec MREF niamodFGEgnidnibmuiclaC esacilehxobHAEDDAEDniamodstEniamodoemoHfitom ahpla eliretS niamodMAS etis yrotibihni aX rotcaFnoitalugaoCrotpecer enomroh raelcun foDBLniamodYRPS niamod htaeDniamodXP niamod HMretropsnart CBAtaeper editpepocirtarteTniamod FASDGlaR noitaicossa saRylimaf nispodohr rotpecer enarbmemsnartniamodHP esanik enisoryt nietorPniamod esanik nietorPniamod tesI nilubolgonummIBnoisufocno gnidoc nietorp rep sniamoDProtein producing oncofusionsProtein kinase or TF containing oncofusionsCsnoisufocnoFT ro esanik nietorp gnitcaretni fo rebmuNLMALOSEMACHTLOHCDAERMVUPRKIMYHTMBGGGLGPCPTCGTDARPCSULDAOCMCKSCSECACLBDAULCRASCSNHCRKIACSECHILACRBVOCCADATSDAAPSCUCECUCBLDHCKIFound in COSMIC cancer gene censusPK oncofusionsTF oncofusionsPKTF oncofusionsOther protein producing oncofusions Percent of unique interactors in COSMIC canger gene census KRTNGAHWYOPXCBURGERFGEAPSHBAHWYRACADHAPSHPECHTRISALERMCMQAHWYFNRXDDNUJLCDCLBVUREAHWYCRSAPRCDCCDAMSMDMCADHPSLMPACPPPNFSAPRROTMBERCDAMSAPSHDNACCADHPCVCDKRPTATSACRBDNCCBDDABUIMBANCPARAROXBFUPNRNHFigure a0 Domain analysis of protein producing fusions A Intact fulllength domains identified in unique protein producing fusions In total intact domains were detected The protein tyrosine kinase domain was the most prevalent with identifications In addition protein kinase domain was detected with copies each Kinase or TF specific domains included and unique domains respectively copies of kinasespecific domains were seen and of TF specific Kinase domains focused more on the two top kinase domains whereas TF domains were a much more evenly distributed group the top TFspecific domain C2H2 type zinc finger having copies B The number of domains per protein coding oncofusion in the TCGA projects C Most common interactors of protein kinaseTF fusions Yaxis describes numbers of unique protein producing fusions where one or both of the fusion partner WT genes interact with the protein Top right inset Proportion of interactors found in COSMIC cancer gene census is higher in both protein kinase and transcription factor fusions and most common in fusions between protein kinase and transcription factor genesScientific RepoRtS 101038s41598020710408Vol0123456789wwwnaturecomscientificreports 0cATranscriptional regulationpathwaysRNA Polymerase II transcriptionGeneric transcription pathwaySignaling by Rho GTPasesMAPK family signaling cascadeGPCR signalingSignaling by NotchTGFβ signalingSignal transductionTranscriptional regulationby TP53Hedgehog pathwaySignaling by Receptor tyrosine kinasesIntegrin signalingIntracellular signaling bysecond messengersWnt signalingDeath receptor signalingNode proteins interact withTF fusionsKinase fusionsBCytokine Signaling in Immune systemDevelopmental BiologyAxon guidanceMetabolismPIP3 activates AKT signalingIntracellular signaling by second messengersPTEN RegulationRegulation of PTEN gene transcriptionOxidative Stress Induced SenescenceHDMs demethylate histonesSUMOylation of transcription cofactorsTranscriptional Regulation by E2F6SUMOylation of chromatin anization proteinsTranscriptional regulation by RUNX1Chromatin anizationChromatin modifying enzymesCellular SenescenceSUMO E3 ligases SUMOylate target proteinsSUMOylationInterferon alphabeta signalingEPHEphrin signalingPhospholipid metabolismEPHephrin mediated repulsion of cellsEPHAmediated growth cone collapsePI MetabolismSynthesis of PIPs at the plasma membraneInterferon SignalingMetabolism of lipidsOncogenic MAPK signalingSignaling by moderate kinase activity BRAF mutantsParadoxical activation of RAF signaling by kinase inactive BRAFSignaling downstream of RAS mutantsrRNA modification in the nucleus and cytosolRHO GTPases Activate ForminsMajor pathway of rRNA processing in the nucleolus and cytosolrRNA processing in the nucleus and cytosolrRNA processingProcessing of Capped IntronContaining PremRNAmRNA SplicingmRNA Splicing Major PathwayMacroautophagyAutophagyToll Like Receptor TLR3 CascadeMyD88independent TLR4 cascade TRIFTICAM1mediated TLR4 signaling Tolllike Receptor CascadesToll Like Receptor TLR4 CascadeSignaling by TGFbeta family membersSignaling by Nuclear ReceptorsTCF dependent signaling in response to WNTSignaling by WNTSignaling by

distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly citedVon Hippel“Lindau disease is an autosomal dominant inherited syndrome predisposing to a variety of highly vascularised tumorsin diï¬erent ans Although bilateral pheochromocytoma was reported in patients with von Hippel“Lindau disease the coexistence of primary hyperparathyroidism is not a common condition We report an observation of a primary hyperparathyroidism secondary to an ectopic secretion of intact parathyroid hormone in a 17yearold girl with von Hippel“Lindau diseaseand bilateral pheochromocytoma She presented with a newly diagnosed diabetes mellitus and a severe arterial hypertensionBlood tests disclosed hypercalcemia with increased intact PTH level Cervical ultrasound and sestamibi scintigraphy were normalTwentyfourhour urinary normetanephrine level was highly elevated pointing to a catecholaminesecreting tumor e abdominal computed tomography showed bilateral adrenal masses MIBG scintigraphy exhibited a high accumulation of the tracerin both adrenal tumors Genetic testing revealed a mutation of the VHL gene e patient underwent a bilateral adrenalectomye postoperative outcome was marked by normalization of blood pressure blood glucose calcium and PTH levels In our casethe elevation of intact PTH and its spontaneous normalization after surgical treatment of pheochromocytomas confirms itsectopic secretion IntroductionPheochromocytomas are uncommon neuroendocrinetumors that arise from chromaffin cells of the adrenalmedulla and produce excessive amounts of catecholamines epinephrine norepinephrine and dopamine []Although most pheochromocytomas are sporadic morethan are associated with an inherited mutation andthis frequency can be as high as if diagnosed before years of age [] In childhood pheochromocytomasare mostly due to genetic causes including von Hippel“Lindau VHL disease multiple endocrine neoplasiatype MEN2 hereditary pheochromocytoma paraganglioma syndrome and rarely neurofibromatosis type [] Compared to adults VHL disease is reported to bethe most frequent genetic disorder causing pheochromocytomasbilateral[] Whileinchildrenpheochromocytomas were reported in patients with VHLdisease the coexistence of primary hyperparathyroidismis not a common condition []Herein we report a case of a VHL disease with bilateralintactpheochromocytoma and an ectopic secretion ofparathyroid hormone in an adolescent girl Presentation of CaseA 17yearold girl was referred to our department for theinvestigation of a newly diagnosed diabetes mellitus and asevere arterial hypertension She was the first child ofconsanguineous parents Her past medical history wasunremarkable Her family history was notable for type diabetes and dyslipidemia but no history of pheochromocytoma paraganglioma unexplained sudden death or 0cCase Reports in Endocrinologycondition that may lead to thinking about VHL disease wasreportedAs symptoms the patient complained of headachespalpitations diaphoresis and hot flashes since one monthShe reported also asthenia and body weight loss of fivekilograms in two weeksOn examination she had a body weight of kg abody height of cm a body mass index of kgm2 ablood pressure of mmHg without orthostatichypotension a regular pulse of bpm and a largeabdominal caf´eaulait spot Figure yroid abdominal and neurological examinations were normalTwentyfourhour blood pressure monitoring confirmed the diagnosis of hypertension and the presence ofpeaks of mmHg Electrocardiogram showed asinus tachycardia Capillary glucose level was glwithout ketosisFundoscopy showed grade hypertensive retinopathywithout any other abnormalitiese results of biological investigations are shown inTable e diagnosis of clinically suspected pheochromocytomawas confirmed by the dosage of urinary methoxylated derivatives at times the upper limit of normal In addition thediagnosis of diabetes mellitus dyslipidemia and primaryhyperparathyroidism were madethe first on thee abdominal computed tomography CT showed tworight measuringadrenal masses — — mm with a spontaneous density of HUheterogeneously enhanced in the arterial time showing areasof necrosis with an absolute washout of the second onthe left measuring — mm with the same characteristicsas the first one Figure ere were neither other localizations norlymph nodes MetaiodobenzylguanidineMIBG scintigraphy exhibited a high accumulation of thetracer in both adrenal tumors with no other localizationCervical ultrasound and 99mTcsestamibi scintigraphy werenormal Cardiac ultrasound was normale diagnosis of multiple endocrine neoplasia type 2awas highly suspected and the patient underwent a molecularinvestigation DNA analysis did not find a RET protooncogene mutation However it showed a missense mutationc191G C pArg64Pro in exon of the VHL gene onchromosome e diagnosis of bilateral pheochromocytoma in the setting of VHL disease was established Abdominal CT scan and craniospinal magnetic resonanceimaging with contrast did not show any cysts or othertumorse patient was treated with an alpha blocker prasozinea calcium channel blocker a beta blocker and insulin at adaily dose of unitskg She underwent a bilateral adrenalectomy in two steps e pathological examinationconfirmed bilateral adrenal pheochromocytomas Replacementtherapy with hydrocortisone was initiated aftersurgerye postoperative outcome was determined by thespontaneous normalization of blood pressure blood glucose calcium and PTH levels e patient remainedasymptomatic with no evidence oflocal recurrence ordistant metastasis during the months of followup efamily screening for VHL has not been performed yet DiscussionVHL disease is a dominantly inherited familial cancersyndrome caused by a germline mutation in the VHL tumorsuppressor gene and predisposing to a variety of benign andmalignant neoplasms most frequently retinal central nervous system and spinal hemangioblastomas renal cell carcinoma RCC pheochromocytoma and pancreatic tumors[ ] While central nervous system and retinal hemangioblastomas are the earliest expressions of the VHL syndrome pheochromocytoma may be the first manifestationof the disease especially in children and adolescents as it wasthe case of our patient [“] e frequency of pheochromocytoma in VHL syndrome is about to []Families with VHL disease have been divided into twosubtypes VHL type and VHL type based on the likelihood of developing a pheochromocytoma e presence ofpheochromocytoma defines types VHL disease is latteris subdivided based on the risk of developing RCC Type 2Aand 2B families have a low and high incidence of RCCrespectively whereas VHL type 2C kinds are characterizedby the development of pheochromocytoma without anyother manifestations of the disease [] However late onsetof other attacks is possible and a followup even spaced isrequired []Genotypephenotype correlations have been documented for this disorder and specific mutations are associated with the appearance of tumors in certain ansWhile most type families were reported to be more likelycarrying a missense mutation in the VHL gene most type families are aï¬ected by truncating or deletion mutations[] In our case the presence of pheochromocytomas andthe missense mutation in VHL gene suggested type VHLdisease Moreover the mutation found in our patientpArg64Pro has been described in patients with isolatedpheochromocytoma associated with RCC or with pancreaticneuroendocrine tumor [“] However our patient didnot present any sign of RCC or pancreatic neuroendocrinetumor during the months of followup is evaluationmay be early in our case as RCC and pancreatic neuroendocrine tumors in patients with VHL disease generallyappear at more advanced ages around “ years []Pheochromocytoma in VHL disease tends to be seen at ayounger age and is more frequently multifocal as in ourpatient and may be extraadrenal [ ] In most publishedcases the mean age at presentation was about years butvery young cases have been described the youngest before years [ ] In addition VHLassociated pheochromocytomas are less likely to be associated with symptoms orbiochemical evidence of catecholamines production compared with those occurring in patients without VHL [ ]In a report of the National Institute of Health about patients with VHL disease and pheochromocytomas a totalof tumors were identified Of these originatedoutside the adrenal gland and of the patients wereasymptomatic without hypertension orevidence of 0cCase Reports in EndocrinologyFasting glucose level mmollGlycated hemoglobin Total cholesterol mmollTriglycerides mmollNatremia mmollKalemia mmollCreatinine mgLCalcium mgLPhosphate mgLAlbumin glIntact PTHˆ— μgLPTH parathormone TSH thyroid stimulating hormone FT4 freeT4 NMN normetanephrines MN metanephrinesˆ—e intact PTH dosage was madeTSH mUILFT4 ngdlCalcitonin ngL hurine NMN μg24 hurine MN μg24 hurine sodium mmol24 h hurine potassium mmol24 h hurine calcium mg24 h hproteinuria g24““using 3rd generation chemiluminescence immunoassayReference ranges‰¤““““““““““““Figure A large abdominal caf´eaulait spotTable Biological parameters before and after surgeryBefore surgeryAfter surgery””””””””Figure Abdominal computed tomography showing the two adrenal masses two white arrowsincreased catecholamines production [] is was not thecase of our patient who had sustained severe hypertensionassociated with the classic symptoms of pheochromocytomapalpitation sweating and hot flashes hypokalemia and asecondary diabetes mellitus In our case as reported in theliterature [ ] a remarkable remission of diabetes 0cCase Reports in EndocrinologySoahighmellitus and an improvement of lipid profile were noticedafter tumor removal confirming the secondary character ofthese two metabolic disorders In fact catecholamine excessaï¬ects insulin secretion decreased glucose uptake in theperipheral tissues and increased insulin resistance leading toimpaired fasting glucose or overt diabetes mellitus []Moreover the increase in catecholamine production may beresponsible for decreased inhibition of lipolysis by insulinand decreased activity of lecithincholesterol acyltransferasean enzyme which breaks down free cholesterol []e risk of malignancy is low Less than of allpheochromocytomas in VHL disease are malignant [] Inour case neither distant metastasis nor lymph nodes werefound but a long term followup should be carried outMeasurement of plasma or urinary metanephrines andnormetanephrines is the gold standard in diagnosingpheochromocytoma and can also provide important diagnostic information [] In fact in a study carried out byEisenhofer [] comparing the clinical and biochemical characteristics of pheochromocytomas in multipleendocrine neoplasia type versus the VHL syndrome andincluding and patients with these disorders respectively VHL patients almost exclusively produced normetanephrinesnormetanephrinestometanephrines ratio is expected in patients with VHL disease as was found in our patient Furthermore in comparison with MEN2 tumors VHL tumors had lower totaltissue contents of catecholamines and expression of tyrosinehydroxylase TH the ratelimiting enzyme in catecholamine synthesis ey also had much lower expression ofphenylethanolamine Nmethyltransferase PNMT the enzyme that converts norepinephrine to epinephrine andtissue stores of epinephrine [] Regarding the histopathological features VHLassociated pheochromocytomas arecharacterized by a thick vascular tumor capsule and are incontrast to MEN not associated with adrenal medullarhyperplasia []While bilateral pheochromocytomas were reported inpatients with VHL disease the coexistence of primary hyperparathyroidism as in our case is not a common condition[] Hypercalcemia associated with pheochromocytoma hasbeen documented and is thought to be caused by severalmechanisms First elevated catecholamines can activate thePTH receptor resulting in catecholamineinduced osteoclastic bone resorption but in contrast to our case the PTHlevel is not elevated [] Secondly hypercalcemia can be dueto the production of PTHrelated peptide PTHrp by thetumor which was doubtless not the case in our patient as thelevel of intact PTH was elevated [] Furthermore intactPTH in our case was quantified using a 3rd generationchemiluminescence immunoassay which does not recognizePTHrpird parathyroid adenoma can be a part of multipleendocrine neoplasia that was however rarely reported inVHL disease [] e negativity of the topographic investigations and the spontaneous normalization of intact PTHafter surgical treatment are against this hypothesis Finallyalthough the exact physiopathological mechanism is notclear the fact that both serum calcium and PTH levels wereelevated before surgery and became normal after the removal of the pheochromocytomas strongly suggests that thetumor itself was secreting PTH or a substance that stimulatesexcessive PTH secretion by the parathyroid glands Only fewcases of ectopic hormonal secretion by pheochromocytomasuch as adrenocorticotropic hormone ACTH calcitoninparathyroid hormone PTH vasoactive intestinal peptideVIP and growth hormonereleasing hormone GHRHwere reported [ ] Unfortunately immunohistochemistry assay for PTH in the tumor tissue was not available toclarify this question in our case Conclusionis report highlights the rare case of ectopic intact PTHsecretion by a bilateral pheochromocytoma in an adolescentgirl with VHL disease We consider that controlling calciumand PTH after adrenalectomy is useful if the topographicassessment of primary hyperparathyroidism is negativeTo the best of our knowledge our patient is the secondyoungest reported childhood VHL case in the literaturepresenting with a bilateral pheochromocytoma secretingectopic intact PTH Genetic testing and a meticulous followup are necessary for the diagnosis ofthe associatedcomorbidities in VHL diseaseData Availabilitye data used to support the findings of this study areavailable from the corresponding author upon requestConflicts of Intereste authors declare that they have no conflicts of interestAcknowledgmentse authors thank Professor Anne Barlier Laboratory ofMolecular Biology Hospital La Conception MarseilleFrance for her help in molecular studyReferences[] J W M Lenders QY Duh G Eisenhofer œPheochromocytoma and paraganglioma an endocrine societyclinical practice guideline e Journal of Clinical Endocrinology Metabolism vol no pp “ [] E Sbardella T Cranston A M Isidori œRoutine geneticscreening with a multigene panel in patients with pheochromocytomas Endocrine vol no pp “ [] S G Waguespack T Rich E Grubbs œA current reviewof the etiology diagnosis and treatment of pediatric pheochromocytoma and paraganglioma e Journal of ClinicalEndocrinology Metabolism vol no pp “[] M Barontini G Levin and G Sanso œCharacteristics ofpheochromocytoma in a to 20yearold population Annals of the New York Academy of Sciences vol no pp “ [] T Arao Y Okada T Tanikawa œA case of von HippelLindau disease with bilateral pheochromocytoma renal cell 0cCase Reports in Endocrinology[] O Mete A S Tischler R D Krijger œProtocol for theexamination of specimens from patients with pheochromocytomas and extraadrenal paragangliomas Archives ofPathology Laboratory Medicine vol no pp “[] N O Atuk T McDonald T Wood œFamilial Pheochromocytoma Hypercalcemia and von HippelLindauDisease a ten year study of a large family Medicine vol no pp “ [] K Takeda N Hara M Kawaguchi T Nishiyama andK Takahashi œParathyroid hormonerelated peptideproducing nonfamilial pheochromocytoma in a child International Journal of Urology vol no pp “ [] J KirkbyBott L Brunaud M Mathonet œEctopichormonesecreting pheochromocytoma a francophone observational study World Journal of Surgery vol no pp “ [] L V Neto G F Taboada L L Corrˆea œAcromegalysecondary to growth hormonereleasing hormone secreted byan incidentally discovered pheochromocytoma EndocrinePathology vol no pp “ carcinoma pelvic tumor spinal hemangioblastoma and primary hyperparathyroidism Endocrine Journal vol no pp “ [] A N A V D HorstSchrivers W J Sluiter R C Kruizinga œe incidence of consecutive manifestations in VonHippelLindau disease Familial Cancer vol no pp “ [] R R Lonser G M Glenn M Walther œvon HippelLindau disease e Lancet vol no pp “[] A D ‡akır H Turan A Aykut A Durmaz O Ercan andO Evliyao˘glu œTwo childhood pheochromocytoma cases dueto von HippelLindau disease one associated with pancreaticneuroendocrine tumor a very rare manifestation Journal ofClinical Research in Pediatric Endocrinology vol no pp “ [] S P Rednam A Erez H Druker œVon hippellindauand hereditary pheochromocytomaparagangliomasyndromes clinical features genetics and surveillance recommendations in childhood Clinical Cancer Research vol no pp e68“e75 [] G F C Fagundes J Petenuci D M Lourenco œNewinsights into pheochromocytoma surveillance of young patients with VHL missense mutations Journal of the Endocrine Society vol no pp “ [] F Hes R V D Luijt A Janssen œFrequency of VonHippelLindau germline mutations in classic and nonclassicVon HippelLindau disease identified by DNA sequencingSouthern blot analysis and multiplex ligationdependentprobe amplification Clinical Genetics vol no pp “ [] T Krauss A M Ferrara T P Links œPreventivemedicine of von HippelLindau diseaseassociated pancreaticneuroendocrine tumors EndocrineRelated Cancer vol no pp “ [] E Wittstr¨om M Nordling and S Andr´easson œGenotypephenotype correlations and retinal function and structure invon HippelLindau disease Ophthalmic Genetics vol no pp “ [] G Eisenhofer M M Walther TT Huynh œPheochromocytomas in von HippelLindau syndrome and multiple endocrine neoplasia type display distinct biochemicaland clinical phenotypes e Journal of Clinical Endocrinology Metabolism vol no pp “ [] M M Walther R Reiter H R Keiser œClinical andgenetic characterization of pheochromocytoma in von HippelLindau families comparison with sporadic pheochromocytomaofpheochromocytoma Journal of Urology vol no pp “ naturalhistoryinsight[] J Cha M Khurram L Gellert P Epstein N Baregamian andC Hendrickson œCase of reversible diabetes mellitus in thesetting of benign Pheochromocytoma Journal of Clinical andTranslational Endocrinology Case Reports vol pp “[] M L Good P Malekzadeh S M Ruï¬ œSurgical resection of pheochromocytomas and paragangliomes is associated with lower cholesterol levels World Journal of Surgeryvol no pp “ [] B Mesmar S PoolaKella and R Malek œe physiologybehind diabetes mellitus in patients with pheochromocytomaa review of the literature Endocrine Practice vol no pp “ givesinto 0c'

predominant male sex hormones drive the development andmaintenance of male characteristics by binding to androgen receptor AR As androgensare systemically distributed throughout the whole anism they affect many tissues andcell types in addition to those in male sexual ans It is now clear that the immunesystem is a target of androgen action In the lungs many immune cells express ARs andare responsive to androgens In this review we describe the effects of androgens and ARson lung myeloid immune cells”monocytes and macrophages”as they relate to healthand disease In particular we highlight the effect of androgens on lung diseases such asasthma chronic obstructive pulmonary disease and lung fibrosis We also discuss thetherapeutic use of androgens and how circulating androgens correlate with lung diseaseIn addition to human studies we also discuss how mouse models have helped to uncoverthe effect of androgens on monocytes and macrophages in lung disease Although therole of estrogen and other female hormones has been broadly analyzed in the literaturewe focus on the new perspectives of androgens as modulators of the immune systemthat target myeloid cells during lung ‚ammationEdited byFlavia BazzoniUniversity of Verona School ofMedicine and Surgery ItalyReviewed byPaola ParronchiUniversity of Florence ItalyTim WillingerKarolinska Institutet SwedenSandra O GollnickUniversity at Buffalo United StatesCorrespondenceNicola HellernhellerjhmieduKeywords androgen androgen receptor monocyte macrophage asthma lung sex difference sex hormoneSpecialty sectionThis was submitted toCytokines and Soluble Mediators inImmunitya section of the journalFrontiers in ImmunologyReceived March Accepted June Published August CitationBecerraDiaz M Song M and Heller N Androgen and AndrogenReceptors as Regulators of Monocyteand Macrophage Biology in theHealthy and Diseased LungFront Immunol 103389fimmu202001698INTRODUCTIONThe immune system is essential for maintaining homeostasis within tissues and ans andprotecting them against threats such as harmful pathogens or cancerous transformation Itcomprises both innate and adaptive components The innate immune system is made up of theinnate lymphoid innate lymphoid cells [ILCs] natural killer cells [NKs] and lymphoid tissueinducers [LTi] and innate myeloid subsets The innate immune system consists of a networkof immune cells and molecules that provide rapid firstline defense against pathogens In contrastthe adaptive immune response made up of B and T lymphocytes takes days or even weeks tobecome established Innate immune cells express pattern recognition receptors that recognizeunique and conserved pathogenassociated molecular patterns such as lipopolysaccharide LPSviral ssRNA and fungal glucan B and T cells have evolved to recognize a finer repertoireof self and nonselfantigens that facilitate pathogenspecific actions immunologic memorygeneration and host immune homeostasis regulation To accomplish this the adaptiveimmune response involves a tightly regulated interplay between T and B lymphocytes andFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyantigenpresenting cells of the myeloid lineage such as dendriticcells DCs monocytes and macrophages Myeloid cells arisefrom the bone marrow The type and magnitude of the immuneresponse is ‚uenced by biological sex and age and thereforediï¬ers between males and females Sex diï¬erences in the functionof the immune system arise from both genetic chromosomalsex diï¬erences and diï¬erences mediated by the action of maleand female sex hormones Because the concentration of sexhormones changes over the lifespan and throughout the courseof the menstrual cycle in women the function of the immunesystem also changes during diï¬erent stages of life Innate myeloidimmune cells like other cell types express sex hormone receptorsand are responsive to sex hormones Sex hormones are synthesized from cholesterol through adefined enzymatic cascade predominately in the gonads and theadrenal glands Sex hormones are also produced in othertissues including the brain placenta mammary glands liver andadipose tissue “ In addition to driving sexual developmentof egg and sperm production sex hormones are responsiblefor the development of male and female secondary sexualcharacteristics like breast development and growth of facial hairthat occur during puberty Androgens include testosteronedihydrotestosterone DHT androstenedione androstenedioland dehydroepiandrosterone DHEA with DHT being the mostpotent The concentration of androgens in circulation isabout sevenfold higher in adult men than in adult women Estradiol and progesterone are the predominantfemale sex hormones synthesized by the ovaries andadrenal glands Both male and female sex hormones are boundto the plasma proteins albumin and sex hormone bindingglobulin SHBG and only a small percentage exists as freehormone “ Thus the bioavailability of sex hormones isregulated by their biosynthesis and also the amount of albuminand SHBGImportantly sex hormones mediate not only anatomicdiï¬erences between women and men but also direct sexdiï¬erences in immune responses leading to diï¬erent risks forimmunologic diseases Overall women have a greaterrisk for autoimmune diseases such as systemic sclerosis andsystemic lupus erythematosus whereas men are morelikely to die of infectious and parasitic diseases Moreovermen have a greater risk of nonreproductive cancers “Both gender and sex are important mediators of these andother health and disease diï¬erences observed between men andwomen While gender refers to the array of socially constructedroles attitudes personality traits and behaviors sex representsa biological characteristic of an individual includingthe hormonal milieu and chromosome complement Ingeneral estrogens are considered to have pro‚ammatoryproperties and androgens are thought to have anti‚ammatoryproperties In the United States and worldwide relevant evidence highlights important epidemiologic sexdiï¬erences in incidence susceptibility and severity of a numberof diseases that aï¬ect the respiratory tract In this reviewwe will focus on how male sex hormones the androgensmodulate the response of myeloid cells in the lung and howthis modulation impacts the outcome of diï¬erent diseases ofthe lungSEX DIFFERENCES IN HUMAN LUNG ANDLUNG DISEASESsex mediates diï¬erencesBiologicalin the incidence andpathophysiology of lung diseases These diï¬erences arise fromsex diï¬erences in the structure and function of the lung itselfand also in the immune cells that populate the lung and arerecruited to it during ‚ammation Before birth the female lunghas several structural advantages over the male lung Surfactantis produced earlier and although the female lung is smaller ithas more alveoli per unit area Neonatal females have higherexpiratory flow rates than do male neonates when corrected forsize Thus male sex is a major risk factor for the developmentof respiratory distress syndrome bronchopulmonary dysplasia inneonates “ and asthma in childhood In addition to the contribution of structural diï¬erences ofthe lung between the sexes sex diï¬erences in lung function andlung diseases are also dependent on the action of sex hormonesWe have summarized some broad concepts that define howtestosterone and estrogen aï¬ectlung macrophage functionand how this may contribute to the outcome of particularlung diseases in Figure As testosterone rises after pubertythe immunosuppressive eï¬ects of this hormone on protectiveimmune responses to infectious diseases in males can worsenpulmonary disease This would be exemplified by tuberculosisor ‚uenza Some of these eï¬ects are a result of androgeneï¬ects on critical ‚ammatory macrophage functions althoughthe eï¬ects on the adaptive immune system also have a significantcontribution to the overall outcome Thus testosterone appearsto play a key immunoregulatory role in lung macrophagesTestosterone™s immunoregulatory properties also appear to bedependent on the amount of cellular expression of AR andon the concentration of the hormone Low concentrations oftestosterone have been noted in patients with asthma COPD andtuberculosis Low testosterone may also be linked to insufficientcontrol of tissuedamaging ‚ammatory responses seen inCOPD and pulmonary fibrosis Estrogen tends to promotewound healing responses in macrophages Dysregulation ofwound healing responses and overactive tissue remodelingmacrophages in the lung could be broadly used to describe theTh2 response in allergic asthma which is worse in womenCancer could also be considered an aberrant wound healingresponse driven by M2like tumor associated macrophages Wehave highlighted here how sex hormones contribute to changesin lung macrophage function that contribute to lung diseaseHowever it should be pointed out that not every sex diï¬erencein lung disease is due to direct eï¬ects on macrophages but on thebroader coordinated immune response as a wholeAsthmaBefore puberty the structural diï¬erences in the lung as wellas gender diï¬erences likely account for the higher incidence ofFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyFIGURE Sex differences in lung diseases discussed in this Review and how they may be connected to the effects of androgens and estrogens on ‚ammatorymacrophages in the lungasthma in boys than in girls With the onset of puberty male andfemale sex hormones and their eï¬ects on the structural cells ofthe lung and on the immune system contribute to the incidenceof asthma The incidence and severity of asthma aregreater in adult women than in adult men and greaterin female than in male mice Female sex hormones suchas estrogen appear to worsen asthma although a straightforwardcorrelation between amount of female sex hormone and asthmasymptoms has not been concluded Androgens have multipleimmunoregulatory and bronchodilatory functions and maycontribute to or be biomarkers for better lung function inmen Accordingly serum testosterone is low in men withmoderate to severe asthma “ In one study each ngdLincrease in serum testosterone correlated with a CI P decrease in the likelihood of having asthma On the other hand high concentrations of testosterone andcyclic AMP in sputum of asthmatic women during the lutealphase of the menstrual cycle were thought to play a role inpremenstrual exacerbations The idea that sex hormonesmay be a causal factor in asthma was significantly strengthenedby a recent study of adults that quantified serum sexhormones and asthma outcomes That study showed thatlow testosterone in both women and men was associated with anincreased incidence of asthma The other interesting finding wasthat higher testosterone was protective against asthma in obesewomen Obesity is a risk factor for asthma “ Thereforehow high body mass index BMI and circulating sex hormonestogether aï¬ect asthma requires further investigationAnother androgen dehydroepiandrosterone DHEA alsoknown as androstenolone is an endogenous steroid hormoneand one of the most abundant circulating steroids in humansIt is a precursor for the synthesis of both testosterone andestrogen DHEA is sulfated at the C3 position into DHEAS by the action ofthe sulfotransferase enzymes SULT2A1and SULT1E1 in the adrenal glands The amount of DHEAS in the circulation is ˆ¼“ times those of DHEADHEA became of interest to the asthma field because womenwith severe asthma had very low concentrations of DHEAS and DHEAS concentration correlated with lung function Interestingly DHEAS is suppressed by oral or inhaledglucocorticoids the mainstay therapy for asthma HumanDHEA peaks at around age and then follows an agedependentdecline until they reach prepubertal concentrations Reducedsecretion of DHEA with age has been related to a numberof ageassociated conditions Replacement of DHEA has beenconsidered as a possible therapeutic that could activate protectiveresponses in an aging immune system DHEA is known todownregulate Th2‚ammatory cytokines while upregulatingIL2 synthesis in concanavalin Astimulated peripheralblood mononuclear cells from adult males with atopic dermatitis Thus it was hypothesized that it would be a usefultreatment for atopic diseases including asthma and the results ofthe clinical trials for DHEA in asthma patients show promiseThe results are discussed in a later section titled œEï¬ects ofandrogen exposure on monocytes macrophages in humans withlung diseaseCOPDSex diï¬erences also have been reported in chronic obstructivepulmonary disease COPD a heterogeneous chronic andprogressive respiratory disorder that includes chronic bronchitisand emphysema Chronic exposure of the airways to insultssuch as cigarette smoke leads to epithelial cell injury destructionof pulmonary capillary vasculature acceleration of epithelial cellsenescence and airway remodeling The loss of lung complianceultimately leads to COPD COPD was previously thoughtto aï¬ect mostly elderly men primarily because of the higherprevalence of smoking in men However as smoking ratesincreased in women the number of COPD cases in womenexceeded that of men These diï¬erences are not only basedon gender as women develop more severe COPD with earlyonset disease years and have greater susceptibility toCOPD with lower tobacco exposure Moreover increasingage in female smokers leads to a faster annual decline inFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyforced expiratory volume in the first second when compared tothat of male smokers even when they smoke fewer cigarettes Similarly pulmonary fibrosis is another lung disease thatmanifests sex diï¬erences with men being more aï¬ectedthan women It is characterized by destruction of thepulmonary parenchyma and deposition of extracellular matrixwith alterations in phenotype of both fibroblasts and alveolarepithelial cells InfluenzaThe lungs are also the target of respiratory viruses such as‚uenza A œï¬‚u respiratory syncytial virus and coronavirusessuch as severe acute respiratory syndrome and the MiddleEast respiratory syndrome The viruses infectthe airwayepithelial cells and cause damage to the epithelial barrierby themselves or as a result ofthe immune response tothe viralinfection Sex diï¬erences have been noted in theimmune response to ‚uenza A virus and to the ‚uenzavaccine In general women have a more robust protectiveimmune response to ‚uenza virus and vaccine than do menAlthough this elevated response is helpful in clearing viruswomen of reproductive age also experience higher mortalityand hospitalizations “ possibly from collateral tissuedamage to the lungs The vigorous immune response in womenalso means that women experience more adverse events aftervaccination Indeed a systems biology approach identifiedthat high testosterone was correlated with a blunted responseto the flu vaccine in men As testosterone wanes in elderlymen mortality increases Since the male immune responseto the virus is also less robustthe incidence of seasonalflu is generally higher in men than in women in developedcountries according to the World Health anization It is not yet known how fluctuations in sex hormones acrossthe menstrual cycle and lifespan aï¬ect the immune system™sresponse to the ‚uenza virus in humans Mouse studieshave revealed that estrogen is protective at high but notlow concentrations On the other hand testosteronereplacement in gonadectomized or aged male mice enhancedsurvival rates Despite these findings in mouse modelsstudies examining the eï¬ect of sex hormones on cellular andmolecular mechanisms in human immune cells during ‚uenzainfection are lackingTuberculosisLike ‚uenza infection tuberculosis TB a lung disease causedby Mycobacterium tuberculosis exhibits notable sex diï¬erencesin the number of cases worldwide with men being almosttwice as frequently aï¬ected than women Both sexand gender diï¬erences impact the incidence of TB AlthoughTB aï¬ects less women than men in adulthood womenin their economically active years “ years old have ahigher TB incidence compared to women in other age groups This indicates that factors associated with gender such asexposure to the bacteria are important in this disease Howeverbecause male predominance does not occur in children thissuggests that biological factors such as male sex hormones alsoplay a significant role This is supported by a study ofmedically castrated men who experienced a significantly smallerproportion of death from TB compared to in intactmen Understanding how androgens lead to the greatersusceptibility of men to TB is critical as TB is still one ofthe leading fatal infectious diseases worldwide and may alsomay favor the development of other diseases such as lungcancer Lung CancerLung cancer is a very complex disease that depends on anumber of variants such as sex gender race and socioeconomicstatus The development of lung cancer is also related toenvironmental factors such as pollution due to industrializationand urbanization An additional genderassociated riskfactor significantly linked to developing lung cancer is cigarettesmoking Historically more men develop lung cancer andsuï¬er lung cancerassociated deaths compared to women However the incidence of lung cancer has changed notably inboth women and men In men lung cancer incidence startedto increase in the 1920s and started to decrease in the early1990s while in women the mortality rates and incidence beganto rise in the 1960s Changes in smoking habits in the lastseveral decades with a rise in the number of women who smokecorrelate with an increase in the incidence of lung cancer in thisdemographic group Smoking is definitely a key factor inthe development of lung cancer however recent studies showa higher incidence of lung cancer in young women comparedto young men even when the prevalence of cigarettesmoking among young women has approached but not exceededthat among men This suggests that the higher incidenceof lung cancer in women is not explained simply by genderdiï¬erences in smoking habits a deeper analysis of diï¬erencesmediated by sex such as greater sensitivity to tobacco smoke inwomen is warranted Furthermore men and women develop diï¬erent specifictypes of lung cancer Malignant mesothelioma is more commonin men while women develop more adenocarcinoma particularly nonsmall cell lung cancer NSCLC Womenhave a superior survival rate for lung cancer compared tomen Tumorassociated macrophages are critical in tumorprogression yet how androgens ‚uence macrophage behaviorin lung cancer and in responses to treatment must be addressedmore deeply to develop better therapies and increase survivalrates in menTHE MYELOID IMMUNE SYSTEM IN LUNGHEALTH AND DISEASEAlveolar MacrophagesThe lungs are a primary interface with the external environmentThe delicate structures needed for gas exchange make themsusceptible to damage from invading pathogens and toxicmolecules Some insults to the lung can lead to the developmentof chronic conditions such as allergic asthma As a protectivemechanism alveolar macrophages clearspace ofinfectious toxic or allergenic ps to maintain homeostasisin the alveoli Thus alveolar macrophages have a dualthe airFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biologyfunction as ‚ammatory cells phagocytosing and killinginhaled bacteria or viruses and also as controllers ofthe‚ammatory immune response minimizing alveolar damageResident alveolar macrophages are seeded embryonically fromyolk sac and fetalliver monocytes “ In asthma andother lung diseases recruited alveolar macrophages derived fromblood monocytes can turn into pathogenic cells worseningthe condition Mouse alveolar macrophages arecharacterized by high surface expression of Siglec F and produceTGF TGF both supports AM development and theirmaintenance of immune homeostasis by induction of Tregs andsuppression of B and T cell proliferation Another importantfunction of AM is the clearance of surfactant AM from male andfemale mice respond diï¬erently to surfactant protein A SPA SPA acts as an opsonin and is important in clearanceof pathogens Sex diï¬erences in AM responses to surfactantcould aï¬ect bacterial clearance and regulate the production ofpro‚ammatory mediators The molecular mechanisms thatmediate these diï¬erences and how sex hormones change thisimportant AM function is an open questionIn the human lung there appears to be more diversity inthe subtypes of lung macrophages compared to mice The maindeterminant of the frequency of subtypes of macrophages inhumans appears to be their anatomicallocation within thelung AM are the predominantimmune cells in the lungairways bronchi and bronchioalveolar space Flow cytometricpanels have employed HLADR CD163 CD169 and CD206to diï¬erentiate between AM IM and monocytes Human AMwere identified as large highly autofluorescent CD14 CD16cells that also express CD206 CD169 and MARCO There appear to be two populations of AM distinguished byeither high or low expression of CD163 More recent approachesto characterize the macrophage populationsin the lunginvolve singlecell transcriptomic analysis Althoughmacrophages show a large variation in the transcriptionalphenotype expression of MARCO CCL18 APOC1 APOEPPARG and MRC1 was found in macrophages from healthydonors while CHI3L1 MARCKS IL1RN PLA2G7MMP9 and SPP1 were highly expressed in macrophages frompulmonary fibrosis patients Thus a second contributor todiversity is likely the activation state of the cells There are nodata that describe sex diï¬erences in human AM responses andthe eï¬ect of sex hormones on these cells From our mouse andhuman MDM studies we would predict that androgens augmentthe immune homeostatic functions of these cells in the malelung Further work is still needed to standardize characterizationof the diï¬erent subpopulations of human lung macrophagepopulations and their role in maintaining healthy lung functionand in diseaseIMsInterstitial MacrophagesInterstitial macrophagesanother macrophagepopulation found in the lung They are a minor populationof monocytederived macrophages which comprise“ of lung macrophages and are localized in the lungparenchyma IMs contribute to maintaining homeostasisthrough the spontaneous release of IL10 a cytokine thataredampens ‚ammation IMs can prevent the developmentof aberranttype allergic responses triggered by inhaledallergens and have been related to reduction of asthma Diï¬erent subpopulations of IMs have been foundin the lung however their characterization has not arrived at aconsensus due to difficulties in their identification and isolationIn the mouse lung diï¬erent subpopulations of IMs have beendescribed based on the expression of surface markers One reportdescribed three diï¬erent subpopulations of IMs based on thediï¬erential expression of pro‚ammatory cytokines chemokineligands MHCII CD11c CD206 and Lyve1 other groupidentified two subpopulations based on similar markers butincluding CX3CR1 Moreover IMs subpopulations canbe also described based on the diï¬erent anatomic locationsthese cells populate inside the mouse lung parenchyma Further work is needed to better characterize and define thediï¬erent IM populations as the diï¬erent subtypes may havediï¬erent functions during the ‚ammatory process Smallerin size than their AM counterparts human IMs express moreof the monocytic marker CD14 than AM perhaps suggestingtheir monocytic origin and have lower expression of CD169than human AM The responses of IM to androgen will dependon their expression of AR which has not been measured Thiswill be a challenge due to difficulties in clearly identifying thispopulation and its subpopulations from the monocytic AMand other myeloid populations in the lungMonocytesMonocytes are produced in the bone marrow along with anumber of other myeloid cells Myeloid cells originate fromcommon pluripotent hematopoietic stem cells and representthe major subset of white cells in circulation These cellscomprise basophils neutrophils eosinophils DCs monocytesand macrophages among others Monocytes are releasedinto circulationthen blood monocytes are recruited into‚amed tissue and can mature into macrophages or dendriticcells There are two main subsets of mouse monocytesœclassical or Ly6Chigh monocytes that originate directly fromLy6C precursors and œnonclassical or Ly6Clow monocytesthat derive from Ly6Chigh monocytes The origin ofLy6C low monocytes was demonstrated by Sunderkotteret al by tracking the maturation of DiIlabeled Ly6Chighmonocytes into DiIlabeled Ly6Clow monocytes Thisprocess depends on the transcription factor Nr4a1 whichregulates the development and survival of Ly6Clow monocytes These two monocyte subsets mirror the human CD14classical and CD16 nonclassical monocyte populationsrespectively Ly6Chigh monocytes highly express thechemokine receptor CCchemokine receptor CCR2 whereasLy6Clow monocytes highly express CX3CR1 ImportantlyCCR2 expression is required for Ly6C monocyte egress fromthe bone marrow into the circulation and entry into non‚amed and ‚amed tissues “ from the blood As monocytes migrate into tissue they mature into macrophagesdeveloping unique tissuedependent morphology and functions They lose expression of Ly6C and gain expression ofMHC class II becoming more efficient antigenpresenting cellsFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage Biology Some authors have proposed the concept of œtissuemonocytes which are monocytes that can enter nonlymphoidans without obligatory diï¬erentiation into macrophagesTherefore monocytes are much more than simply precursorsfor macrophagesIn human lungs monocytes which can be both beneficialand pathogenic in a variety of pulmonary diseases arepresent at steady state Multiplecolor cytometric analysison cells obtained from diï¬erent anatomical locations of the lungof healthy subjects nonsmokers with normal lung function andabsence of disease or infection revealed that while intermediatemonocytes CD14CD16 are more frequent in the airwaysclassical monocytes CD14CD16ˆ’ are more frequent in blood Moreover the diï¬erent monocyte subsets produced TNFα to diï¬erent degrees upon stimulation with TLR ligands and Thus the anatomic location where samples are obtainedshould be considered and reported when working with humanbronchoscopies as this may alter the type and abundance ofmonocytes and macrophages found Accurate identification ofmonocytes in the lung compartments in humans has been achallenge because monocytic œcontamination from the bloodvessels Overcoming this challenge Desch et alperformed a flow cytometric phenotyping study and identifiedtwo additional lung monocyte populations by analyzing lungsobtained from donors who died of nonpulmonary causes CD14 CD206ˆ’ CD1cˆ’ CD1aˆ’ intravascular monocyteswere similar to CD14 blood monocytes and CD14 CD206CD1cˆ’ CD1aˆ’ monocytes were described as tissue œmonocytesThese studies highlightthe beginningof understanding the complexity of lung monocyte subtypesand their functions depending on the ‚ammatory state ofthe lungthat we are just atOther myeloid populationslike DCs occupy the lungparenchyma at steady state and their relative numbers changeduring ‚ammation We refer readers to previous excellentreviews in this journal that cover the importance of DCs inimmune responses in the lung and how they are aï¬ectedby sex diï¬erences Therefore we will not discuss DCs here “Macrophage ActivationPolarization is a very important eï¬ector characteristic observedin monocytes and macrophages Polarization refers to the changein phenotype and function of monocytes and macrophagesas they are exposed to diï¬erent‚ammatory milieus orfactors in the tissue microenvironment To understand theeï¬ects of the diï¬ering ‚ammatory or tissue environments onmonocytemacrophage phenotype and function researchershave used cytokines and other factors in vitro to mimic diï¬erent‚ammatoryand tissue microenvironments Monocytesand macrophages stimulated with interferonγ LPS TNFαinterleukin IL12colonystimulating factor promote a pro‚ammatory macrophagephenotype denoted as M1 polarization The activation state wasalso known as œclassical activation M1polarized macrophagesmediate immunity to intracellular infections such as viruses andand granulocytemacrophagebacteria and they are generally considered tumoricidal “ M1 macrophages accomplish these functions by inducingproduction of nitric oxide reactive nitrogen intermediatesreactive oxygen species and hydrogen peroxide “ Incontrast activation of macrophages with IL4 or IL13 as inextracellular parasitic infections and allergic reactionsleadsto M2 polarization or œalternative activation of macrophages M2 macrophages produce ‚ammatory mediatorsand chemokines such as chitinaselike proteins IL13 CCL17 CCL18 CCL22 and CCL24 which activateTh2 cells and promote eosinophil ltration into the lungs In allergic asthma a Th2‚ammatory response to inhaledallergens drives lung macrophages toward an M2 phenotypeIncreased number and percent of M2 macrophages havebeen correlated with asthma severity and a decline in lungfunction in humans and mouse models “ SimilarlyM2 macrophages are the predominant subset seen in pulmonaryfibrosis and are responsible for fibrogenesis During COPDthe number of macrophages in airwayslung parenchymabronchoalveolar lavage fluid and sputum increases This increase may occur as a result of enhanced monocyterecruitment from circulation in response to chemokines suchas CCL2 and CXCchemokine ligand1 which are increased inthe sputum and bronchoalveolar lavage fluid of patients withCOPD Unlike in allergic asthma and pulmonary fibrosismacrophages in COPD are polarized toward an M1 profile In addition to aï¬ecting men and women diï¬erently anothercommonality of COPD is that macrophages both in the alveolarspace and in lung tissue present an altered activation phenotypeDiï¬erent concentrations of cytokines TNFα IL1 IL6 IL IL12 and chemokines CCL2 CCL5 CCL7 CCL13 CCL22IL8 CXCL9 and CXCL10 are found comparing smokers tohealthy subjects “ Thus the external provoking stimulusuniquely shapes macrophage phenotype and functionWhile the M1M2 designations are useful for in vitro studieswith stimulation with defined cytokines the in vivo phenotypeof macrophages exists on a spectrum somewhere in betweenthese two welldefined opposing phenotypes or does not fitthe paradigm at all For example M1 and M2 markers canexist simultaneously within the same cell in some cases “ The key factors dictating the macrophage phenotypeor activation state are the stage ofthe immune responseand the soluble factors and interactions in a particular tissuemicroenvironment For example the lung environment is richin GMCSF TGF and PPARγ and is critical for developmentof mature AMs after birth in both mice “and humans “ Furthermoreinteractions betweenCD200 on type II alveolar epithelial cells and CD200R on thesurface of the AM deliver regulatory signals to the AM toprevent pro‚ammatory signaling and macrophage activation Thus macrophage nomenclature has evolved as ourunderstanding of the phenotypes and functions of diï¬erenttypes of tissue resident macrophages recruited monocytes andmonocytederived macrophages advances Indepth studies ofthe eï¬ects of androgens and other sex hormones on tissuemacrophage plasticity and phenotype have yet to be carried outFrontiers in Immunology wwwfrontiersinAugust Volume 0cBecerraDiaz et alAndrogenAR in Lung MonocyteMacrophage BiologyMECHANISMS OF ANDROGEN SEXSTEROID ACTIONEFFECTS OF ANDROGEN EXPOSURE ONMONOCYTES MACROPHAGES IN VITROBecause androgens are lipophilic steroid hormones they caneasily diï¬use across cell membranes withoutthe need forreceptormediated import Androgens in circulation arefound mostly bound to sex hormonebinding globulin andalbumin Free unbound steroid sex hormones can signalthrough two diï¬erent mechanisms the classical ARlocate

" prognostic markers play an essential role in the proper management of communityacquiredpneumonia this research work aimed to evaluate the association of rdw and or mpv with mortality andmorbidity in patients with cap to improve the yield of already used prognostic scoresresults the current study enrolled patients with communityacquired pneumonia cap out of them patients improved while died it was noticed that each of delta mpv and rdw p hadpositive significant correlation with psi and curb65 delta mpv and rdw was significantly higher in patients whodied ± vs ± p for delta mpv and ± vs ± p for rdw initial rdw and rising mpv during hospitalization for cap is associated with more severe clinicalcharacteristics and high mortality moreover the use of rdw and delta mpv in patients admitted with cap canimprove the performance of prognostic scaleskeywords communityacquired pneumonia red cell distribution width mean platelet volume communityacquired pneumonia cap is the fourthleading cause of death all over the world and plays animportant role of morbidity and mortality [“] scoringsystems have an essential role in the management of patients with cap and currently there are several severityscores in use such as pneumonia severity index psicurb65 however these severity scores have some limitations and variations for example the curb65 andcrb65 are crude scores for rapid assessment of thehighrisk patients while psi is believed to be useful foridentifying lowrisk patients therefore there is effort toimprove the prognostic value of these severity scores correspondence randaezzeldin98gmailcom32561department of chest diseases faculty of medicine assiut university assiutegyptfull list of author information is available at the end of the several biomarkers have been checked and verified foruse in cap which could improve the prognostic performance of severity scores [ ] however some ofthese biomarkers are nonspecific and not sensitiveothers are expensive and are not always available immediately mean platelet volume mpv is done as a routine laboratory test that is measured in complete bloodcount and it is considered a marker of platelet functionand activation [ ] a single elevated mpv measurement has been found to be associated with increasedmorbidity and mortality in various patient populations[ ] patients hospitalized with communityacquiredpneumonia cap are found to be at an increased risk ofdeath in the hospital and following discharge [“]the prognostic significance of mpv has been studied inonly two small studies on cap patients which werebased on single mpv determinations [ ] the clinical characteristics and prognosis oftimedependent the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40 0cfarghly the egyptian of bronchology page of mpv changes have not been investigated in the cappopulation we hypothesized that the mpv may reflectplatelet activity and may be associated with an impairedhost response according to this hypothesis an increasing mpv may be associated with poor outcomes andmay predict inhospital mortality in icu patients withsevere pneumonia to test our hypothesis we examinedmpv alterations in patients with severe pneumonia whohad been admitted or transferred to the icured cell distribution width rdw is defined as a coefficient of variation of circulating red cells it is affectedby changes of red cell volume and is measured in theroutine complete blood count cbc few years earlyrdw used in the clinical practice to diagnose differenttypes of anemia moreover elevated rdw had a prognostic role in the outcome of some diseases like cardiovascular disease rheumatoid arthritis colon cancer andmetabolic syndrome [ ] furthermorefew researches have reported rdw as a prognostic predictorof mortality in different populations the exactmechanism of variation in rdw is still unknown but itis mostly associated with the process of oxidative stressand inflammation which reflects the prognostic role ofrdw to our knowledge rdw use does not implyany additional cost because it is routinely provided aspart of the whole blood count by hemocytometryseveral studies support the hypothesis that rdw maybe a useful parameter for gathering either diagnostic orprognostic information on a variety of cardiovascularand thrombotic disorders [ ] although the link between rdw and cardiovascular disease is unclear in recent years rdw has been associated with capoutcomes especially with 30day and 90day mortalityand complicated hospitalization [ ] this researchwork aimed to validate the role of rdw and mpv aspromising markers of mortality and morbidity in patients with cap to improve the yield of already used severity scoresmethodsthis prospective study included adult years ofage patients admitted to chest department and ricuof assiut university hospital with a diagnosis of capbetween october and october patients wereprospectively recruited within h of their arrival capwas defined as an acute disease with a radiological infiltrate that was not previously present and not due to another known cause and was associated with symptomsof lower respiratory tract infection exclusion criteriawere severe immunodepression hiv infection or severe hematological diseasesimmunosuppressivetherapy prednisone or equivalent dose of mg dailyfor weeks or any immunosuppressive regime therapy azathioprine cyclosporine cyclophosphamide andor other immunosuppressant drugs leukopenia leukocyte per mm3 or neutropenia neutrophils per mm3 andor chemotherapy in the previousyear pulmonary abscess radiological cavitation aspiration pneumonia and obstructive pneumoniapossible nosocomial origin days from hospital discharge and known active neoplasia all patientswere followed up during their hospital stay and thosewith a definitive diagnosis other than cap were excluded all of the patients were followed up until beingdischarged our study primary outcome variable was inhospital mortality of patients with cap the secondaryfig outcome of patients with cap 0cfarghly the egyptian of bronchology page of table baseline characteristics of both groupsimproved n died n ± ± p valuetable correlation between curb65 and psi in correlation todelta mpv and rdwdelta mpvrppsicurb65 rdwrp date expressed as r strength of correlation p significance of correlation pvalue was significant if mpv mean platelet volume rdw red celldistribution width psi pneumonia severity indexage yearsexmalefemalesmokingcurrent smokingnonestopped smokerexsmokerscomorbid diseasescurb65psiclassiiiiiivvlaboratory datardw delta mpvwbcs — 109lplatelets — 109lpao2 fio2hospital stay dayneed to mvtransfer to icuradiological findings ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ph pao2 urea nainhaled or oral corticoids and antibiotics were recordedon admission the following data were also recordeddays of duration of disease cap signs and symptomsfever cough sputum dyspnea pleural pain vital constants at the ed temperature respiratory and heartrates arterial pressure and oxygen saturation analyticdataglucose hematocrithemoglobin red cell distribution width rdw andmean platelet volume mpv number of lobes involvedand type of radiological condensation alveolar interstitial or mixed and complications respiratory cardiologic renal neurologic digestive and others noninvasive mechanical ventilation nimv need of icuand invasive mechanical ventilation imv psi andcurb65 scores were calculated for all patients all patients were admitted to the hospital for ‰¥ hsample collection and processingblood was collected from anticubital fossa by experienced phlebotomists using a standardized atraumaticprotocol using clean venipuncture and minimum tourniquet pressure needles used were “ gauge specimens were maintained at room temperature “ °cnot placed on ice refrigerator or water bath tubes keptcapped upright at room temperature not exposed to vibration excessive mixing or agitation specimens containing any evidence of clotting were discarded twosamples of ml of venous blood in standard tubes contain ethylenediamine tetraacetic acid edta anticoagulant for complete blood count cbc the first one atthe time of admission and the second one after daysof admission the cbc sample was examined within has recommended by bcsh guideline to avoidbias due to excessive platelet swelling and to minimizevariation due to sample aging mpv and other bloodcount parameters were measured by an automatedanalyzer advia 2120i jermany with lh controlsystem in our laboratory the range of normal mpvvalues is “ fl for analysis of timedependentmpv changes patients were categorized according toδmpv mpv on discharge minus mpv on admissioninto patients with no rising mpv δmpv fl andpatients with rising mpv δmpv ‰¥ fl rdw wasunilobar pneumonia multilobar pneumonia effusion positive blood culture positive sputum culture data expressed as mean sd frequency percentage p value was significantif mv mechanical ventilation icu intensive care unitoutcome variables were length of hospital stay intensivecare unit icu admission and mechanical ventilator requirement this study protocol was approved by thelocal ethics committee and informed consent was obtained from all patients or next of kindata collectionage sex tobacco and alcohol consumption comorbidconditions diabetes liver chronic kidney hearth andcerebrovascular diseases nonactive neoplasia bronchiectasis and previous cap and previous therapies 0cfarghly the egyptian of bronchology page of fig correlation between delta mpv and psireported as a part of the cbc results rdw is the standard deviation of mcv and was measured in as percentage a single rdw had been recorded from cbc ofpatients on admission in our laboratory the range ofnormal rdw values is statistical analysisdata was collected and analyzed those using spss statistical package for the social science version ibmand armonk ny continuous data was expressed inform of mean ± sd or median range while nominaldata was expressed in form of frequency percentageindependent risk factors of mortality had been determined by multivariate regression analysisreceiver operator curve was used to determine cutoffof rdw and delta mpv for prediction of inhospitalmortality in patient with communityacquired pneumonia pearson correlation was used to determine correlation between psi and curb65 with rdw and deltampv level of confidence was kept at hence pvalue was significant if resultsthe currentstudy included with communityacquired pneumonia cap out of them patients improved while died as shown in fig the characteristic data of the enrolled patients are summarized in table mean age of improved patients was ± years majority of them were malesand of them were smokers mean age of patients who died was ± years and ofthem were females it was noticed that patientswho died were smokers it was noticed that patients who improved and patients who diedhad coexisting comorbidities with significant differencesbetween both groups it was noticed that class of psi wasii iii iv and v in and respectively of improved patients and in and of patients died respectively mean psi was significantly higher in patientswho died ± vs12339 ± p moreover curb65 was significantly higher in patientswho died in comparison to improved patients ± vs ± p fig correlation between delta mpv and curb65 0cfarghly the egyptian of bronchology page of fig correlation between rdw and psidelta mpv and rdw was significantly higher in patients who died ± vs ± p fordelta mpv and ± vs ± p for rdw our research also detected that comorbiddiseases transfer to icu and need for mechanical ventilation were highly frequent in patients who died moreover patients who died had longer duration of hospitalstay on radiological findings pleural effusion and unilobar pneumonia were presented in and ofpatients who died vs and of improved patients respectively while multilobar pneumonia wasmore frequent in patients who died blood culture waspositive only in of patients who improved vs of patients who diedthe current study also discovered that each of δ mpvand rdw had positive significant correlation with psiand curb65 p as shown in table and figs and based on the current study table the following variables were predictors of inhospital mortality in patients withcap with adjusted r2 was curb65 or ci “ p psi or ci “ p rdw or ci “ p delta mpv or ci “ p it was worthwhile to notice that rdw at cutoff point had sensitivity and specificity for predictionof mortality in patient with cap with area under curve while delta mpv at cutoff point had sensitivityand specificity for prediction of death in patient withcap with area under curve as shown in table andfig discussionin the recent few years cap had been considered one ofthe leading causes of death worldwide thereforefig correlation between rdw and curb65 0cfarghly the egyptian of bronchology page of table predictors of inhospital mortality in patients with capp valueodd™s ratioage confidence interval“““““““sexcomorbiditiescurb65psirdwdelta mpvp value was significant if mpv mean platelet volume rdw red celldistribution width psi pneumonia severity index capcommunityacquired pneumoniaaugmentation of the conventional severity scoreslikethe psi and curb65 became a must to identify patients with high risk for a complex course as the predictive performance of these scores alone may be limitedseveral researches have detected that discovering newbiomarkers could augment the validity of these severityscores [ ]in this prospective study we planned to assess the validity of the mean platelet volume change and rdw asbiomarkers for assessing the severity of cap it wasworthwhile to know that no previous study has beendone at assiut university hospital to assess those twobiomarkers in patients with cap the main potentialmechanism for rising mpv in patient population is severe inflammation caused by pneumonia in severe infection increased release of thrombopoietin and variousinflammatory cytokines such as interleukin1 and and tumor necrosis factorα result in increased thrombopoiesis and enhanced expression of younger largeplatelets into the blood circulation [ “] on theother hand rising mpv may be attributed to increasedthrombocyte consumption in the peripheral tissue andspleeninduced by severe inflammatory status [ ] communityacquired pneumonia is an infectiousdisease that results in inflammatory and oxidative stresstable performance of rdw and delta mpv in prediction ofmortality in capsensitivityspecificitypositive predictive valuenegative predictive valuecutoff pointrdw delta mpv area under curvep valuep value was significant if rdw red cell distribution width mpv meanplatelet volume cap communityacquired pneumonia to the host if these stresses are severe mortality will beincreased the underlying pathophysiological mechanisms for a relationship of rising mpv with poor prognosis and mortality are not fully understood the mainpotential explanation can be increased platelet activation[ ] larger thrombocytes are known to be functionally metabolically and enzymatically more active thansmaller ones the greater activation of enlarged plateletsresults in increased release of procoagulant substancessuch as thromboxane a2 βthromboglobulin and surface proteins [ ] consequent hyperaggregability ofplatelets extended vasoconstriction and endothelial dysfunction may contribute to an increased shortterm riskof cardiovascular thrombosis and death in patients withrising mpv [ ]our research detected that a high rdw and risingmpv were significantly related with increased risk ofdeath in patients with cap as delta mpv and rdw wassignificantly higher in patients who died ± vs ± p for delta mpv and ± vs ± p for rdw our results are inagreement with braun who detected thatrdw was associated with high risk of death and disability in young patients admitted with cap in this retrospective study brawn in a cohort of patients of years or older hospitalized due to cap demonstratedthat elevated rdw was independently associated with complicated hospitalization length of stay days and admission to icu and 90day mortality irrespective of hemoglobin levels in line with the results of our study lee also identified a high rdwis a prognostic factor for 30day cap mortality ageis significant prognostic factor in various diseasesincluding cap in our study the mean age of improvedpatients with cap was ± years while meanage of patients who died was ± years thusour findings support rdw as a significant prognosticfactor in patients with cap across all ages these resultsare in agreement with braun their finding is similar to our results however they only includedpatients who were younger than years which theycited as a limitation of their study this study revealedthat each of psi and curb65 had positive significantcorrelation with delta mpv and rdw p ourresults are also in line with gorelik whostated that rising mpv during hospitalization for cap isassociated with a more severe clinical profile and mortality than no rise in mpv they found that mpv valuesabove the cutoff at discharge were associated with anincreased risk of mechanical ventilation and death during hospitalization and shortened survival following discharge based on the current study the following variableswere predictors of inhospital mortality in patients with 0cfarghly the egyptian of bronchology page of fig diagnostic performance of delta mpv and rdw in prediction of inhospital mortalitycap with adjusted r2 was curb65 or ci “ p psi or ci “ p rdw or ci “ p delta mpv or ci “ p indeed in our patient population a risein mpv was associated with higher pneumonia severityscoresin our study the mortality prediction of both the psiand curb65 was improved by the addition of rdwand delta mpv as severity criteria the exact mechanisms of an association of rdw with the mortality ofcap need to be determined it has been suggested thatinflammation and oxidative stress affect red cell homeostasis a previous study revealed that rdw displayed astrong graded association with inflammatory biomarkersin general outpatient populations and anotherstudy indicated that serum antioxidant levels includingselenium and carotenoids were associated with rdw inolder women in our data patients with a higherrdw had a tendency toward higher severity indexscores and the overall mortality was also higher in patients with a higher rdwthere are some limitations of our research work firstthe study included one group of cap patients who wereadmitted in our assiut chest department and ricuthus it cannot be generalized to all patients with capsecond larger studies of large numbers of patients needto be done to investigate the value of mean plateletvolume change and rdw as prognostic markers incommunityacquired pneumoniasrdw and rising mpv during hospitalization for cap isassociated with more severe clinical characteristics andhigh mortality we suggestthat repeated mpv andrdw determination throughout hospitalization may improve risk stratification for cap patientsabbreviationscap communityacquired pneumonia cbc complete blood count crb confusion respiratory rate blood pressure age ‰¥ curb65 confusionurea respiratory rate blood pressure age ‰¥ δmpv mean platelet volumechange ed emergency department icu intensive care unit rdw blood celldistribution widthacknowledgementsto all work team who do their best to do this research in a perfect way andto all patients involved in this study and their parentsauthors™ contributionssf and ra carried out historytaking and physical examination of all participants in addition to obtaining blood samples participated in the sequencealignment and drafted the manuscript they also analyzed and interpretedthe patients™ data re and da carried out laboratory investigations and participated in the revision of the manuscript all authors read and approved thefinal manuscriptfundingno fund was taken from any institute or companyavailability of data and materialsdata and materials are available 0cfarghly the egyptian of bronchology page of ethics approval and consent to participatethe study obtained approval from the ethical committee at the faculty ofmedicine assiut university and a written consent was taken from theparticipants no reference number is usually given for approved studies inour universityconsent for publicationconsent for publication was taken from all authorscompeting intereststhe authors declare that they have no competing interestsauthor details1department of chest diseases faculty of medicine assiut university assiutegypt 2department of clinical pathology faculty of medicine assiutuniversity assiut egyptreceived april accepted august references mandell la wunderink rg anzueto a infectious diseasessociety of americaamerican thoracic society consensus guidelines on themanagement of communityacquired pneumonia in adults clin infect dis44suppl 2s27“s72almirall j bolibar i vidal j epidemiology of community acquiredpneumonia in adults a populationbased study eur respir j “armstrong gl conn la pinner rw trends in infectious diseasemortality in the united states during the 20th century jama “ menendez r martinez r reyes s biomarkers improve mortalityprediction by prognostic scales in communityacquired pneumonia thorax“lee jh kim j kim k albumin and creactive protein haveprognostic significance in patients with communityacquired pneumonia jcrit care “chu sg becker rc berger pb mean platelet volume as apredictor of cardiovascular risk a systematic review and metaanalysis jthromb haemost “noris p melazzini f balduini cl new roles for mean platelet volumemeasurement in the clinical practice platelets “ar©valolorido jc carreterog³mez j ¡lvarezoliva a guti©rrezmonta±o cfern¡ndezrecio jm najarrodiez f mean platelet volume in acutephase of ischemic stroke as predictor of mortality and functional outcomeafter 1year j stroke cerebrovasc dis “ wasilewski j desperak p hawranek m prognostic implicationsof mean platelet volume on short and longterm outcomes among patientswith nonstsegment elevation myocardial infarction treated withpercutaneous coronary intervention a singlecenter large observationalstudy platelets “ dabbah s hammerman h markiewicz w relation between redcell distribution width and clinical outcomes after acute myocardialinfarction am j cardiol 105312e331sangoi mb da silva sh da silva je relation between red bloodcell distribution width and mortality alter acute myocardial infarction int jcardiol 146278e280 montagnana m cervellin g meschi t the role of red blood celldistribution width in cardiovascular and thrombotic disorders clin chemlabmed 504635e641lee jh chung hj kim k red cell distribution width as aprognostic marker in patients with communityacquired pneumonia am jemerg med 31172e79 braun e domany e kenig y elevated red cell distribution widthpredicts poor outcome in young patients with community acquiredpneumonia crit care 154r194 harrison p mackie i mumford a briggs c liesner r winter m machin s guidelines for the laboratory investigation of heritable disorders ofplatelet function br j haematol “ bello s fandos s lasierra ab minchole e panadero c simon al de pabloog menendez r torres a red blood cell distribution width [rdw]and longterm mortality after communityacquired pneumonia acomparison with proadrenomedullin respir med 1091193e1206 ware j corken a khetpal r platelet function beyond hemostasis andthrombosis curr opin hematol “ becchi c al malyan m fabbri lp marsili m boddi v boncinelli s mean platelet volume trend in sepsis is it a useful parameter minervaanestesiol “kitazawa t yoshino y tatsuno k ota y yotsuyanagi h changes inthe mean platelet volume levels after bloodstream infection haveprognostic value intern med “kamath s blann ad lip gy platelet activation assessment andquantification eur heart j “ gorelik o tzur i barchel d almozninosarafian d swarka m feldman iblcohen n izhakian s a rise in mean platelet volume duringhospitalization for communityacquired pneumonia predicts poorprognosis a retrospective observational cohort study bmc pulmonarymedicine lippi g targher g montagnana m relation between red bloodcell distribution width and inflammatory biomarkers in a large cohort ofunselected outpatients arch pathol lab med “semba rd patel kv ferrucci l serum antioxidants andinflammation predict red cell distribution width in older women thewomen's health and aging study i clin nutr “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations musher dm thorner ar communityacquired pneumonia n engl jmed “jain s self wh wunderink rg communityacquired pneumoniarequiring hospitalization among us adults n engl j med “eurich dt marrie tj minhassandhu jk tenyear mortality aftercommunityacquired pneumonia a prospective cohort am j respir critcare med “karadagoncel e ozsurekci y kara a karahan s cengiz ab ceyhan m the value of mean platelet volume in the determination ofcommunity acquired pneumonia in children ital j pediatr golcuk y golcuk b bilge a irik m dikmen o combination of meanplatelet volume and the curb65 score better predicts 28day mortality inpatients with communityacquired pneumonia am j emerg med “felker gm allen la pocock sj red cell distribution width as anovel prognostic marker in heart failure data from the charm programand the duke databank j am coll cardiol “ ani c ovbiagele b elevated red blood cell distribution width predictsmortality in persons with known stroke j neurol sci ““ patel kv semba rd ferrucci l red cell distribution width andmortality in older adults a metaanalysis j gerontol a biol sci med sci“ 0c"

as one of the most common gynecological malignant tumors cervical cancer is the fourth leadingcause of cancerrelated death among women worldwide although eï¬orts including periodiccancer screening prompt surgical treatment chemotherapy and radiotherapy have been madeto decrease the mortality of cervical cancer the prognosis of patients is still poor and cervicalcancer remains an important public health issue the pathogenesis of cervical cancer has notbeen clearly illustrated but it is confirmed that the activation of tumorpromoting genes and theinactivation of tumor suppressor genes participate in the progression of cervical cancer toscreen for novel abnormally expressed genes functioning in cervical cancer may provide potentialprognostic markers and therapeutic targets for treatmentedited byihab youniscarnegie mellon university inqatar qatarreviewed byweifeng dingnantong university chinamassimo brogginimario negri pharmacologicalresearch institute irccs italycorrespondencelin xuxulin83njmueducnemei lulem13705179888sinacnbinhui renrobbishren163com these authors have contributedequally to this workspecialty sectionthis was submitted tocancer geneticsa section of the frontiers in oncologyreceived april accepted june published august citationzhu b wu y luo j zhang qhuang j li q xu l lu e and ren b mnx1 promotes malignantprogression of cervical cancer viarepressing the transcription ofp21cip1 front oncol 103389fonc202001307frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancermnx1 motor neuron and pancreas homeobox also knownas hb9 hlxb9 is a member of homeobox gene family andencodes a nuclear protein the homeobox genes are agroup of genes containing homeobox a base pairs longdna sequence and encode homeodomain proteins that actas transcription factors many homeobox genes have beenproved to be implicated in various human cancers acting asoncogenes or tumor suppressors “ mnx1 was firstly foundto be expressed in lymphoid and pancreatic tissues and definedas a novel human homeobox gene in early studiesshowed that mnx1 was involved invertebrate and pancreaticdevelopment and motor neuronal diï¬erentiation defects in this gene result in currarino syndrome an autosomicdominant congenital malformation in followup studymnx1 was found to be abnormally expressed in several cancertypes including prostate cancer hepatocellular carcinoma andacute myeloid leukemia “ furthermore recent studiesconfirmed that mnx1 played oncogenic roles in colorectalcancer breast cancer and bladder cancer “the aim of this study is to identify the expression andfunction of mnx1 in cervical cancer our results revealedthat mnx1 was significantly upregulated in cervical cancerand correlated with poorer prognosis the knockdown oroverexpressed mnx1 inhibited or promoted aggressiveness ofcervical cancer including proliferation migration and invasioncapacities by enhancing or repressing the transcription of p21cip1thus regulating the g2m cell cycle transition these findingssuggested that mnx1 might be a potential diagnostic marker andtherapeutic target for cervical cancermaterials and methodsbioinformaticsthe tcga dataset termed tcga_cesc_exp_hiseqv22015 was downloaded from the ucsc cancer browser genomecancerucscedu to evaluate the expression ofmnx1 in cervical cancer and adjacent normal tissues gepiagene expression profiling interactive analysis httpgepiacancerpku cnindexhtml was used to analyze the expressionof mnx1 with disease free survival dfs of cervical cancerpatients the cbioportal website httpwww cbioportal was utilized to obtain highly coexpressed genes with mnx1totally genes highly correlated with mnx1 pearson score table s1 were submitted to david bioinformaticsresources httpdavidabccncifcrfgov for geneontology go kyoto encyclopedia of genes and genomeskegg and reactome pathway analysis and we analyzed thebinding site of mnx1 and p21cip promoters through the jaspardatabase httpjaspardevgeneregnet human cervical cancer cell linesthe human normal cervical celllines hacat and cervicalcancer cell lines hela siha caski and c33a were purchasedfrom american type culture collection atcc usa helasiha c33a and hacat cells were incubated in dmem mediumkeygen nanjing china and caski cells were cultured inrpmi1640 keygen nanjing china medium containing fetal bovine serum gibcobrl invitrogen carlsbad causa and cultured at —¦c in a humidified incubator containing co2human cervical cancer tissuesthe pairs of cervical cancer tissues and adjacent tissues wereselected from the affiliated cancer hospital of nanjing medicaluniversity and informed consent was obtained from all subjectsall tumors and paired nontumor tissues were confirmed byexperienced pathologists and no patients received chemotherapyor radiotherapy before surgery the mrna expression ofmnx1 and p21cip1 in cervical cancer tissues was detected byqrtpcr collection of human tissue samples was conductedin accordance with the international ethical guidelines forbiomedical research involving human subjects cioms thisstudy was approved by the ethics committee of the nanjingmedical university affiliated cancer hospitaltissue microarrayspaired cervical cancer tissue microarrays were obtained fromshanghai outdo biotech co ltd cat no odctrputr03 and odctrputr03006 totally pairs of paraffinembedded human cervical cancer sections were analyzed formnx1 expression all tissues were examined by two experiencedpathologists and the tnm stage was confirmed in each patientwith blinded methods the sections were incubated with an antimnx1 primary antibody abcam ab79541 the ihcscores were calculated according to intensity and percentage ofpositive cells the staining intensity was evaluated as the basis offour grades negative staining 1weak staining moderatestaining or strong staining the product percentage ofpositive cells and respective intensity scores was used as the finalstaining scores a minimum value of and a maximum valueof rna preparation reverse transcriptionand qrtpcrtrizol reagent invitrogen carlsbad ca usa was used toextract total rna from tissue samples or cultured cells accordingto the manufacturer™s protocol a reverse transcription kittakara cat rr036a keygen was utilized to generate cdnaqrtpcr was performed with sybr select master mix appliedbiosystemscat keygen nanjing china and primersare shown in table s2western blottinglysis buï¬er ripa keygen containing protease inhibitorspmsf keygen was used to extract protein of cells andtissues and protein concentration was detected with a bcakit keygen protein samples µg were loaded into sodium dodecyl sulfate polyacrylamide electrophoresissdspage gels and transferred onto a pvdf membraneafter electrophoresis the membrane was blocked with nonfatmilk for h and incubated overnight with antibodies againstrespective antibodies mnx1 abcam ab79541 p21cip1cell signaling technology pthr161cdk1 cellsignaling technology cdk1 cell signalingfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancertechnology p27kip1 cell signaling technology cyclinb1 abcam ab72 cycline1abcam ab3927 cycline1 abcam ab3927 cyclind1santa cruz biotechnology sc246 actinabcam ab15265 sirna and plasmid transfectionthe sirnas targeting mnx1 and p21cip1 were conductedand purchased from ribobio guangzhou china all sirnasequences are shown in table s3 the fulllength cdnaof human mnx1 were pcramplified and cloned intothe expression vector pgpu6gfpneo vigene biosciencesshandong china the sirnas and overexpression plasmidswere transfected into cervical cancer cells according to thelipofectamine reagent invitrogen carlsbad ca usaprotocol nonsense rnai sinc and empty plasmids oencwas used as negative controls²analysiscell proliferation assaythe cell proliferation assays were performed h aftertransfection for real timexcelligencesystemrtca cells100 µl were seeded in eplates and theplates were locked into the rtca dp device in the incubator tocalculate the œcell index value in colony formation assay a totalof cells were placed in afresh 6wellplate and the cells werestained with crystal violet solution after “ days for5ethynyl2deoxyuridine edu assay keyfluor488 clickitedu kit ribobio guangzhou china the transfected cells wereplaced in 96wellplates cellswell overnight in a co2incubator then cells were incubated with µlwell of µmedu for h at —¦c and fixed with µl paraformaldehydecontaining pbs for min at room temperature subsequentlythe cells were cultured for min with µl of mgmlglycine and then washed with µl bsa in pbs afterpermeabilization with triton x100 for min the cellswere cultured with × clickit reaction solution for minat room temperature in dark conditions after that cells wereincubated with µlwell of × hoechst solutionsfor min at room temperature in the dark after washing with µl of pbs the cells were then imaged using fluorescencemicroscopy and proliferation cell ratios were counted fromfive random fields in every well each experiment was repeatedthree times a total of cells in a fresh sixwellplates weremaintained in medium containing fbs the medium wasreplaced every or days after weeks the cells were fixedwith paraformaldehyde and stained with crystal violeteach experiment was repeated three timesmigration and invasion assayfor wound healing assay cells were growing on the 6wellplate then artificial scratch on a confluent monolayer of cellswas created with a µl pipette tip the medium wasreplaced with the serumfree and cells imaged h later fortranswell and matrigel assay totally transfected cells wereadded to the upper chamber of transwell assay inserts µmpet 24well millicell or a matrigel coated membrane bdbiosciences containing µl serumfree dmem media thelower chambers were filled with µl dmem media containing fbs after a 24h migration assay or 48h invasionassay incubation the cells were fixed with polyformaldehydestained with crystal violet and imaged migration and invasionwere assessed by counting cell nuclei from five random fields onevery filter each experiment was repeated three times rtcawas also used to evaluated the ability of migration and invasioncimplates installation and baseline measurement was carriedout according to the instructions add µl of mixed serumfree cell suspension × cells to the upper chamber in cimplates and the plates were locked into the rtca dp device in theincubator to calculate the œcell index valuecell cycle analysiscells were digested with trypsinedta and fixed with ethanol for h at —¦c the ethanolsuspended cells werecentrifuged and stained with pi staining solution for minin the dark at —¦c a facscalibur flow cytometer was usedto detect cell cycle distribution the percentage of the cells ing0“g1 s and g2“m were counted and comparedchromatin immunoprecipitation chipcells were crosslinked in paraformaldehyde and the reactionwas quenched with glycine after two washes with cold pbscells were added with precooling pbs containing cocktailhalttm protease inhibitor cocktail thermo scientific and scraped into a centrifuge tube the cells were centrifugedfor min at g at —¦c then added with µl celllysis buï¬er containing µl cocktail and incubated onice for min cells were then centrifuged for min at × g —¦c and cell precipitates were resuspended in µlnucleus lysis buï¬er containing µl cocktail the cellswere sonicated amplitude on ice for min and solublechromatin was obtained by centrifuging for min at g at—¦c five micrograms of antimnx1 antibody sigmaaldrichsab2101494 coupled to magnetic beads resin m2 sigmashanghai china was used to immunoprecipitate the dnaprotein complex and the igg antibody was used as a negativecontrol the immunoprecipitation products were washed with µl low salt buï¬er high salt buï¬er lici buï¬er and tebuï¬er successively all for min at —¦c the chip elution buï¬ercontaining proteinase k was used for dna purification thebeads were wiped out on a magnetic frame and the dna waseluted with elution buï¬er c from adsorption column chipdna samples were subjected to pcr amplification with primersspecific to p21cip1 promoter region pcr products were then usedfor agarose gel electrophoresis the sequence of primers used areshown in table s4 and gapdh was used as a controlluciferase reporter assaythe p21cip1 cdkn1a promoter region ˆ’ bp wasamplified and cloned into luciferase reporter plasmid pgl3basic the p21cip1 promoter wildtype plasmids or mutanttype plasmids were cotransfected with cmvmnx1 expressionplasmids in hek293t cells and cmvempty vectors were usedas a negative control relative luciferase activity was corrected forfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerrenilla luciferase activity of pgl3basic and normalized to theactivity of the controlxenograft modelall animal studies were conducted in accordance with nihanimal use guidelines and protocols were approved by nanjingmedical university animal care committee sixteen femalenude mice “ weeks old were purchased from nanjingmedical university school of medicine™s accredited animalfacility the mice were randomly divided into two groupsusing random number generator in each group × exponentially growing cervical cancer cells were injected inaxilla subcutaneously before tumor transplantation cells weretransfected with shrnas or overexpression plasmids thetransfection was performed by transient transfection accordingto the specification of lipofectamine invitrogen carlsbadca usa the shnc and empty vector pcdna31 were usedas controls and totally µg plasmid vectors were transfectedinto cells for each group the sequences of shrnas are shown intable s5 tumors were harvested at weeks after injection theweight of tumor was measured on the scale and tumor volumewas estimated using calipers [length × width2] and tissueswere immunohistochemically stained with mnx1 abcamab79541 ki67 abcam ab79541 and p21cip1abcam ab109520 western blotting was performed aspreviously described no blinding was done in the animal studiesstatistical analysisresults are presented as the mean ± standard deviation sdstatistical analyses were performed using spss statistics version chicago ill and graphpad prism software graphpadsoftware inc la jolla ca usa p was consideredstatistically significantresultsoverexpression of mnx1 correlates withpoorer prognosis and more aggressiveclinical featuresanalysis of tcga dataset revealed that the mrna expressionof mnx1 was remarkably upregulated in cervical cancer tissuescompared with paratumor tissues p figure 1a ingepia gene expression profiling interactive analysis websitepatients with higher expression of mnx1 bore a worse diseasefree survival nhigh nlow p figure 1b theexpression of mnx1 in cervical cancer tissues were significantlyhigher than adjacent tissues in of cervicalcancer patients p figures 1cd ihc assays based ontissue microarrays tmas were performed to detect the proteinexpression of mnx1 in paired human cervical cancer tissuesand paratumor tissues and results showed that staining scoresof mnx1 were higher in cancer tissues p figure 1ecombined with the patients™ clinical information the expressionof mnx1 was higher in patients with more advanced tnm stagestage i“ii vs iii“iv p figure 1f t stage t1 vst2“t3 p figure 1g and n stage n0 vs n1 p figure 1h moreover mnx1 staining scores were linkedto higher pathological grade level ii vs iii p figure 1iand larger tumor maximum diameter d vs ‰¥ cm p figure 1j and ihc images of two patients with diï¬erentclinical stages were presented figure 1kknockdown of mnx1 inhibited progressionof cervical cancer in vitroto evaluate the expression of mnx1 in cell lines qrtpcr andwestern blotting were performed and results showed that mnx1was generally upregulated in cervical cancer cell lines comparedwith normal human cervical cell lines hacat figures 2ab tofurther investigate the biological function of mnx1 in cervicalcancer two specific sirnas targeting mnx1 were transfectedinto hela and siha cells both two sirnas showed favorablesuppression efficiency in hela figures 2cd and siha cellsfigures 2ef the rtca proliferation assay figure 2g eduassay figure 2h and colony formation assay figure 2ishowed that knockdown of mnx1 inhibited the proliferationability of cervical cancer in hela and siha cells moreover rtcamigration assay figure 2j transwell assay and matrigel assayfigure 2k and wound healing assay figure 2l revealed thatsilencing mnx1 inhibited the ability of cervical cancer cells tomigrate and invade these results suggest that mnx1 plays a vitalrole in the malignant phenotype of cervical cancerectopic expression of mnx1 enhancedaggressiveness of cervical cancer in vitroto further verify the biological role of mnx1 in cervical cancer apcdna31 plasmid to overexpress mnx1 was constructed andtransfected into c33a and hela cells the plasmid eï¬ectivelyupregulated the expression of mnx1 confirmed by qrtpcrand western blotting figures 3ab consistently our resultsshowed that ectopic expression of mnx1 promotes proliferationmigration and invasion figures 3c“g of cervical cancer cellssimnx1 induced g2m cell cycle arrestand upregulated the expression of p21cip1two hundred and eight genes highly correlated with mnx1were used for go kegg and reactome pathway analysisresults showed that mnx1 may participate in œtranscriptionand œmetabolism pathway figure 4a cell cycle detectionshowed that knockdown of mnx1 induced g2m cell cyclearrest in hela and siha cells figure 4b furthermore weexamined the eï¬ect of mnx1 on the expression of cell cyclekeyrelated genes including p15ink4b p16ink4a p21cip1 p27kip1cdk1 cdk2 cdk4 cyclinb1 cyclind1 and cycline1 bothin hela and siha cells knockdown of mnx1 upregulated theexpression of p21cip1 which has been confirmed as a tumorsuppressor gene in multiple cancers figure 4c and westernblotting results suggested that knockdown of mnx1 increasedthe expression of p21cip1 while decreased the expression ofphosphorylated cdk1 pthr161cdk1 a downstream eï¬ectorof p21cip1 figure 4d consistently with these results ectopicexpression of mnx1 decreased the expression of p21cip1 whileincreased the expression of pthr161cdk1 in c33a and helacells figures 4ef it suggested that mnx1 might exerted itsbiological function via modulating the expression of p21cip1frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 is upregulated in cc tissues and positively correlates with aggressive clinical characteristics a mnx1 is upregulated in cc tissues compared withadjacent normal tissues in tcga dataset p b patients with high expression of mnx1 have poor disease free survival dfs in cc p cd themrna expression of mnx1 in cervical cancer tissues was significantly higher than that in adjacent normal tissues in patients p e the mnx1staining score was upregulated compared with that in adjacent normal tissues p f the mnx1 staining score was positively correlated with tnm stage p g t stage p h lymph node metastasis p i tumor differentiation p and j local primary tumor diameter p incc patients k representative ihc staining images in tmas were shown error bars represent the mean ± sd values ns no significance represents p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 suppressed the proliferation migration and invasion in cc cells ab mnx1 mrna and protein level are upregulated in cc celllines c“f two specific sirna si1 and si2 of mnx1 were designed and the transfection efficiencies of sirnas in hela and siha cells were analyzed by qrtpcrand western blot g“i the proliferation abilities were evaluated by xcelligence system assay edu incorporation assay and colony formation assay were inhibitedafter knockdown of mnx1 in hela and siha cells j the xcelligence system assay k transwell and matrigel assay and l wound healing assay indicated thatmigration and invasion capacities were suppressed after simnx1 in hela and siha cells error bars represent the mean ± sd values of three independentexperiments p p p ns no significancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure ectopic expression of mnx1 enhanced aggressive abilities in c33a and hela cells ab the pcdna31mnx1 was synthesize and the transfectionefficiencies were analyzed by qrtpcr and western blot the proliferation functions were measured by c the xcelligence system assay d colony formationassays and e edu incorporation assays were elevated in oemnx1 c33a and hela cells f the transwell assay and matrigel invasion assay g wound healingassay also showed that oemnx1 strengthened migration and invasion capacities error bars represent the mean ± sd values of three independent experiments p p p ns no significancemnx1 suppressed the expression ofp21cip1 via binding to its promoter regionour previous results showed that knockdown or ectopicexpression of mnx1 altered the expression of p21cip1 to furtherverify the mechanism we analyzed the correlation betweenmnx1 and p21cip1 in cases of cc samples and the resultswere shown that mnx1 and p21cip1 had a negative correlationn p figure 5a as transcription factors usuallybind to sequencespecific dna to regulate transcription weutilized jaspar database to predict the binding site betweenmnx1 and the promoter region upstream bp of codingregion of cdkn1a the gene symbol of p21cip1 it turnedout that mnx1 was predicted to have four binding sites withthe promoter region of cdkn1a of which “ bpfrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown of mnx1 expression induced g2m phage arrest by regulating the p21cip1 expression a many genes were enriched in regulation oftranscription by go analysis most of the genes were enriched in the metabolic pathways by kegg and reactome pathway analysis b knockdown of mnx1generated g2m stage arrest in hela and siha cells were measured by flow cytometry cf the p21cip1 mrna levels were upregulated or downregulated after si oroemnx1 in cc cell lines de the protein level of p21cip1 was upregulated or downregulated while the expression of pthr161cdk1 was decreased or increased afterknockdown or ectopic mnx1 of cc cells the expression of cdk1 ccne1 ccnd1 and ccnb1 had no obvious changes error bars represent the mean ± sdvalues of three independent experiments p p p ns no significanceaacaataaat and “ bp gcccattaat showedhigher combination scores figure 5b accordingly the wildcdkn1a promoter region and mutant types 226mt and1371mt were generated and cloned into luciferase reportervector pgl3basic figure 5c and in luciferase reporterassay overexpression of mnx1 inhibited the transcriptionalactivity of the wild cdkn1a promoter but not mutant typefigure 5d moreover chip assay also revealed that mnx1bound to the p21cip1 promoter region in hela and sihacells figures 5effrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure mnx1 bounds to the p21cip1 promoter region and suppresses p21cip1 transcription a the expression of mnx1 and p21cip1 is negatively correlated in cervical cancer tissues p b the jarspar database indicates that mnx1 has several binding sites with the promoter region of p21cip1 c schematicdiagram shows that the two sites with the highest score of mnx1 on p21cip1 promoter and the mutant p21cip1 promoter were selected d overexpression of mnx1remarkably decreased wild type but not mutant p21cip1 promoter luciferase activity p21cip1226 p p21cip11371 p e chromatinimmunoprecipitation chip assays using normal igg or antimnx1 demonstrated that mnx1 directly binding to p21cip1 promoter region f the results of chippcrproduct electrophoresis were showed that a clear band was observed in the antimnx1 group while almost no band was detected in the igg control groupp p frontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure downregulation of p21cip1 partially recovered the malignant phenotypes of simnx1 cells a the transfection efficiency of p21cip1 was determined byqrtpcr and si1p21cip1 was chosen to further experiments b“d the proliferative abilities were partially rescued after knockdown p21cip1 in simnx1 hela cellswere measured by the xcelligence system assay colony formation assay and edu incorporation assay ef the invasion and migration capacities have also beensignificantly improved after knockdown p21cip1 in simnx1 cells compared with simnx1 alone cells g the protein level of p21cip1 and pthr161cdk1 were partiallyreversed when knockdown of p21cip1 in simnx1 compared with simnx1 alone error bars represent the mean ± sd values of three independent experiments p p p ns no significancefrontiers in oncology wwwfrontiersinaugust volume 0czhu mnx1 enhances progression of cervical cancerfigure knockdown or overexpression of mnx1 inhibited or promoted tumor growth in vivo ab the transfection efficiency of shmnx1 was measured byqrtpcr and western blot c a total of eight nude female mice were sacrificed and xenograft tumors were collected after injection with shmnx1 cells weeksde tumor volume and weight were reduced in the shmnx1 group compared with those in the shnc group f the expression of mnx1 and ki67 wasdownregulated and p21cip1 was upregulated in shmnx1 xenograft tumors analyzing by ihc staining g the protein level of mnx1 pthr161cdk1 weredownregulated and p21cip1 was upregulated in shmnx1 mouse xenograft tumors analyzed by western blot h a total of eight nude female mice were sacrificed andxenograft tumors were collected after injection with oemnx1 cells weeks jk tumor volume and weight was increased in the oemnx1 group compared withthose in the oenc group i the expression of mnx1 and ki67 was upregulated and p21cip1 was downregulated in oemnx1 xenograft tumors analyzing by ihcstaining error bars represent the mean ± sd values p p p ns no significancesilencing p21cip1 rescued the function ofsimnx1to determine whether the function of mnx1 was relied onp21cip1 we designed three sirnas table s3 to knockdownthe expression of p21cip1in hela cells the si1p21cip1showed the best transfection efficiency figure 6a and it wasused for the following experiment rtca proliferation assaycolony formation assay edu assay transwell assay matrigelassay and would healing assay revealed that silencing p21cip1partially rescued the decreased proliferation migration andinvasion ability of hela cells caused by knockdown of mnx1figures 6b“f and western blotting showed that the proteinlevel of p21cip1 and pthr161cdk1 were partially reversed bysilencing p21cip1 figure 6gmnx1 promoted tumor growth of cervicalcancer in vivothe xenograft models were used to explore the function ofmnx1 in vivo the shrnamnx1 shrnanc as control wastransfected into hela cells and the knockdown efficiency wasconfirmed by qrtpcr and western blotting figures 7abresults showed that knockdown of mnx1 inhibited tumrowth measured by tumor weight and volume in vivofigures 7c“e ihc staining and western blotting of harvestedtumors revealed that knockdown of mnx1 upregulated theprotein level of p21cip1 and downregulated ki67 and pthr161cdk1 in vivo figures 7fg moreover ectopic expression ofmnx1 promoted tumor growth and altered the expression ofp21cip1 and ki67 in vivo figures 7h“kfrontiers in oncology wwwfrontiersinaugust volume 0czhu discussionin this study we identified mnx1 a transcription factor ofhomeobox family was significantly upregulated and involvedin the progression of cervical cancer the overexpression ofmnx1 correlated with advanced clinical stages and poorerprognosis of cervical cancer patients furthermore mnx1exerted its oncogenic role via modulating the expression ofp21cip1 especially by targeting the promoter region of p21cip1thus to repress its transcriptionin accordance with ourfindings a recent showed that mnx1 had a role in theprogression of cervical cancer partially through upregulating cellcycle regulators ccne1 and ccne2 and mnxas1 theantisense lncrna of mnx1 was also reported to promote theinvasion and metastasis of gastric cancer through repression ofcdkn1a all this results indicated that mnx1 played acritical role in cancer growth and cell cycle progression andmnx1 might serve as a useful diagnostic and treatment targetfor cervical cancermnx1is a member of homeobox gene family which allcontain a homeobox a dna sequence around basepairs long and encode homeodomain protein products astranscription factors this cluster of genes has beenidentified to participate in the regulation of development andmorphogenesis in animals fungi and plants for examplecdx1 which is stably expressed in the human intestine playsan important role in embryonic epicardial development and the protagonist of our study mnx1 participates inmotor neuron development and neurodegenerative processesof zebrafish and moreover controls cell fate choice inthe developing endocrine pancreas in recent years moreand more researches uncovered the role of developmentrelatedhomeobox genes in carcinogenesis and these genes show greatapplication prospect in tumor diagnosis and prevention asthe role of carcinoembryonic antigen cea in gastroenterictumors and alpha fetal protein afp in liver cancer “for instance pdx1 is a key regulator in pancreatic developmentand cell function and meanwhile dynamically regulatespancreatic ductal adenocarcinoma initiation and maintenance hoxc13 a highly conserved transcription factor involvedin morphogenesis of all multicellular anisms is aberrantlyexpressed and associated with cancer progression in esophagealcancer lung adenocarcinoma and liposarcomas likewise mnx1 has been reported to promote sustainedproliferation in bladder cancer by upregulating ccne12 and to act as a novel oncogene in prostate cancer and in ourstudy mnx1 was also confirmed to be upregulated in cervicalcancer and enhance the progression of cervical cancerin terms of mechanism we found that mnx1 promotedtumor growth of cervical cancer via accelerating the progressionof the cell cycle especially by modulating the expression ofp21cip1 cell cycle is a vital process by which a cellleadsto duplication and disorders of the cell cycle regulation maylead to tumor formation the cell cycle progress isdetermined by two types of regulatory factors cyclins and cyclindependent kinases cdks active cyclincdk complexesphosphorylate proteins to elevate the expression levels of cyclinsmnx1 enhances progression of cervical cancerand enzymes required for dna replication converselythe cell cycle progression can be prevented by inhibitors bybinding to and thus inactivating cyclincdk complexes suchas p21cip1 p27kip1 p16ink4a and so on the p21cip1also known as cyclindependent kinase inhibitor cdkn1ahas been identified as a regulator of cell cycle and a tumorsuppressor in multiple kinds of cancers our results provedthat mnx1 repressed the transcription of p21cip1 by directlytargeting its promoter region and furthermore promoted thephosphorylation of downstream cdk1 the mnx1p21cip1pthr161cdk1 axis played crucial roles in the progression ofcervical cancer and meanwhile provided new evidence forthe pathogenesis of cervical cancer moreover the associationbetween cervical cancer and hpv has long been identified as asexually transmitted agent hpv are involved in transformationand maintaining of transformed status many studies havereported that hpv can also alter the expression of p21 “thus we searched the geo dataset to seek for information aboutthe relationship between mnx1 and hpv viral infection weanalyzed the gse dataset and found that there were nosignificant changes in the expression of mnx1 nm_005515 inhacat cells infected with hpv11e6 or hpv18e6 in gse3292gds1667 hpv positive or negative head and neck squamouscell carcinoma hnscc showed no expression diï¬erences ofmnx1 figure s1 this information suggests that mnx1 mightnot be directly involved in hpv carcinogenesis and furtherinvestigations are needed in the future in addition cervicalcancer i

continuously increasing with development of the economy and the environment [“] the prognosis for hcc patients remains extremely poor although significant progress has been achieved strategies for early diagnosis are urgently needed because the majority of patients with hcc are diagnosed in very late stages however the molecular mechanism of hcc has not been clearly defined circular rnas circrnas are a new class of rna molecules that have functions as regulators of parental gene transcription in alternative splicing and as mirna sponges through use of rna deep sequencing gtechnology numerous circrnas have been identified as the predominant regulatory elements in diseases moreover accumulating evidence shows that circrnas play pivotal roles in many diseases in particular abnormally expressed circrnas are involved in tumor progression including cell proliferation migration and invasion [“] in addition some research indicates that circrnas level are closely correlated wit specific phenotypes and tumorigenesis in hcc [“] nevertheless the research concerning circrnas is frankly in its infancy which greatly hinders the application of circrnas as biomarkers for diagnosis of hcc in clinicsrelated research shows that circrnas possess great potential to be used for diagnosis of hcc recent studies have found that hsa_circ_0067934 plays oncogenic roles by accelerating cell proliferation and metastasis in glioblastoma gbm circsmarca5 was significantly elevated and thereby suppressed cell apoptosis and arrested cell cycle in prostate cancer in addition previous studies have shown that downregulation of hsa_circ_0005986 facilitated cell proliferation by promoting the g0g1 to s phase transition in hcc similarly alteration in expression of circrnas correlated with development and metastasis of malignant tumors these data suggest that circrnas may be of greater benefit in clinical diagnosis of hcc however reliable circrna biomarkers for hcc are still lacking therefore this review synthetically integrates available data on the role of circrna in hcc progression and attempts to provide crucial clues for investigating the molecular mechanism regarding hccoverview of circrnacircrnas are a category of singlestranded closedcircle molecules which take part in multifaceted biological regulation recently research has verified that the majority of circrnas are synthesized by backspliced exons and that others are formed from intron intergenic and untranslated regions utr therefore biogenesis of circrnas can be divided into eicirnas exonintron circrnas ecircrnas circular exonic rnas and cirnas circular intronic rnas meanwhile over circrnas have been identified and this type of transcript has been considered a new form of gene expression generally the structure of the transcription is inverted and the order of genomic exons is altered and these exons are spliced over time the biological functions of circrnas gradually have been recognized including roles in embryonic development maintainenance of homeostasis and promotion of tumor progression figure properties of circrnascircrnas recently have attracted great attention related to their pathological role in disease development compared with linear rnas circrnas have special properties including biological roles and clinical use circrnas are mainly enriched in certain body fluids comprising blood saliva and urine they are covalently closed loop structures degradation of most rna is highly dependent on rna exonuclease or rnase hence circrnas remain highly stable based on their high resistance to enzyme degradation moreover studies have shown that expression of circrnas is tissuespecific and correlated with different phases of development and they exhibit different expression patterns at different developmental stages roles of circrnasaccumulating evidence shows that circrnas play a crucial role in the pathogenesis of diseases as a result of their complex biological functions generally the molecular functions of circrnas mainly include being sponges of mirna acting as rnabinding proteins performing alternative splicing of premrnas regulating transcription and translation and potentially encoding proteins these properties are described in detail belowsponges of mirnathe different types of circrnas have different mirna binding sites some circrnas negatively regulate mirnas by absorbing and specifically binding to mirnas then decreasing mirna activity and elevating expression of mirnarelated target genes researchers have shown that cirs7 inhibits mir7 function and positively mediates mir7 target genes acting as a molecular sponge in addition functional analyses have indicated that circrnas constitute an entire molecular regulatory network which specifically regulates degradation of mirnas as mirna sponges this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238322indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832premrna™e1e2abcbasepairinge3dguriche4™ecrichpretrnarna bindingproteinsrbp™™™™™™™™gnicilpskcablariat splicingaecircrnaelcirnacirnatrnatricrnafigure1 biogenesis of circular rnas a lariatdriven circularization the ™ hydroxyl of the upstream exon reacts with the ™ phosphate of the downstream exon to form a covalent linkage then producing a lariat including exons and introns the ™ hydroxyl of the ™ intron interacts with the ™ phosphate of the ™intron to form an ecircrna following an interaction between the ™ hydroxyl of the ™ exon and the ™ phosphate of the ™ exon b rnabinding protein rbpdriven circularization rbps accelerate interaction of the downstream intron and upstream intron thereby promoting formation of ecircrna c basepairingdriven circularization the downstream introns and upstream introns are paired depends on inverserepeatingcomplementary sequences formation of ecircrnaeicirna was derived from the introns are removedretained d biosynthesis of cirna formation of cirnas mainly based on a 7nt gurich element and an 11nt crich element to escape debranching and exonucleolytic degradation e formation of tricrna trna splicing enzymes divide pretrna into two parts tricrnas are generated by a ™“™ phosphodiester bond and the other part generates trnascircrnasbinding proteinsrna binding proteins rbps are a broad class of proteins involved in gene transcription translation and interaction studies suggest that distribution of rbps is widespread in many tissue types furthermore rbps participate in development of disorders by regulating posttranscriptional regulation of rnas rbps assemble ribonucleoprotein complexes to bind rna sequences thereby affecting the function of the target rnas previous research has shown that circrnas serve as protein decoys to harbor binding sites of specific proteins and block protein activity circfoxo3 induces cell cycle arrest resulting in defective cdk2 gene function by binding to p21 and cdk2 moreover circrna ciacgas binds to cgas protein and suppresses enzymatic activity of cgas thereby preventing cgas from recognizing selfdna e9238323indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832circrnas regulate alternative splicing transcription and translationcellular localization of most circrnas is cytoplasmic which is the basis for the biological function of mirna and protein decoys several studies have suggested that circrnas participate in rna splicing assembly and biosynthesis recently research has shown that circrnas may play pivotal roles in regulating alternative splicing transcription and translation in addition the exon of the splicing factor may form a circrna by affecting formation of linear rna eicirnas interact with the u1 small nuclear ribonucleoproteinsnrnp thereby regulating parental gene transcription by binding to rna polymerase ii interestingly translation of circrnas is mediated by ires and n6methyladenosine m6a and translation efficiency of circrna is regulated by the level of m6a modification moreover circfbxw7 effectively inhibits glioma proliferation and cell cycle progression by antagonizing usp28induced cmyc stabilization potential to encode proteinscircrnas are implicated in numerous physiological processes and pathogenesis of diseases strong evidence indicates that circrnas can encode proteins by mimicking dna rolling circle amplification related studies indicate that circrna circppp1r12a plays a key molecular role by encoding a functional protein circppp1r12a73aa which promotes proliferation migration and invasion of colon cancer circanril interacts with pescadillo zebrafish homolog pes1 to mediate ribosome biogenesis and prerrna processing in vascular macrophages and smooth muscle cells these studies have significantly increased the knowledge base about the biological functions of circrnascircrnas in diseasescircrnas are involved in processes that lead to development of various disorders such as neuronal and cardiovascular diseases and cancers circrnas participate in regulating gene transcription and protein expression and are indirectly and directly associated with time and regionspecific variations as mentioned previously abnormal expression of circrnas is implicated in neurological disorders atherosclerosis and ribosomal rna maturation reportedly are regulated by circanril simultaneously some studies have suggested that circrnas upregulation significantly affects sprouting and proliferation of vascular endothelial cells and elicits vascular dysfunction recently several experiments have implicated circrnas in pathogenesis of cancer via activation of a series of cascade reactions however the underlying mechanism for the effect of circrnas in initiation and progression of tumors has not been fully clarified to date related studies have revealed that certain circrnas are highly expressed in tumor tissues and overexpression of circrnas promotes tumor proliferation and deterioration an investigation revealed that hsa_circ_002059 was downregulated in gastric cancer while hsa_circ_0004018 was upregulated in hcc meanwhile tumorspecific circrnas candidates were screened in lung adenocarcinoma tissue by microarrays and circrnas were identified downregulated and upregulated of the circrnas hsa_circ_0013958 clearly was positive correlated with lymph node metastasis and tnm stage these findings indicate that circrnas have important roles in tumor progression and may have potential for broad applicatoins in medicine scienceoverview of hcchcc is one of the most prevalent tumors worldwide with diagnoses and approximately deaths annually epidemiological survey data indicate that morbidity and mortality from hcc are gradually increasing risk factors for hcc include diabetes mellitus obesity smoking alcohol consumption older age male sex chronic hbv liver cirrhosis and chronic hepatitis c virus hcv the primary risk factors include liver cirrhosis viral hepatitis alcohol intake and obesity worldwide approximately hcc patients are infected with hepatitis b virus hbv or hcv in addition alcohol abuse is a crucial factor for onset of hcc [“] obesity hypertension and diabetes are closely linked with development of hcc but specific correlations remain unknown moreover regular screening has been widely applied for early detection and to ensure effective treatment of hcc most commonly good results have been achieved with regular screening with ultrasonography blood alphafetoprotein content testing mri and ct generally surgical resection and chemotherapy are mainstays of therapy in patients with hcc yet some tumors cannot be fully removed which results in tumor growth invasion and metastasis local and systemic metastases are the main reasons for the unsatisfactory prognosis in patients with hcc therefore more effective therapeutic approaches need to be developedroles of circrnas in hccnumerous studies have documented the important role that circrnas play in tumorigenesis metastasis and invasion research has shown that circrnas are localized in the nucleus and interfere with transcription and promote alternative this work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd e9238324indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review s 0czou rc research progress on circrnas in hcc med sci monit e923832hsa_circ_0001649circzkscan1circitchwntbetacatenincircmto1mir9p21hsa_circ_00059836mir1295phmgb1 ragenfκbmir7hsa_circ_101368hsa_circ_001569cdr1ashsa_circ_0000673figure the function of circrnas in hcc carcinogenesis this graph demonstrates the role of circrnas in hcc carcinogenesis including positive and negative effects respectivelytable brief summary of circrnas as biomarkers for hccnamediseaseconclusiondoicirs7hsa_circ_0003570hsa_circ_0005075hepatocellular carcinomahepatocellular carcinomahepatocellular carcinomacirs7 was one of the independent factors and may be a promising biomarker for hepatic mvi and a novel therapy target for restraining mvi101007s0043201622567hsa_circ_0003570 expression levels were associated with hcc clinicopathological characteristics101002jcla22239hsa_circ_0005075 promotes proliferation migration and invasiveness of hcc via mir431 regulation101016jbiopha201801150splicing circpvt1 is overexpressed in gastric cancer tissues compared with nontumor tissues and circpvt1 acts as an oncogene to mediate expression of mir4975p however studies concerning the role of circrnas in development and progression of hcc remain in their infancytumor inhibitioncurrently circrnas are considered promising diagnostic biomarkers and ideal therapeutic targets for hcc studies have revealed that circitch inhibits tumor proliferation by suppressing the wntbetacatenin pathway expression of circitch has been positively correlated with good survival outcome in patients with hcc analysis of the circrnas expression profile in human hcc tissues showed that circmto1 was markedly decreased in hcc tissues and that expression of circmto1 was positively correlated with survival rate circmto1 reportedly inhibits hcc progress by sponging mir9 and thereby increasing p21 expression meanwhile overexpression of hsa_circ_0001649 negatively affects invasion and proliferation and promotes apoptosis of hcc cells downregulation of zkscan1 and circzkscan1 enhances cell proliferation and promotes progression of hcc tumor promotionin patients with hcc cdr1was more abundant in tumor specimens than in adjacent normal tissues cdr1as effectively suppresses the invasion and proliferation of hcc cells by targeting mir7 some reports have shown that hsa_circ_0000673 is significantly upregulated in hcc tissues and hsa_circ_0000673 downregulation markedly inhibits proliferation and invasion of hcc cells in vitro meanwhile a positive correlation was found between circ_001569 expression level and tumor size advanced tnm stages and unfavorable prognosis in patients with hcc circrna101368 was abundantly expressed in hcc tissue which correlated with poorer prognosis in addition circrna101368 inhibited cell migration by reducing protein levels in nfkb rage and hmgb1 figure e9238325indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0czou rc research progress on circrnas in hcc med sci monit e923832biomarkerconclusionsprevious studies have shown that circrnas are closely related to development of tumors clinicopathological features in patients with hcc are correlated to with levels of expression of cirs7 and its targeted mrnas global circrna expression profile analysis showed that hsa_circ_0005075 exhibited significant differences in tumor tissue versus adjacent tissues in patients with hcc expression of hsa_circ_0005075 also was related to tumor proliferation and metastasis therefore an increasing number of circrnas have been identified as diagnostic markers as summarized in table given the high incidence and mortality fo hcc worldwide it is one of the most serious diseases threatening human health increasing attention is being paid due to this serious situation evidence is increasing to support the close association between circrnas 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“ yu l gong x sun l the circular rna cdr1as act as an oncogene in hepatocellular carcinoma through targeting mir7 expression plos one e0158347 jiang w wen d gong l circular rna hsa_circ_0000673 promotes hepatocellular carcinoma malignance by decreasing mir7673p targeting set biochem biophys res commun “ liu h xue l song c overexpression of circular rna circ_001569 indicates poor prognosis in hepatocellular carcinoma and promotes cell growth and metastasis by sponging mir4115p and mir4325p biochem biophys res commun “ li s gu h huang y circular rna 101368mir200a axis modulates the migration of hepatocellular carcinoma through hmgb1rage signaling cell cycle “ “ shang x li g liu h comprehensive circular rna profiling reveals that hsa_circ_0005075 a new circular rna biomarker is involved in hepatocellular carcinoma development medicine baltimore e3811 yao t chen q shao z circular rna as a new biomarker for hepatocellular carcinoma metastasis j clin lab anal e22572e9238327indexed in [current contentsclinical medicine] [sci expanded] [isi alerting system] [isi s master list] [index medicusmedline] [embaseexcerpta medica] [chemical scas]review sthis work is licensed under creative common attributionnoncommercialnoderivatives international cc byncnd 0c'

" it is well established that retrieved lymph node rln counts were positively correlated with betteroverall survival in gastric cancer gc but little is known about the relationship between rln count and shorttermcomplications after radical surgerymethods a total of consecutive gc patients between january and december at nanjing drumtower hospital were retrospectively analyzed univariate analyses were performed to elucidate the associationbetween rln count and postoperative complications we further identified clinical factors that might affect the rlncountresults among all of the patients postoperative complications occurred in patients the mean rlncount was and patients were diagnosed with lymph node metastasis univariate analyses showedno significant difference between rln count and postoperative complications both overall and stratified by cdcgrade univariate and multivariate analyses further revealed that type of resection tumor invasion and lymph nodemetastasis were associated with rln counts the current study demonstrated that rln count was not associated with postoperative shorttermcomplications following gastrectomy of gc which provided a rationale for the determination of a proper rlncount of curative gastrectomykeywords retrieved lymph nodes postoperative complications gastric cancer there are approximately one million new cases of gastriccancer gc each year worldwide and half of them occurin eastern asia including china japan and south korea despite advances in early screening and comprehensive treatment of gc it remains the third most commoncause of cancerrelated death in the world for advanced gc a consensus has been reached of radical gastrectomy with d2 lymphadenectomy however there correspondence medguanwenxian163com wangmeng001263net feng sun song liu and peng song contributed equally to this workdepartment of gastrointestinal surgery nanjing drum tower hospital theaffiliated hospital of nanjing university medical school nanjing chinais still controversy over the number of retrieved lymphnodes rlns for accurate pathological stagingseveral studies have reported that rln count waspositively correlated with better overall survival in gceven in lymph nodenegative gc [“] an rln countof ‰¥ has been recommended by the 8th edition tnmclassification for gc to guarantee the accurate pn stage moreover okajima suggested an optimal rlncount of ‰¥ for nodal staging recently by stratumanalysis of patients deng proposed an optimal rln count of ‰¥ for lymph nodenegative gc and for lymph nodepositive gc these abovestudies are all conducted by comparing the rln count the authors open access this is licensed under a creative commons attribution international licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the creative commons licence and indicate ifchanges were made the images or other third party material in this are included in the 's creative commonslicence unless indicated otherwise in a credit line to the material if material is not included in the 's creative commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder to view a copy of this licence visit httpcreativecommonslicensesby40the creative commons public domain dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0csun world of surgical oncology page of table demographic and clinical features of patientscharacteristicsage yearsgender nn ± malefemalebmi kgm2preoperative comorbidities nprevious abdominalsurgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp glasa ‰¥ mode of surgical approach nlaparoscopicopentype of resection ndistal gastrectomyproximal gastrectomytotal gastrectomyoperation time minblood loss mltumor invasiont1“t3“tumor sitecardiafundusbodypylorusantrumrln countlymph node metastasispositivenegativelnrloddsptnm stage iiiiiiivlauren subtypeintestinaldiffusemixedunknownpostoperative complicationspositive ± ± ± ± ± ± ± ˆ’ ± table demographic and clinical features of patientscontinuedcharacteristicsnegativepostoperative stay daystotal hospital charges ¥n ± ± bmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratio loddslog odds of positive lymph nodeswith longterm survival but little is known about the relationship between the rln count and shortterm complications after radical surgerypostoperative complications of gc pose a significantimpact on the length of postoperative stay and hospitalcharges which further affect the quality oflife thereforeinvestigating the relationship between rlncount and postoperative shortterm complications wouldprovide more comprehensive evidence for selecting theappropriate rln countmethodspatientsa total of consecutive gc patients between january and december at nanjing drum tower hospital were retrospectively reviewed all patients underwent curative r0 gastrectomy and were histologicallyconfirmed the exclusion criteria were as follows multivisceral resection patients accepting preoperative radiotherapy or chemotherapy patients with previous stomach surgery and patients with incompleteclinical data this study was approved by the ethicscommittee of nanjing drum tower hospitalcharacteristicsfor preoperativedata collectiondataintraoperativeindex and postoperative features were extracted preoperative characteristics included age gender body massindex bmi comorbidities and laboratory data the intraoperative index involved the american society of anesthesiologists asa grade surgical approach type ofresection operation time and blood loss postoperativefeatures included depth of tumor invasion tumor site retrieved lymph node count lymph node metastasis lymphnode ratio lnrlog odds of positive lymph nodeslodds ptnm stage lauren subtype shortterm complications postoperative stay and total hospital chargeslnr was defined as the ratio of positive to retrievedlymph nodes lodds was calculated by log [positivelymph nodes 05total lymph nodes ˆ’ positive lymphnodes ] the postoperative shortterm complications occurring in the hospital or within days werecollected all complications were evaluated according tothe claviendindo classification system 0csun world of surgical oncology page of statistical analysisstatistical analyses were conducted by spss chicago il usa continuous variables were shown asmeans ± sd student™s t test was applied for normallydistributed data mannwhitney u test was applied fornonnormally distributed data categorical variable datawere presented as numbers and analyzed using the chisquared test or the fisher exact test univariate andmultivariate analyses were performed to analyze the riskfactors associated with the postoperative complicationsor retrieved lymph node count the optimal cutoffvalues of lnr and lodds were determined by receivertable univariate and multivariate analyses of characteristics associated with postoperative complicationscharacteristicsunivariateormultivariateor ci““p“reference“““age ‰¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ‰¥ glasa ‰¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3“rlnslymph node metastasislnr lodds ˆ’ ptnm stage ‰¥ iiilauren subtypeintestinaldiffusemixedunknown ci““““““““““reference““““reference““““““““reference“““p 0csun world of surgical oncology page of operating characteristic roc analysis all statisticaltests were conducted twosided and statistical differences were termed as p value resultspatient characteristicsthe characteristics of the patients enrolledin this study were presented in table there were gc patients in all including men and women the median age was years with arange from to years a total of patients underwent open gastrectomy while underwent laparoscopic surgery the type of resectionwas distal gastrectomy in patients proximalgastrectomy in and total gastrectomy in the mean operation time was min and themean intraoperative blood loss was ml pathologicalresults were stage iiiiiiiv in patientsrespectively the mean rln count was range “ and patients were tested with lymphnode metastasis overall postoperative shortterm complications occurred in patients the meanpostoperative stay was days and the mean total hospital charges were × ¥association between perioperative characteristics andpostoperative complicationsas presented in table univariate and multivariate analyses indicated that postoperative shortterm complications were significantly correlated with age gender levelof preoperative serum albumin and operation timestratified analyses by type of resection revealed thatcomplications occurred frequently in proximal gastrectomy compared with total gastrectomy while there wasno significant difference between distal gastrectomy andtotal gastrectomy no significant association was observed between rln count and overall postoperativecomplicationsimpact of rln count on postoperative complicationsof the patients developed complications of encountered a single complication and of encountered multiplecomplications the details of patients with shorttermcomplications based on the claviendindo classification are for grade i for grade ii frade iii for grade iv and for grade vthe rate of major complications cdc grade ‰¥ iiiwas the median rln count in this study was so we divided all patients into two groups basedon the median rln count univariateanalysesshowed no significant difference between rln countand postoperative complicationsboth overall andstratified by cdc grade table table univariate analyses of postoperative complicationsassociated with rln countcharacteristicsallrln count ‰¥ overall ngrade i nfever °cemesispainabdominopelvic collectionpleural effusiongrade ii nblood transfusionsearly postoperative bowel obstructiongastroparesisliver function abnormalitieswound infectionpneumoniaintraabdominal infectionsurinary tract infectionenteritisbacteremiagrade iii nanastomotic leakagelymphatic leakagepancreatic fistulabiliary fistulableedingabdominopelvic collectionpleural effusionintraabdominal abscesswound disruptiondelayed wound healinggastroparesisearly postoperative bowel obstructionsplenic necrosisgrade iv nheart failurekidney failurebrain infarctionmodspvaluegrade v ngrade ‰¥ iii nrlns retrieved lymph nodes mods multiple an dysfunction syndrome 0csun world of surgical oncology page of factors associated with rln countwe further explored the potential factors associated withrln count univariate analyses revealed that preoperative serum albumin type of resection tumor invasionlymph node metastasis and ptnm stage were associatedwith rln count p table stratification bytype of resection showed that rln count in either distalgastrectomy or proximal gastrectomy was significantlyin total gastrectomy multivariatelowerthan thatanalyses further indicated that type of resection tumorinvasion and lymph node metastasis were still significantly associated with rln count p table discussionnodal involvement significantly affected the prognosis ofgc patients because it is the major root of tumor relapse after surgery [ ] thus standardized lymphnode dissection is the basic requirement for curativetable univariate and multivariate analyses of factors associated with rln count ‰¥ characteristicsunivariateormultivariateor cipreference““ ““age ‰¥ gendermalefemalebmi kgm2preoperative comorbiditiesprevious abdominal surgerydiabetes mellitushypertensionpreoperative laboratory dataserum albumin glcrp ‰¥ glasa ‰¥ mode of surgical approachlaparoscopicopentype of resectiontotal gastrectomydistal gastrectomyproximal gastrectomyoperation timeblood losstumor sitecardiafundusbodypylorusantrumtumor invasion t3“lymph node metastasisptnm stage ‰¥ iiilauren subtypeintestinaldiffusemixedunknown ci““““““““““reference““““reference“““““reference“““p bmi body mass index crp creactive protein asa american society of anesthesiologists rlns retrieved lymph nodes or odds ratio ci confidence interval 0csun world of surgical oncology page of r0 gastrectomy curative gastrectomy with d2 lymphadenectomy has been considered as the standard fashionfor decades in eastern asia especially in japan [ ]this procedure has been gradually accepted by westerncountries in recent years [ ] as for the rln countthe 8th edition tnm classification for gc recommendeddissecting at least lymph nodes moreover emergingevidence revealed the positive correlations between rlncount and overall survival of gc patients [ ] bycomparing rln count to survival time okajima suggested an optimal rln count of ‰¥ deng proposed an optimal rln count of ‰¥ for lymphnodenegative gc and for lymph nodepositive gcby stratum analysis of patients sano reported that rln count preferably achieved or moreby a multicenter study enrolling patients additionally lnr and lodds were also reported to[“] thesebe associated with gc prognosisabove studies mainly focused on the relationship between rln count and longterm prognosis howeverlittle is known aboutits effects on postoperativeshortterm complicationsin this study we concentrated on the association betweenrln count and shortterm prognosis univariate analysesshowed no significant difference between rln count andpostoperative complications both overall and stratified bycdc grade therefore more lymph nodes were encouragedto be dissected from the perspective of shortterm prognosisalthough curative gastrectomy with d2 lymphadenectomy is considered a pivotal strategy for advanced gcthere are international and institutional differences in thenumber of rln count [ ] various factors were reported to influence the rln count including the confidence and enthusiasm of doctors both surgeons andpathologists surgical situation and innate lymph nodecount in each patient [ ] in our study we concludedthat rln count was related to the type of resection tumorinvasion and lymph node metastasis of note rln countwas positively correlated with the lymph node metastasisrate which underlined the importance of rln count foraccurate stagingactuallyfor a thorough pathological examinationrlns should be individually divided from a completetissue sample after surgery owing to much time andeffort was required during this procedureit has notbeen widely implemented clinically therefore the examined lymph node count by pathologists might belower than the dissected lymph node count multipleattempts have been conducted to improve the detection rate of lymph nodes [“] li elucidatedthat the mean number of rlns could be significantlyelevated by injecting carbon nanops before surgery compared with controls vs markl and colleagues reported a twofold lymph nodepick up rate utilizing methylene blue staining thanunstained groups vs several dye materials were also used to increase the number of lymphnodes dissected during surgery such as fluorescentindocyanine green icg and 5aminolevulinic acid5ala [ ]we acknowledge that this study had some potentialit was a retrospective singlecenterlimitations firststudy so the results might be flawed because of residualconfounding factors second the rln count was closelyrelated to the quality of surgeons and pathologists theperioperative variables might differ in different doctorstherefore multicenter studies are needed to confirmour resultssin the current study demonstrated thatrlns\\ count was not associated with postoperativeshortterm complications following gastrectomy of gctherefore our analysis encouraged more lymph nodesto be dissected for accurate pathologic stagingabbreviationsbmi body mass index crp creactive protein asa american society ofanesthesiologists rlns retrieved lymph nodes lnr lymph node ratiolodds log odds of positive lymph nodesacknowledgementsthe authors gratefully acknowledge all the investigators for theircontributions to the trialauthors™ contributionsfs worked on the study design collected the data and drafted themanuscript sl contributed to the study design and data collection ps wasinvolved in the data collection and extraction cz helped collect the datawg was involved in the study design and data extraction mw revised themanuscript all authors have read and approved the final manuscriptfundingthere is no funding supporting this workavailability of data and materialsaccess to the data and the calculation method can be obtained from theauthors by email medsunfeng163comethics approval and consent to participatethis retrospective study was approved by the ethics committee of nanjingdrum tower hospital medical school of nanjing university due to theretrospective nature the requirement for informed consent was waived bythe irbs from nanjing drum tower hospital medical school of nanjinguniversityconsent for publicationnot applicablecompeting intereststhe authors declare that they have no competing interestsreceived june accepted august referencesstewart b wild cp world cancer report public health 0csun world of surgical oncology page of degiuli m de manzoni g di leo a ™ugo dd galasso e marrelli d gastric cancer current status of lymph node dissection world jgastroenterol “son t hyung wj lee jh kim ym kim hi an jy clinical implication ofan insufficient number of examined lymph nodes after curative resectionfor gastric cancer cancer “li z ao s bu z wu a wu x shan f clinical study of harvestinglymph nodes with carbon nanops in advanced gastric cancer aprospective randomized trial world j surg oncol markl b kerwel tg jahnig hg oruzio d arnholdt hm scholer c methylene blueassisted lymph node dissection in colon specimens aprospective randomized study am j clin pathol “ aoyama t yoshikawa t morita s shirai j fujikawa h iwasaki k methylene blueassisted technique for harvesting lymph nodes after radicalsurgery for gastric cancer a prospective randomized phase iii study bmccancer he m jiang z wang c hao z an j shen j diagnostic value of nearinfrared or fluorescent indocyanine green guided sentinel lymph nodemapping in gastric cancer a systematic review and metaanalysis j surgoncol “koizumi n harada y murayama y harada k beika m yamaoka y detection of metastatic lymph nodes using 5aminolevulinic acid inpatients with gastric cancer ann surg oncol “publisher™s notespringer nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliationsbray f ferlay j soerjomataram i siegel rl torre la jemal a global cancerstatistics globocan estimates of incidence and mortality worldwidefor cancers in countries ca cancer j clin “van cutsem e sagaert x topal b haustermans k prenen h gastric cancerlancet “zhang w zhangyuan g wang j jin k liu y wang f effect of lymphnodes count in nodepositive gastric cancer j cancer “chu x yang zf impact on survival of the number of lymph nodes resectedin patients with lymph nodenegative gastric cancer world j surg oncoljiang l yang kh guan ql zhao p chen y tian jh survival and recurrencefree benefits with different lymphadenectomy for resectable gastric cancera metaanalysis j surg oncol “deng j yamashita h seto y liang h increasing the number of examinedlymph nodes is a prerequisite for improvement in the accurate evaluationof overall survival of nodenegative gastric cancer patients ann surg oncol“amin mb greene fl edge sb compton cc gershenwald je brookland rk the eighth edition ajcc cancer staging manual continuing to build abridge from a populationbased to a more œpersonalized approach tocancer staging ca cancer j clin “okajima w komatsu s ichikawa d kosuga t kubota t okamoto k prognostic impact of the number of retrieved lymph nodes in patients withgastric cancer j gastroenterol hepatol “ deng j liu j wang w sun z wang z zhou z validation of clinicalsignificance of examined lymph node count for accurate prognosticevaluation of gastric cancer for the eighth edition of the american jointcommittee on cancer ajcc tnm staging system chin j cancer res “kim th suh ys huh yj son yg park jh yang jy the comprehensivecomplication index cci is a more sensitive complication index than theconventional claviendindo classification in radical gastric cancer surgerygastric cancer “ wang j hassett jm dayton mt kulaylat mn the prognostic superiority oflog odds of positive lymph nodes in stage iii colon cancer j gastrointestsurg “ dindo d demartines n clavien pa classification of surgical complicationsa new proposal with evaluation in a cohort of patients and results of asurvey ann surg “ hirabayashi s kosugi s isobe y nashimoto a oda i hayashi k development and external validation of a nomogram for overall survivalafter curative resection in serosanegative locally advanced gastric cancerann oncol “tóth d bíró a varga z török m árkosy p comparison of different lymphnode staging systems in prognosis of gastric cancer a biinstitutional studyfrom hungary chin j cancer res de steur wo dikken jl hartgrink hh lymph node dissection in resectableadvanced gastric cancer dig surg “ maruyama k kaminishi m hayashi ki isobe y honda i katai h gastric cancer treated in in japan data analysis of nationwide registrygastric cancer “liang h deng j evaluation of rational extent lymphadenectomy for localadvanced gastric cancer chin j cancer res degiuli m sasako m ponti a vendrame a tomatis m mazza c randomized clinical trial comparing survival after d1 or d2 gastrectomy fastric cancer br j surg “sano t coit dg kim hh roviello f kassab p wittekind c proposal ofa new stage grouping of gastric cancer for tnm classification internationalgastric cancer association staging project gastric cancer “ zhao e zhou c chen s prognostic nomogram based on log odds ofpositive lymph nodes for gastric carcinoma patients after surgical resectionfuture oncol “ alatengbaolide lin d li y xu h chen j wang b liu c lu p lymph noderatio is an independent prognostic factor in gastric cancer after curativeresection r0 regardless of the examined number of lymph nodes am jclin oncol wang j dang p raut cp pandalai pk maduekwe un rattner dw comparison of a lymph node ratiobased staging system with the 7th ajccsystem for gastric cancer analysis of patients from the seer databaseann surg “ 0c"

complex disease caused by coordinated alterations of multiple signaling pathways TheRasRAFMEKERK MAPK signaling is one of the bestdefined pathways in cancer biology and its hyperactivation isresponsible for over human cancer cases To drive carcinogenesis this signaling promotes cellular overgrowthby turning on proliferative genes and simultaneously enables cells to overcome metabolic stress by inhibitingAMPK signaling a key singular node of cellular metabolism Recent studies have shown that AMPK signaling canalso reversibly regulate hyperactive MAPK signaling in cancer cells by phosphorylating its key components RAFKSRfamily kinases which affects not only carcinogenesis but also the outcomes of targeted cancer therapies againstthe MAPK signaling In this review we will summarize the current proceedings of how MAPKAMPK signalingsinterplay with each other in cancer biology as well as its implications in clinic cancer treatment with MAPKinhibition and AMPK modulators and discuss the exploitation of combinatory therapies targeting both MAPK andAMPK as a novel therapeutic interventionKeywords RasRAFMEKERK signaling AMPK signaling Interplay Tumorigenesis Cellular metabolism RAFMEKERKinhibitors AMPK inhibitors AMPK activators Autophagy Targeted therapyIntroductionThe RasRAFMEKERK MAPK signaling is a fundamental pathway in cell biology and its alteration causeshuman cancers or developmental disorders Given its crucial roles in physiology and pathology this pathway hasbeen extensively studied for over two decades Unfortunately the regulation of MAPK signaling remains ambiguous till now by virtue of its intrinsic complexity anddiverse crosstalks with other signalings Here we focus onthe complicated interplays between the MAPK and theAMPK signalings in cellular carcinogenesis and their implications in current targeted cancer therapies We hopethis review would provide a conceptual framework for Correspondence yuanjiminszhospitalcom hujianchengnccscomsg1Department of Urology Shenzhen People™s Hospital The Second ClinicalMedical College Jinan University The First Affiliated Hospital SouthernUniversity of Science and Technology Shenzhen Guangdong China4Cancer and Stem Cell Program DukeNUS Medical School College RoadSingapore SingaporeFull list of author information is available at the end of the developing more effective therapeutic approaches againsthyperactive MAPK signalingdriven cancersThe RasRAFMEKERK MAPK signaling and itsaberrant activation in cancersThe RasRAFMEKERK MAPK signalingThe RasRAFMEKERK MAPK mitogenactivated protein kinase signaling is a central pathway that regulatescellular proliferation differentiation and survival Thissignaling pathway was discovered in the 1970s“1980swhen Ras small GTPases were identified as first oncogenes from sarcoma viruses [“] Later studies on viraloncogenes had also led to the discovery of a Nterminaltruncated version of RAF SerThr kinase RAF1 or CRAF[“] In contrast the other two components of this signaling pathway MEK mitogenactivated protein kinasekinase and ERK mitogenactivated protein kinase wereidentified as cytoplasmic protein kinases activated by mitogens in the 1990s [“] Following these discoveries The Authors Access This is licensed under a Creative Commons Attribution International Licensewhich permits use sharing adaptation distribution and reproduction in any medium or format as long as you giveappropriate credit to the original authors and the source provide a link to the Creative Commons licence and indicate ifchanges were made The images or other third party material in this are included in the 's Creative Commonslicence unless indicated otherwise in a credit line to the material If material is not included in the 's Creative Commonslicence and your intended use is not permitted by statutory regulation or exceeds the permitted use you will need to obtainpermission directly from the copyright holder To view a copy of this licence visit httpcreativecommonslicensesby40The Creative Commons Public Domain Dedication waiver httpcreativecommonspublicdomainzero10 applies to thedata made available in this unless otherwise stated in a credit line to the data 0cYuan Journal of Hematology Oncology Page of RAF was identified as the upstream kinase of MEK in and the first direct effector of Ras in [ ]resulting in the delineation of the whole MAPK signalingpathway which is considered as a milestone in our understanding of how cell senses external stimuliThe first component of MAPK signaling Ras smallGTPases have three gene isoforms Hras Kras and Nras that encode four proteins with splicing isoforms ofKras giving rise to Kras4A and Kras4B Although allRas proteins possess highly homologous sequences theyhave quite different activities tissue expression patternsand effector preferences which lead to their differentialphysiological and pathological functions [“]The downstream of Ras small GTPases is the RAFMEKERK kinase cascade [] The first kinases in thiscascade RAFKSR kinase suppressor of Ras family kinases include three RAF isoforms ie CRAF BRAF andARAF and two close pseudokinases ie KSR1 and KSR2All RAF isoforms have highly homologous sequences andsimilar structures with three conserved regions conservedregion CR1 contains RASbinding domain RBD anda Cysrich domain [ ] conserved region CR2 ischaracterized by a SerThrrich sequence conserved region CR3 comprises of a putative kinase domain with aNterminal acidic motif NTA [“] and a Cterminalregulatory tail [“] Nevertheless RAF isoforms havevariable kinase activities with an order as BRAFCRAFARAF likely by virtue of their distinct NTA motifs andAPE motifs that contribute to the dimerizationdriventransactivation of RAFs [“] In contrast to RAF isoforms KSR proteins replace the RBD at the Nterminusfused sterile αmotif and Prorichwith a coiledcoilstretch that are responsible for recruiting proteins to theplasma membrane upon stimulation and lack the catalyticlysine in VAIK motif of kinase domain which impairs theircatalytic activity [ ] Given their associations withMEK and ERK as well as low kinase activity KSR proteinshave been thought as scaffold proteins in a long termHowever recent studies have indicated that KSR proteinscan also function as allosteric activators to stimulate thecatalytic activity of RAF proteins through dimerization[ “] The sidetoside dimerization of RAFKSRfamily kinases is critical not only for their activation butalso for their catalytic activity towards downstream kinases [ “] MEKs MEK1 and MEK2 are the second kinases of the RAFMEKERK kinase cascade whichhave both redundant and nonredundant functions [] These two dualspecific kinases comprise a shortregulatory Nterminus and a canonic kinase domain TheNterminal regulatory region of MEK12 contains a docking site for substrate ERKs a nuclear export sequence thatcontrols the cytoplasmicnuclear shuttling of proteins anda negative regulatory sequence that forms a helix andlocks kinase in an inactive conformation [ ]Further through its kinase domain MEK12 forms a facetoface heterodimer with RAFKSR or a homodimerheterodimer with itself which is indispensable for its activation stimulated by RAF and for its activity towards ERKs[ ] Like MEKs the terminal kinases of MAPKsignaling ERKs also include two highly homologousmembers ERK1 and ERK2 which have a central kinasedomain flanked by short N and Cterminal tails Thesetwo isoforms also have redundant functions albeit different expression patterns [“] However unlike RAFs andMEKs that have very limited substrates ERKs recognizeand phosphorylate numerous substrates that include transcription factors protein kinases and phosphatases andother functional proteins [“]It should be noted that active Ras also turns on othersignaling pathways such as PI3KAKTmTORC whichregulate different cellular functions [] In this reviewwe focus only on the MAPK signaling given its dominant role in cancer biologyHyperactive RasRAFMEKERK MAPK signaling incancersThe MAPK signaling plays a crucial role in cell biologyand is tightly regulated in normal cells Upon engagement of receptor tyrosine kinases RTKs or other stimulations Ras small GTPases are activated by GTPGDPexchange factors GEFs which in turn recruit RAFMEK complexes to the plasma membrane and triggerthe RAFMEKERK kinase cascade through facilitatingRAFRAF or KSR RAFMEK and MEKMEK interactions as well as subsequent phosphorylations [] ActiveERKs are further translocated into the nuclei or stay inthe cytoplasm where they phosphorylate a number ofsubstrates that regulate cell functions [“ ]On the other hand active MAPK signaling also turns onsome negative feedback loops which help cells return toquiescent status [“] An aberrant activation ofMAPK signaling frequently induces human cancers ordevelopmental disordersthough an extremely highMAPK signaling may induce cell death or senescenceunder some conditions [“]its upstream activators or componentsHyperactive MAPK signaling exists in over ofcancers which is caused directly by genetic alterationsofincludingRTKs Ras and BRAF or indirectly by those independent of Ras or RAF [“] and significantly promotesdisease progression [] Since genetic alterations ofRTKs in cancers have been extensively reviewed in recent years [“] here we focus on oncogenic mutations of Ras and BRAF As a small GTPase Ras cyclesbetween active GTPbound status and inactive GDPbound status which is regulated by GEFs and GTPaseactivating proteins GAPs Oncogenic Ras mutationscan be mainly classified into two groups mutations 0cYuan Journal of Hematology Oncology Page of on glycine or G1213 that impair GAP associations and mutations on glutamine Q61 that diminish the intrinsic GTPase activity of Ras [] both ofwhich lead to an extended halflife of GTPloaded RasOncogenic Ras mutations have both isoform andcancertype preferences Kras is mostly mutated in allcancers followed by Nras and Hras and its mutations prevailin pancreatic cancers whilethose of Nras in myeloma and melanomas and Hrasin adrenal gland cancers [ ] This phenomenon mayreflect underlying fundamental signaling landscapes andRAS mutants interplay with these landscapes As thedownstream effector of Ras RAF is another dominanttarget of oncogenic mutations in the MAPK signalingpathway Similarly RAF mutations have isoform preference in cancers as Ras mutations with BRAF CRAF ARAF which may arise from their different basal activities Overall a single point mutation that converts Val into Glu in the activation loop of BRAF accounts for cases [] Although BRAF V600E exists only in of all cancers it is highly prevalent in some tissuespecific cancers such as melanoma thyroid cancer and histiocytosis [“] albeit theunderlying molecular mechanisms remains unknown Incontrast to Ras and RAF MEK and ERK have rare mutations in cancers though their mutations have been shownto be responsible for some RAF inhibitor RAFiresistantcases in current cancer therapies [“]Targeting the RasRAFMEKERK MAPK signalingpathway for cancer therapy promising but challengingGiven their high prevalence in cancers great efforts havebeen made to develop specific inhibitors against oncogenicRas and RAF mutants in the last decades These inhibitorsthat have been approved for clinic treatment of RasRAFmutated cancers or under clinical trials are listed in Table However none of these inhibitors can effectively target thelarge portion of Ras mutants in cancers Since having no attractive docking sites suitable for designing highaffinity andselective small molecule inhibitors Ras mutants have beenthought as œundruggable cancer drivers in a long term Untilrecently a group of covalent small inhibitors that are dockedinto a previously unknown pocket of GDPbound Ras andare linked to the adventive cysteine of RasG12C have beendeveloped and achieved encouraging outcomes for treatingRasG12Cdriven cancers as a single agent in clinical trials[“] Fig To further enhance their efficacy theseRasG12C inhibitors are also undergoing clinical evaluationwhen combined with SHP2 Src homology region domaincontaining phosphatase2 inhibitors that block the pathwayreactivation caused by the relief of negative feedback loops[ ] Clinical Trial NCT04330664 In addition these inhibitors have also been further developed into RasG12C degraders by conjugating with ligands of ubiquitin E3 ligaseswhich effectively deplete Ras mutant proteins in cancer cells[ ] though their efficacy in vivo remains unknown Unlike RasG12C the majority of Ras mutants remain œundruggable at present []It has been shown that Ras activates downstream effectors through direct interactions Therefore disruptingRaseffector interactions might be an alternative approach that can effectively block cancer growth drivenby Ras mutations Such a type of small moleculeblockers include rigosertib sulindac and MCP110 andamong which the therapeutic efficacy of rigosertib combined with nivolumab for Rasmutated cancers is beingdetermined by phase III clinical trials currently []Clinical Trial NCT04263090 Howeverit has to benoted that these inhibitors impair the MAPK signalingin both Rasmutated cancers and normal tissues andthereby their therapeutic index may not be highGenetic studies have revealed that the ablation of theRAFMEKERK kinase cascade but not other effectorpathways is a most efficient approach to inhibit thegrowth of Rasmutated cancers [] which leads to extensive developments of specific inhibitors against thiskinase cascade for treating Rasmutated cancers Moreover these inhibitors should be also effective for treatingRAFmutated cancers Indeed a number of RAFMEKERK inhibitors have been developed and applied to clinical trials for treating RasRAFmutated cancers [ “] At present three RAF inhibitors and three MEKinhibitors have been approved to treatlatestageBRAFV600Eharboring cancers as a single agent or incombination with other chemotherapeutics and exhibited excellent efficacies [ “] Fig HoweverRasmutated cancers possess intrinsic resistance to bothRAF and MEK inhibitors [] and even BRAF V600Eharboring cancers develop acquired resistance after “months treatment [ ] Mechanistic studies haveshown that active Ras facilitates the RAF dimerizationon plasma membrane which leads to both intrinsic andacquired resistance to RAF inhibitors [“] Toovercome the drug resistance arising from enhancedRAF dimerization the secondgeneration RAF inhibitorssuch as PLX8394 BGB283 TAK580 and CCT3833have been developed and are undergoing clinical evaluations Clinical Trials NCT02428712 NCT02610361NCT03905148 NCT02327169 NCT02437227 Thesenovel RAF inhibitors reduce the RAF dimerizationdriven resistance through distinct mechanismsPLX8394 and BGB283 impair RAF dimerization uponloading on RAF proteins [“] TAK580 bindsto and inhibits both protomers in RAF dimers [] CCT3833 inhibits both RAF and upstream kinases ofRas and thereby prevents the activation of Ras by the relief of negative feedback loops [ ] Besides thesesecondgeneration RAF inhibitors a unique RAFMEK 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancersTargetKRasG12CDescriptionPhase I results showed ORR of nonsmall cell lung cancer NSCLC harboring KRas G12CCompoundAMG510Development stagesPhase IIINCT04303780MRTX849JNJRigosertibRasPhase IIINCT03785249Phase IIINCT04330664Evaluation of clinical activity of MRTX849 alone and combined with TNO155SHP2 inhibitor in KRas G12C mutated cancersPhase I NCT04006301 Safety and PK of JNJ74699157Phase IIINCT04263090Evaluation of safety and clinical efficacy of Rigosertib plus Nivolumab PD1 Abin KRas mutated NSCLCBRAFVemurafenib ApprovedLatestage or unresectable melanoma expressing BRAF V600E in ErdheimChester disease ECD with BRAF V600E mutation in DabrafenibApprovedEncorafenibApprovedLatestage or unresectable melanoma expressing BRAF V600E in Combination with trametinib for the treatment of unresectable or metastatic melanoma withBRAF V600EK in Combination with trametinib for the treatment of metastatic NSCLC with BRAF V600E in Combination with trametinib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with trametinib for the treatment of anaplastic thyroid cancer ATC that cannotbe removed by surgery or has spread to other parts of the body with BRAF V600E in Combination with binimetinib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Combination with cetuximab EGFR Ab for the treatment of metastatic colorectal cancerwith BRAF V600E in PLX8394BGB283TAK580Phase IIINCT02428712PLX8394 with cobicistat CYP3A inhibitor was well tolerated and showed promising activityin BRAFmutated refractory cancersPhase I NCT02610361Phase IIINCT03905148Evaluation of safety and PK of BGB283 alone and combination with mirdametinibPhase I NCT02327169Phase I NCT03429803TAK580 is the inhibitor of BRAF V600E and dimersTreatment in pediatric lowgrade gliomaCCT3833Phase I NCT02437227 CCT3833 is a panRAF inhibitor of mutant BRAF CRAF and SRC kinasesRAFMEKRO5126766Phase I NCT00773526Phase I NCT03681483Phase I NCT03875820Phase I NCT02407509RO5126766 is a dual inhibitor for both RAF and MEKTreatment of advanced KRasmutant lung adenocarcinomasEvaluation of safety and PK of RO5126766 with VS6063 FAK inhibitor or everolimusmTOR inhibitorRO5126766 showed activity across Ras and RAFmutated malignancies with significantresponse in lung and gynecological cancersMEK12TrametinibApprovedCobimetinib ApprovedPhase IIINCT03989115BinimetinibApprovedSelumetinibApprovedMirdametinib Phase IINCT03962543Phase IINCT02022982Phase IIINCT03905148A singleagent oral treatment for unresectable or metastatic melanoma with BRAFV600EK in Combination with dabrafenib for the treatment of unresectable or metastatic melanomawith BRAF V600EK in Combination with dabrafenib for the treatment of metastatic NSCLC with BRAF V600E in Combination with dabrafenib for the adjuvant treatment of melanoma with BRAF V600EK in Combination with dabrafenib for the treatment of ATC that cannot be removed by surgeryor has spread to other parts of the body with BRAF V600E in In combination with vemurafenib to treat advanced melanoma with BRAF V600EK in Doseescalation of combination of RMC4630 SHP2 inhibitor and cobimetinibCombination with encorafenib for the treatment of patients with unresectable or metastaticmelanoma with BRAF V600EK in Selumetinib was approved for neurofibromatosis type with symptomatic inoperable plexiformneurofibromas according to NCT01362803Evaluation of mirdametinib in the treatment of symptomatic inoperableneurofibromatosis type1 NF1associated plexiform neurofibromas PNsCombination of mirdametinib with palbociclib in the treatment of KRasmutant nonsmall cell lung cancer NSCLCEvaluation of safety and PK of BGB283 alone and combination with mirdametinibSHR7390Phase I NCT02968485 Evaluation of safety and PK of SHR7390 0cYuan Journal of Hematology Oncology Page of Table Summary of small molecule inhibitors approved and under clinical trials for treating RasRAFmutated cancers ContinuedTargetDescriptionEvaluation of safety and PK of CS3006CompoundCS3006Development stagesPhase I NCT03516123Phase I NCT03736850ERK12UlixertinibMK8353LY3214996ASTX029ATG017KO947Phase IIINCT01781429Phase I NCT04145297Phase IINCT03698994Phase I NCT03454035Phase I NCT01358331Phase I NCT03745989Phase I NCT02972034Phase I NCT04081259Phase I NCT04391595Phase I NCT02857270Phase IINCT04386057Phase IIINCT03520075Responses to ulixertinib in NRas BRAF V600 and nonV600 BRAF mutant cancersEvaluation of ulixertinib alone or combined with hydroxychloroquine palbociclibCDK46 inhibitor in MAPK mutated cancersMK8353 was optimized from SCH772984 for better pharmacokinetics and exhibitedinhibition of BRAF V600 mutant cancersEvaluation of combination of MK8353 with selumetinib or pembrolizumab PD1 Abin advanced malignanciesEvaluation of treatment of MK8353 alone or combined with abemaciclibCDK46 inhibitorHydroxychloroquine in advanced malignanciesEvaluation of safety and PK of ASTX029Phase I NCT04305249 Evaluation of safety and PK of ATG017Phase I NCT03051035 Evaluation of safety and PK of KO947dual inhibitor RO5126766 has been developed and exhibited a strong potential against both Ras and RAFmutated cancers in phase I clinical trials [“] Thisallosteric inhibitor docks on MEK and prevents the release of MEK from RAF as well as the subsequent phosphorylation of MEK by RAF [] which gives it muchmore advantages than all other known RAF inhibitorsaccording to the regulatory mechanism of the RAFMEKERK kinase cascade [] As to small molecule inhibitors that target the terminal kinase ERK although anumber of them have been developed and are undergoing clinical trials [ ] their therapeutic values fortreating RasRAFmutated cancers remain unknownLike MEK inhibitorsthese ERK inhibitors may notachieve a good therapeutic index as single agents byvirtue of their inhibitory role in both malignant and normal tissues However they may contribute to antiRasRAF cancer therapy as synergetic agents combined withRasRAF inhibitorsOveralltargeting hyperactive MAPK signaling hasachieved exciting outcomes for treating RasRAFmutated cancers However although some effective smallmolecule inhibitors have been developed and applied toclinical treatment drug resistance and side effects remain remarkable challenges and there is still a long wayto develop a longeffective approach with manageableside effects for treating RasRAFmutated cancersAlthough hyperactive MAPK signaling has a dominantrole in cancer biology it is finetuned by other signalingssuch as PI3KAKTmTORC and AMPK during diseaseprogression [] These signaling interplays have important impacts on both cancer progression and clinicaltreatment based on MAPK inhibition In this review wewill focus on the crosstalk between MAPK and AMPKsignalingsAMPK signaling and its roles in cancer biologyAMPK signaling and cellular metabolismAMPK AMPactivated protein kinase is an energy sensorthat monitors the AMPADPATP ratio in eukaryotic cellsThis atypical protein kinase was firstly discovered as acontaminant during the purification of acetylCoA carboxylase ACC a wellstudied substrate of AMPK for fattyacid FA synthesis nowadays [“] Fig Howeverthe phosphorylation of ACC by AMPK in response to thehigh AMPATP ratio had not been revealed until a decadelater [] and the enzyme was thus named as AMPKthereafter [] Fig Biochemical studies have shownthat AMPK consists of three subunits including the catalytic α subunit and the regulatory β and γ subunits [“] Fig In mammals AMPK subunits are encoded asseveral isoforms α1 α2 β1 β2 γ1 γ2 γ3 which arepreferentially expressed in specific tissues or anisms[ ] For instance the α2 subunit associatesonly with β1 in type I muscle fibers while it binds to bothβ1 and β2 in type II muscle fibers [ ] Also theliver formulation of AMPK subunits differs among speciesas that α1β2γ1 is dominant in human whereas α1β1γ1and α2β1γ1 in dog and rat respectively [] Althoughan isoform replacement of AMPK subunits may not extensively affect the basal activity of AMPK as adaptive reit alters AMPK™ssponses such as exercise do []subcellular locations and sensitivity as well as interactionswith other signaling pathways [] The anismtissue 0cYuan Journal of Hematology Oncology Page of Fig Target hyperactive RasRAFMEKERK MAPK signaling for cancer therapy The RasRAFMEKERK MAPK signaling functions downstream ofreceptor tyrosine kinases RTKs Upon engagement by their ligands RTKs activates guanine exchange factors Sos proteins which load GTP to RasGTPases Then GTPbound Ras GTPases recruit RAFMEK heterodimers in cytosol to plasma membrane where they form transient tetramers throughthe sidetoside dimerization of RAFs The RAF dimerization not only turns on RAFs but also loosens RAFMEK heterodimerization and facilitates MEKhomodimerization on RAF dimer surface which leads to the activation of MEKs by RAFs Once MEKs are activated they phosphorylate ERKs and thenactive ERKs phosphorylate a number of downstream effectors In cancer cells hyperactive RasRAFMEKERK MAPK signaling arising from geneticmutations of Ras GTPases and BRAF can be targeted by small molecular inhibitors of Ras G12C BRAFV600E MEK and ERKstagespecific selectivity of subunit isoforms complicatesAMPK™s regulationAs a key sensor of cellular energy stress the activity ofAMPK is predominantly regulated by cellular AMPADPATP that competitively binds to the γ subunit of AMPKand thus promotes or inhibits the phosphorylation ofThr172 on α subunit by the tumor suppressor liver kinaseB1 LKB1 or the dephosphorylation of this site by phosphatases [ ] Fig Besides adenine nucleotidesintracellular calcium ions activate AMPK through calciumcalmodulindependent protein kinase kinase CAMKK2 also called CAMKKβFig which acts downstream of the hormoneactivated receptors such as muscarinic receptors and ghrelin receptor onendothelial cells or neuron cells [“] On the otherhand AMPK can be inhibited by a metabolite of glucosefructose 16bisphosphate FBP which binds to the aldolase and prevents the interaction of AMPK with LKB1 inglucoserich environments [] Fig Active AMPK[“]has more than downstream substrates that regulatethe metabolism of lipids cholesterol carbohydrates andamino acidsActive AMPK promotes the oxidation of fatty acidsand inhibits the synthesis of fatty acids and cholesterolwhich involves largely in acetylCoA AMPK phosphorylates and inhibits HMGCoA reductase HMGR that requires acetylCoA in its reduction reaction [ ] Fig Also AMPK phosphorylates ACC that converts acetylCoA to malonylCoA and therefore slowsdown the de novo fatty acid FA synthesis and increasesthe FA oxidation [] Fig Alternatively AMPK regulates the lipid metabolism through altering the mitochondria structure and function In the mitochondriaAMPK phosphorylates Akinase anchoring protein AKAP1 a key scaffold protein for protein kinase APKA and facilitates the phosphorylation of a mitochondriafusion factor dynaminrelated protein DRP1 by PKA which promotes mitochondrial fusion 0cYuan Journal of Hematology Oncology Page of Fig AMPK signaling and its downstream effectors AMPK is activated by liver kinase B1 LKB1 or calciumcalmodulindependent protein kinasekinase CAMKK2β through phosphorylation on Thr172 of α subunit and is inactivated through dephosphorylation of this site by proteinphosphatases in response to changes of cellular AMPADPATP ratio Downstream effectors activated by AMPK are indicated as arrows and thoseinhibited by AMPK are shown as barheaded linesand oxidative phosphorylation [] Moreover AMPKaccelerates the mitochondria biogenesis likely throughphosphorylating and activating the transcriptional activator proliferatoractivated receptor gamma coactivator1alpha PGC1α [ ] Fig However upon energy stress AMPK plays an opposite role in mitochondria biology Under this condition AMPK is essential forthe fragmentation of mitochondria AMPK phosphorylates mitochondrial fission factor MFF on Ser129 andthereby facilitates the translocation of DRP1 from cytosol to mitochondria membrane in energy stressdrivenmitochondria fission [ ] Then AMPK promotesthe clearance of damaged mitochondria through autophagy In this process AMPK binds directly to and phosphorylates the unc51like autophagy activating kinase ULK1 Autophagyrelated gene ATG9 and Beclin which triggers the autophagosome formation [“] Fig Active AMPK directly regulates the carbohydrate metabolism or indirectly through altering the fatty acid metabolism as described above Activation of AMPKstimulates the expression and plasma membrane translocation of solute carrier family member GLUT proteins and thereby facilitates glucose import [ “] Fig Intracellularly AMPK phosphorylates andactivatesis6phosphofructo2kinasePFK2thatfructose 26bisphoresponsible for the synthesis ofsphate a potent stimulator of glycolysis and thus accelerates glycolysis []Fig Furthermore AMPKappears to phosphorylate and inhibit glycogen synthasein the liver which dampens glycogen synthesis and thusindirectly enhances glycolysis []Active AMPK maintains cellular amino acid homeostasis mainly by controlling the activity of mammaliantarget ofrapamycin complex mTORC1 ThemTORC1 is a central sensor of cellular amino acids thatsamples amino acids in both cytosol and lysosome [] Upon activation by amino acids mTORC1 stimulates protein synthesis by phosphorylating ribosomalprotein S6 kinase B1 S6K and eukaryotic translationinitiation factor 4E binding protein 4EBP1 whichenhances the consumption of cellular amino acidsMoreover active mTORC1 blocks cellular autophagy byphosphorylating ULK1 and impairs the recycling ofamino acids [] Both effects of mTORC1 lead to a remarkable drop of cellular amino acid reservoir ActiveAMPK has been shown to inhibitthe activity ofmTORC1 direct and indirectly upon energy stresswhich limits the expenditure of amino acids Alternatively active AMPK can restrict protein synthesis byphosphorylating and thereby inhibiting eukaryotic translation elongation factor eEF2 kinase a key regulator 0cYuan Journal of Hematology Oncology Page of of protein synthesis [] To restore cellular amino acidreservoir active AMPK stimulates cellular autophagy asdiscussed above which degrades surplus or dysfunctional proteins into amino acids [] In addition it isworth noted that cellular amino acids can affect theactivity of AMPK reversely Dependent on conditionscontexts either amino acids may inhibit or stimulate theactivity of AMPK though underlying molecular mechanisms remain ambiguous [“]YAPactivity which impairsAMPK signaling in cancer biologyIt is well known that AMPK is a putative substrate oftumor suppressor LKB1 [ ] Fig Therefore AMPK has been generally considered as a key effector that mediates the tumorsuppressive function ofLKB1 Indeed a genetic ablation of the AMPK α subunitin mice accelerates Mycdriven lymphomagenesis throughfacilitating a metabolic shift to aerobic glycolysis []Simultaneously AMPK inhibitorsAMPKi promoteepithelialtomesenchymal transition EMT in breast andprostate cancers [] These studies validate AMPK as atumor suppressor under certain circumstances Furthermechanistic studies have demonstrated that AMPK prevents cancers through phosphorylating multiple targetsthat play indispensable roles on different layers of diseaseprogression AMPK phosphorylates angiomotin like AMOTL1 an adaptor protein in the HippoYap pathway and thus blocks Yes1 associated transcriptional regucells™latorproliferation and survival [] AMPK also phosphorylates TSC complex subunit TSC2 and regulatory associated protein of MTOR complex Raptor and therebyinactivates mTORC1 [ ] which in turn elevatescellular autophagy activity and inhibits cancer initiationTo bypass this inhibitory effect cancer cells can activatethe MAGE family member A MAGEA36tripartitemotif containing TRIM28 ubiquitin ligase complexthat targets the AMPK α subunit for degradation and thusreactivates mTORC1 to restrict cellular autophagy []Moreover AMPK is able to phosphorylate enhancer ofzeste polycomb repressive complex subunit EZH2and thereby disrupts the polycomb repressive complex PRC2 which relieves the epigenetic silence of tumorsuppressors in cancers [] Alternatively AMPK phosphorylates and stabilizes another epigenetic master regulator Tet methylcytosine dioxygenase TET2 whichfunctions as a putative tumor suppressor to preventtumorigenesis [] Altogether these findings indicatethat AMPK has a pronounced antitumor activity as itsupstream kinase LKB1 doescancerkillsandLICsstressleukemia TALL oncogenic Notch signaling induces ahigh level of aerobic glycolysis which needs to be restrained by AMPK and loss of AMPK results in energystressdriven apoptosis of leukemic cells and slows downdisease progression [] Similarlyin acute myeloidleukemia AML metabolic stress elevates the ROS leveland induces DNA damage in leukemiainitiating cellsLICs and AMPK confers metabolic stress resistance toLICs [] AMPK knockout or pharmaceutical inhibitionunder metabolicinhibitsleukemogenesis Moreover AMPK plays a determinantrole in maintaining the NADPH homeostasis in cancercells upon energy stress which is critic