The questionnaire was answered by 47 paediatric oncologists from 15 countries. iCHG progressing during first line therapy with carboplatin-vincristine would be considered for treatment with alternative chemotherapy by 17 (36%) and with surgery plus chemotherapy by 27 respondents (58%). Components suggested for second line were vinblastine (62%), cisplatin (34%) and cyclophosphamide (26%). For third line therapy bevacizumab (BVZ) was considered as suitable by respondents in 53% (often with irinotecan 40%) and vinblastine by 34% respectively. Experience with BVZ in CHG is shown by 53% of respondents regarding at least 95 patients (median treated 1–5 patients per respondent at any age) with a median BVZ administration over 12 months. Effectiveness was reported varying between stable disease and regression while complications were rarely stated . BVZ would be available to 85% of respondents as therapeutic option for iCHG patients. Multiple anti-neoplastic drug regimens are applied for progressive iCHG, partly considered in combination with surgery if safely feasible. BVZ is commonly used at a satisfactory level in third line, mainly combined with irinotecan.Treatment of infant hypothalamic chiasmatic glioma (iCHG) is challenging, about 30% of the children progress during chemotherapy. Despite subsequent treatments the 5 year overall-survival rate is only 70%. This study investigates treatment strategies currently applied for progressive iCHG. A web-based questionnaire was sent out to the members of the SIOPE Brain Tumour Group asking for current second and third line strategies at progression The online version of this article (doi:10.1007/s11060-017-2630-6) contains supplementary material, which is available to authorized users. Low-grade gliomas (LGG) are the most common brain tumours in childhood, accounting for approximately 35% during the first year of life and up to 50% in older children . The chiThe present survey was conducted following intense discussions among the SIOPE LGG working group on possible treatment strategies for infants with progressive CHG. The survey therefore aimed at evaluating treatment strategies currently adopted for progressive iCHG, i.e. progressive tumour in children diagnosed during the first year of life. Following primary reports on the efficacy of bevacizumab (BVZ) in patients with LGG –23, and http://www.thesistools.de . Per definition, tumours were considered as infant CHG if diagnosed during the first year of life. Treatment at progression or at relapse after the end of intial therapy (independent of the age at relapse) was addressed in the present survey. The survey took around 15 min to fill in and consisted of questions regarding treatment strategies, drugs applied for systemic therapy and experience with bevacizumab in CHG patients. Additional questions were asked on characteristics of the participating medical specialist: profession, country of origin, years of clinical experience, yearly amount of children treated with a brain tumour and the subgroups of LGG, CHG as well as iCHG specifically. As the survey aimed at assessing general treatment strategies, it did not collect data on patient characteristics .A web-based questionnaire on experiences and preferences on relapse treatment for iCHG (during or following first line treatment with carboplatin/vincristine) was created at The invitation to participate in the survey with a directly accessible link was sent to specialists in the field of paediatric neuro-oncology consisting of the 220 members of the multidisciplinary SIOPE Brain Tumour Group via email in February 2014 and repeated after 3 weeks.The questionnaire was answered by 47 paediatric oncologists from 15 countries . Two neuro-surgeons were omitted from the analysis as they indicated not to be involved in the decision on systemic treatment approaches. The professional experience of the respondents was reflected by the fact that 85% worked in a clinic with > 20 new brain tumour patients per year. Their years of experience in neuro-oncology were as follows: <5 years n = 5, 5–10 years n = 8, 10–20 years n = 18, > 20 years n = 15 and missing n = 1. 81% treated 1–6 patients with newly diagnosed CHG yearly, 19% seven or more patients per year. The reported rate of iCHG was low, during a 5 year period 57% of respondents cared for 1–3, 23% for 4–6, while 20% for more than six patients with iCHG.Considering the high rate of patients progressing after chemotherapy, the survey asked on the opinion of prolonging first line chemotherapy (see Table during first line therapy with carboplatin - vincristine would be treated with different chemotherapy by 17 (36%) and with surgery plus chemotherapy by 27 respondents (58%). Vinblastine was selected as single chemotherapeutic agent in 62% followed by cisplatin (34%) and cyclophosphamide (26%) in multi-agent combinations.Therapeutic strategies adopted and systemic therapies administered at progression in patients with iCHG are summarised in Table after the end of combined carboplatin—vincristine chemotherapy 54% of respondents would favour vinblastine monotherapy while about 44% would choose a platinum derivative, 35% vincristine and 20% cyclophosphamide, mostly in combination therapy. In addition to systemic therapy 38% of respondents would consider a neurosurgical option (if safely feasible) in combination with further chemotherapy.In case of progression In third line respondents would use BVZ in 53%, in 19/25 (76%) combined with irinotecan. Vinblastine is considered by 34%.The actual experiences in application of BVZ are listed in Table Radiotherapy was not considered an option in second and third line therapy  and exhibits a poor overall survival of 70% –6. Due tAccording to the current recommendations of the SIOPE LGG group, carboplatin and vincristine are mostly administered over a period of 18 months in first line therapy, but a third of responding experts would consider prolongation of first line treatment to ameliorate PFS.The present survey underlines that systemic therapies are considered the mainstay of relapse therapy in iCHG. Even if neurosurgery is (re-)considered, subsequent systemic therapy approaches are deemed necessary to sustain a possible tumour reduction achieved by neurosurgery. Accordingly none of the respondents would select a stand-alone neurosurgical approach at first tumour progression and 6% at second progression.Radiotherapy was not considered an option in second and third line therapy by respondents of this survey. The vast majority would even only consider radiotherapy after failure of multiple lines of systemic therapy. This reasoning will probably be based on the possible devastating side effects of cranial radiotherapy in small children , 11. AlsThe survey revealed that a wide variety of anti-neoplastic agents are applied in subsequent therapy lines.At first progression vinblastine is chosen most often as 2nd line treatment, 62% for progression during carboplatin-vincristine and 54% for progression after the end first line treatment. The excellent tolerability and efficacy of vinblastine in LGG has been documented earlier and its efficacy was also reported in young children with iCHG and those with diencephalic syndrome, either alone or in combination with carboplatin , 24, 25.A wide variety of drugs is used in case of progression during and/or after first line chemotherapy. We could not define specific subgroups of professionals for instance by nationality and/or years of experience, who provided specific alternative chemotherapy regimens. Only the application of cisplatin in combination with etoposide was reported by Italian neuro-oncologists as published by Massimino et al. , 28 AlthThe antiangiogenic agent BVZ, a monoclonal antibody targeting vascular endothelial growth factor is approved for oncologic treatment in adults and was consecutively applied in a variety of paediatric CNS malignancies. In childhood LGG several studies demonstrated benefit of BVZ, specifically in patients with CHG. Packer et al. were the first to report clinical and radiologic responses in ten cases with multiple relapsed LGG with a combination of two-weekly BVZ and irinotecan . Kalra eIndeed application of BVZ for progressive iCHG seems to be a hopeful strategy as shown in the experiences of 53% of our respondents reflecting experience in at least 95 patients. The previously published results of BVZ in LGG incited us to investigate the current practice of BVZ application in progressive CHG in the clinical practice of paediatric neuro-oncology. The results of the survey suggest that while vinblastine is widely applied in second line, BVZ was indicated as most prevalent choice in third line therapy with a majority of clinical responses despite multiple progressions of their LGG.BVZ is not approved for the use in children with LGG and expensive compared to conventional chemotherapy. Considering its effectiveness in LGG , 21 and Following the increasing understanding of molecular pathways involved in LGG pathogenesis , a novelSince a majority of patients with LGG become long term survivors, more insight is needed in the late outcome by gathering data in a common registry . This isOur survey provides insights in the opinions of a broad group of experienced paediatric neuro-oncologists and reflects clinical practice from many different countries in the choices made in treatment of progressive iCHG. The information regarding BVZ in clinical practice refers to at least 95 patients, extending the information on the use of BVZ, so far limited to reports published on 25 CHG patients treated with BVZ. Due to the nature of an online survey there was the possibility that multiple respondents from one treatment centre may have answered the questionnaire. Indeed, two IP-addresses were used by two respondents each. All four could be identified as independent respondents; one of them was neurosurgeon and left out from analysis (see above). Only 47/220 persons invited via email have responded to the survey, maybe distorting the results. As the mailing list of SIOPE also included neurologists, radiotherapists, neurosurgeons, neuro-pathologists, psychologists, endocrinologists, statisticians, basic scientists and ophthalmologists who were not addressed by the present survey, the percentage of paediatric neuro-oncologists responding is much higher than suggested by this number.The efficacy of other targeted drugs, such as specification of MEK inhibitors were not implicated in our survey yet. The survey allowed however to name alternative drugs and the application of a MEK inhibitor was indicated only twice by the respondents. The group of MEK inhibitors deserves specific attention in a possible follow-up survey in the future.Although clinical response to BVZ is reported by the majority of respondents our survey did not extrapolate on the responses in vision and the differences between children with or without neurofibromatosis type 1.Multiple different drug regimens are applied for progressive iCHG. Also in case of a neurosurgical approach subsequent anti-neoplastic treatment is widely applied. The application of targeted drugs emerges as novel treatment strategy. While vinblastine is the most common drug used in second line treatment, BVZ is often used in third line, mostly in combination with irinotecan. Prospective data on the efficacy of BVZ in LGG are needed and should include the infant population. Whereas the present findings cannot replace the lack of prospective trials for this small group of patients with progressive iCHG, they clearly reflect current strategies applied by an international community of paediatric neuro-oncologists. Still, treatment decisions have to be made on a patient per patient basis by an experienced interdisciplinary paediatric neuro-oncology team as depicted by the wide variety of possible therapeutic strategies. We advocate an international registry for children with (i)CHG to prospectively gather information on the multiple progressions and subsequent treatment strategies.Below is the link to the electronic supplementary material.Supplementary material 1 (PDF 1278 KB)


Despite the remarkable progress that has been made to reduce global malaria mortality by 29% in the past 5 years, malaria is still a serious global health problem. Inadequate diagnostics is one of the major obstacles in fighting the disease. An automated system for malaria diagnosis can help to make malaria screening faster and more reliable. We present an automated system to detect and segment red blood cells (RBCs) and identify infected cells in Wright–Giemsa stained thin blood smears. Specifically, using image analysis and machine learning techniques, we process digital images of thin blood smears to determine the parasitemia in each smear. We use a cell extraction method to segment RBCs, in particular overlapping cells. We show that a combination of RGB color and texture features outperforms other features. We evaluate our method on microscopic blood smear images from human and mouse and show that it outperforms other techniques. For human cells, we measure an absolute error of 1.18% between the true and the automatic parasite counts. For mouse cells, our automatic counts correlate well with expert and flow cytometry counts. This makes our system the first one to work for both human and mouse. Parasite-infected red blood cells (RBCs) lead to symptoms, such as fever, malaise, seizures, and coma, in severe cases. Fast and reliable diagnosis and early treatment of malaria is one of the most effective ways of fighting the disease, together with better treatments and mosquito control.,,––Although both thick and thin blood smears are commonly used to quantify malaria parasitemia, many of the computer-assisted malaria screening tools currently available rely on thin blood smears.We present an end-to-end automated detection system for identifying and quantifying malaria parasites  in thin blood smears of both human and mouse. The main difference between human and mouse malaria parasites is that in mice, all the stages of the parasite can be seen in the peripheral blood, whereas in humans, the mature stages, such as trophozoites and schizonts, are mostly sequestered. Another difference is that P. falciparum has elongated, banana-shaped gametocytes and takes around 10 to 12 days until complete maturation, whereas the gametocytes in mouse are round and maturate faster. This makes our software robust to different visual patterns of parasite stages. In resource-limited settings, where research labs have no access to flow cytometry or other cell counting means, our software can help expedite research experiments on mice models, taking the manual cell counting load from researchers. Moreover, flow cytometry is too expensive for field-use and requires a technical person to prepare, acquire, and analyze samples.Our automated malaria parasite detection system consists of four main steps, as illustrated in We develop an efficient RBC detection and segmentation technique that uses a multiscale Laplacian of Gaussian (LoG) cell detection method as input to an active contours-based segmentation scheme named coupled edge profile active contours (C-EPAC) to accurately detect and segment individual RBCs and highly overlapping cells with varying annular and disk-like morphologies and textural variations . Ersoy eThen, we use a combination of color and texture features to characterize segmented RBCs. We develop an offline feature evaluation framework using manually annotated cells to select the most discriminative features, reduce feature dimensionality, and improve classification performance . The feaFinally, we use a linear support vector machine (SVM) to classify infected and uninfected cells because of its simplicity, efficiency, and easy translation to a smartphone . We alsoThe main contributions of this work are summarized as follows: •The fusion of LoG filter with C-EPAC enables us to efficiently detect and segment individual RBCs, including highly overlapping cells with varying annular and disk-like morphologies and textural variations. We achieve a superior cell detection F1 score of 94.5% and 95% for human and mouse respectively, including a better performance in splitting touching or overlapping cells. We compute Jaccard indices of 92.5% for human cells and 81% for mouse cells.•We use a combination of low-level complementary features to encode both color and texture information of RBCs. Features are selected through an offline evaluation framework to optimize the classification performance using manually annotated cells.•We are the first to present a robust system for both human and mouse blood smears, including evaluation of the overall system performance in terms of precision, recall, accuracy, and F1 score. For human, we measure an average absolute error of 1.18% between the true and the automatic parasite counts. For mouse, we are the first to compare automatic cell counts with flow cytometry counts, measuring a high correlation.•On average, our system can process about We organize the remainder of the paper as follows: Sec. 2We use blood slide images for both human and mouse provided by the National Institute of Allergy and Infectious Diseases (NIAID) to evaluate our system. All experiments are approved by the NIAID Animal Care and Use Committee (NIAID ACUC). The approved Animal Study Proposal (Identification Number LIG-1E) adheres to the regulations of the Animal Welfare Regulations and Public Health Service Policy on Human Care and Use of Laboratory Animals.2.12.1.1in vitro in the conditioned media comprising of RPMI 1640. The culture was maintained in a mixed gas environment with 5% Whole blood from Interstate Blood Bank was processed to remove all the white blood cells by passing it through SEPACELL R-500 II leukocyte reduction filter from Fenwall. The processed blood was used to culture Plasmodium falciparum 2.1.2C57BL/6 female mice (7 to 10 weeks old) were obtained from The Jackson Laboratories. Mice were infected with PbA by injecting i.p. 2.1.3Peripheral blood parasitemia was determined by flow cytometry using a modification of a previously described method.In the following sections, we will refer to the acquired slide images and annotations as the human-NIAID and mouse-NIAID datasets since the human and mouse blood slides have been provided by NIAID.2.2Blood slide images were acquired with the Zeiss Axio Imager, an upright research microscope platform, using a magnification of Cells were manually annotated by an expert as either infected or uninfected, using our Firefly online annotation tool [We evaluate the performance of features and classifiers on 70 human-NIAID images  with the most popular methods including: (i) Otsu-thresholding combined with morphology operations (Otsu-M),3.2.1The accuracy of cell segmentation relies on the performance of the cell detection algorithm in detecting individual and touching cells. Therefore, we first evaluate the performance of the LoG-based cell detection algorithm and compare it to cell detection results of Otsu-M and Chan–Vese active contour methods. otations . TherefoTo combine the precision and recall performance of the detection algorithm and report the overall performance, 3.2.2We compute the region-based Jaccard indexJaccard index is one of the most popular segmentation evaluation metrics that measures the similarity between a computed segmentation mask 4Once the cells have been detected and segmented from the whole image, in the next step, we extract all segmented cells and characterize them by their color and texture information to distinguish infected cells from normal cells within a learning framework. We have studied different features for describing normal and abnormal cells, and evaluated their performance using SVM and ANN classifiers to select the most discriminative feature set. We evaluated the performance on both SVM and ANN to show that the best feature set outperforms other features independent from the classifier used. In feature evaluation experiments, we used ground truth annotations to extract cells and decouple the performance of features and classifier from our automatic segmentation results. The color feature set includes YCbCr, normalized green channel from RGB color model (NG), a combination of three discriminative normalized channels from different color models: To select the most discriminative feature set, we measure precision, recall, accuracy, and F1 score of SVM and ANN classifiers using the described features in TP is the number of cells that are truly classified as infected and TN is the number of cells that are truly identified as normal cells. FP and FN report the number of cells that are being misclassified.5In the last step of our processing pipeline, we use a SVM classifier with a linear kernel, a two-layer ANN feedforward network with a sigmoid transfer function in the hidden layer, and a softmax transfer function in the output layer to classify cells into two classes: infected and uninfected. We evaluate the system pipeline performance on a set of 14 thin blood slides, each containing 5 images, from human-NIAID dataset  using a 10-fold cross-validation scheme to train and test the classifiers. In each fold, 63 images are used for training and 7 images are used for testing.To quantify the malaria infection, we compute the infection ratio as follows: 6To evaluate our system systematically, we monitored the malaria infections of two mice identified as 2805 and 2808, during a course of several days. We compared the counts of human experts with the automatic counts provided by our system. In addition, we compared our counts with automatic counts produced by flow cytometry and with the counts of a layperson, who received a brief introduction into the art of cell counting by expert slide readers. 7We have developed an image analysis system that can automatically quantify a malaria infection in digitized images of thin blood slides. The system’s image processing pipeline consists of three major steps: cell segmentation, feature computation, and classification into infected and uninfected cells. The most challenging task of the pipeline is the segmentation phase, which needs to be fast and accurate in splitting any clumped cells to avoid miscounting and misclassification in the last stage of the pipeline. We use a combination of multiscale LoG filter and C-EPAC level-set scheme to detect and segment cells, which is capable of identifying individual cells in a clumped cell cluster of touching cells and outperforms other methods. For feature computation, we use a combination of NRGB and JAMBP texture features. The color feature picks up the typical color of stained parasites and the texture feature detects cell texture information including the cytoplasm of parasites. This feature combination works well in our experiments and helps to avoid FPs due to staining artifacts. In the classification step, we evaluate the linear SVM and ANN classifiers performance on human and mouse slides. The ANN classifier achieves F1 score of 90% in correctly identifying infected cells on human-NIAID dataset. We measure an average absolute error of 1.18% between the true and the automatic parasite counts for human. For mouse cells, our automatic counts correlate well with expert and flow cytometry counts, making this the first system that works well for both human and mouse. Compared to human counting, our system is much faster and can process


Their biological profile was also inspected in vitro on the HL-60 cell line using different flow cytometric techniques . The compounds exhibited a profound inhibitory effect on topoisomerase activity (e.g. compound 22 inhibited type I topoisomerase at 1 µM concentration). The treatment of HL-60 cells with the studied compounds showed inhibition of cell growth especially with hybrids containing thiourea (14–17) and urea moieties (21 and 22). Moreover, treatment of human dermal fibroblasts with the studied compounds did not indicate significant cytotoxicity. The observed results suggest beneficial selectivity of the heterodimers as potential drugs to target cancer cells.A combination of biochemical, biophysical and biological techniques was used to study calf thymus DNA interaction with newly synthesized 7-MEOTA-tacrine thiourea  In the next step, 1 was treated with phosphorus oxychloride to give a quantitative yield of 9-chloro-7-methoxy-1,2,3,4-tetrahydroacridine 2,2 with appropriate 1,ω-diamines in the presence of phenol then provided the desired intermediates N-alkane-1,ω-diamines 3–8 (71–93%). 1,2,3,4-Tetrahydroacridin-10H-9-one 9 was formed directly from the neat reaction of cyclohexanone with N-methylanthranilate (65%). Chlorination of 9 was carried out by refluxing with phosphorus oxychloride to obtain 9-chloro-1,2,3,4-tetrahydroacridine 10 in quantitative yield. 9-Isothiocyanato-1,2,3,4-tetrahydroacridine 11 was obtained by refluxing 10 in the dark with silver thiocyanate in anhydrous toluene12–17 were formed by reaction of the two synthons 11 and diamines 3–8 (65–78%). The oxo-analogs were obtained using 2,4,6-trimethylbenzonitrile N-oxide to afford 7-MEOTA-THA ureas . Structural determination and signal assignments of thiourea hybrids 12–17 and urea hybrids 18–23 were accomplished by application of the usual combination of 1H and 13C NMR spectra. Unequivocal assignments were performed by homo- and hetero-correlated two-dimensional NMR experiments . The infrared spectrum was obtained only for molecule 11 to observe isothiocyanate group vibrations.12–22, their influence on the catalytic activity of Topo I/II, their ability to affect cell cycle distribution, and their effect on the mitochondrial membrane potential (MMP) and the metabolic activity and viability of the HL-60 cell line. The results acquired from these in vitro studies should enhance understanding of the effects pertinent for drugs targeting cancer. The urea heterodimer 23 was not tested due to its low solubility.In this work, we have studied DNA binding of novel THA-7-MEOTA dimers 12–22, we prefer to use multiple assays that reflect real physiological/pathological changes in more detail and on single cell level . It is generally known that most of the chemotherapeutic agents exert their cytotoxic effect either by induction of cancer cell apoptosis or by cell cycle arrest at a specific pointm) followed by release of pro-apoptotic molecules into the cytoplasm, which leads to programed cell death12–22 affect mitochondrial physiological processes, HL-60 cells were treated with the studied compounds for 24, 48 and 72 h and labeled with TMRE. In general, mitochondria with normal MMP retain the dye, resulting in strong fluorescence. Compounds inducing a collapse in MMP allow flow of the dye from mitochondria to cytoplasm leading to depolarization of the mitochondrial membrane and fluorescence decrease. The results from MMP analysis clearly show that 14–17 and 21 at 15 µM and 22 at 5 µM were able to evoke MMP dissipation in more than approximately 60% of cells after 24 h treatment and caused cell death with an efficiency between 80 and 100% at 15 µM for urea analogs and 25 µM for thioureas after 24, 48 and 72 h . A simultaneous analysis of viability (staining with PI) and the activity of cellular metabolism showed a very similar trend to acquired changes in MMP analysis identifying the effectivity of compounds 14–17 and 21, 22 to kill cancer cells  and 22 (at 5 µM) having five and six methylenes in the linker being found to exert the most cytotoxic profile. The higher cytotoxic effect presumably reflects easier cell membrane penetration. Interestingly, the cytotoxic effect against HL-60 also correlated well with the ability to inhibit activity of topoisomerases. It is known that inhibitors of Topo I, such as camptothecin, exhibit cell cycle arrest induced apoptosisFor biological effect analysis of novel compounds and 72 h . Their eand 72 h . The other cells . IC50 frFigure 2.14–17 (5–25 μM), ureas 20–22 (1–15 μM) and THA (5–10 μM) on changes in A: MMP and B: viability in HL-60 cells. Cells were analyzed 24, 48 and 72 h after treatment with studied compounds. The results were calculated as mean ± SD from three independent experiments. Statistical significance p < .05 (*), .01 (**), and .001 (***) for the particular experimental group compared to untreated control (C).Effect of 7-MEOTA-THA thioureas 12–22 was also determined on cultured human dermal fibroblasts by measuring MMP. As shown in Supplementary Figure S1, all the studied compounds were less toxic to non-cancer cells compared to cancer cells at given concentrations after 72 h treatment, which underlies their advantage in development of anticancer drugs.The cytotoxicity of 12–22 to impair cell proliferation, flow cytometry analysis of the cell cycle distribution was hence used. Based on the results from MMP dissipation and viability analyzes, HL-60 cells were treated with different concentrations of 12–17 (5–25 µM) and 18–22 (1–15 µM) for 24, 48 and 72 h. After 48 h incubation, results for urea derivatives 21 (15 µM) and 22 (10 µM) clearly demonstrated a significant accumulation of cells in G1 phase ). In comparison, thiourea analogs 14–17 revealed a concentration effect ). These compounds significantly altered cell cycle progression, inducing block of the cell cycle in S phase after just 24 h treatment at 25 µM concentration. THA and acridine were used as controls. Despite the relatively high concentration of THA, it did not exert any effect in this experiment ).In order to address the effect of compounds 12–22 was analyzed on the model of A549 adherent cancer cells and human dermal fibroblasts by a simple approach. After 24 h incubation with the derivatives, the cells were washed in order to dispose of unbound compounds, so that only compounds that were able to permeate into the cells could be visualized. The fluorescence of each sample was examined in each channel but images were captured using only two channels: (1) blue excitation range [BP excitation filter 450–490 nm (cube I3) and LP suppression filter 515 nm]—when heterodimers were present in the samples they exhibited positivity only in this channel; (0) brightfield mode was also applied in order to capture the cells themselves, visualizing single cells regardless of fluorescence of the studied compounds. According to the signal in channel 1 (I3 filter cube), compounds 14–17 (5 µM) showed abundant accumulation after 24 h incubation and a strong fluorescence signal in the A549 cells. All these heterodimers were visible as dark spots even in brightfield mode  whereas urea heterodimers 18, 19 did not (data not shown). It was also observed that ureas 20–22 accumulated neither in A549 cells nor in fibroblasts , it is important to consider whether they are also able to inhibit some of the cell nucleus enzymes involved in DNA operations, and which alter its topology through decatenation and relaxation of supercoiled DNA, as for example Topo I/II. Moreover, Topo I/II are also employed in other cell functions, such as replication, transcription, recombination and chromosomal segregationGiven that the studied 7-MEOTA-THA thio-/urea hybrids 12–22 on the catalytic activity of Topo I by measuring the Topo I-mediated relaxation of supercoiled plasmid pUC19. Due to significant and dose-dependent inhibition of Topo I catalytic activity all the compounds under study reduced the amount of relaxed DNA, while simultaneously increasing the amount of supercoiled DNA. 7-MEOTA-THA thioureas 12–17 inhibited the catalytic activity of Topo I at 60 µM . Compounds 14–17 were able to inhibit the catalytic relaxation ability of Topo I on pUC19 already at 5 µM, and hence further testing was carried out at concentrations of 1–10 µM . The most effective from this subset was compound 17 due to strong inhibition at 1 µM . Intriguingly, compound 22 was effective at 1 µM concentration , inhibit ATP binding (e.g. novobiocin), or stabilize the noncovalent DNA-Topo II complex . Topo II poisons are able to stabilize the covalent DNA-Topo II cleavage complex and are clinically used for their antitumor activity 12–22 to affect the decatenation of catenated kinetoplast DNA (kDNA) from the insect trypanosome Crithidia fasciculata by Topo II was examined . ATP-dependent decatenation assay is a specific assay for detection of compounds with potential Topo II inhibition activity. Topo II-targeted compounds are able to interact with (at least) one step of the topoisomerase catalytic cycle. The decatenation of kDNA by Topo II generates different products  which move easily into the agarose gel compared with the larger-sized catenated kDNA12–22 at 5 µM compared to 200 µM etoposide, 100 µM ellipticine and 100 µM mAMSA (Topo II poisons/inhibitors used as standards). All the compounds revealed the same pattern of inhibition of catalytic activity of Topo II at 50 and 100 µM concentrations. Vispe et al.The ability of mAMSA) are able to stabilize the DNA-Topo II complex and thus form linear DNA due to the simultaneous cleavage of both strands of double-stranded DNA. Plasmid DNA pHOT1 (the DNA substrate) used in the cleavage assay contains a single high affinity Topo II cleavage site12–22 . These results indicated that 12–22 are characterized with Topo II catalytic inhibition, but not as Topo II poisons. Topo II poisons were found to be important for activating secondary leukemias involving certain chromosomal translocations. On the other hand, Topo II catalytic inhibitors are able to change the cytotoxic effect of poisons and overcome multidrug resistance,,Topo II poisons . The observed changes suggested that studied compounds 12–22 are able to interact with ctDNA, although from the small bathochromic shifts we can rather point out to groove binding or electrostatic interaction, rather than intercalation. The DNA-binding characteristics of the studied 7-MEOTA-THA thio-/ureas 12–22 are summarized in K of the studied compounds in complex with ctDNA ranged between 0.5 and 8.0 × 106 M−1 and were comparable with the measured K of acridine and greater than the literature-cited K for acridin-3,6-diyl dithiourea hydrochlorides (2.9–7.6 × 105 M−1)5 M−1)5 M−1)4–106 M−1 and they are usually lower than binding constants typical for groove binders (range from 105 to 109 M−1).Several binding modes have been identified between native and/or synthetic double-stranded DNA (dsDNA) and low molecular-weight compounds acting as chemotherapeutic agents or gene regulators. The non-covalent DNA interactions are represented mainly by intercalation between the base pairs of dsDNA; and binding to minor/major grooves of dsDNA, stabilized by a mix of electrostatic, hydrophobic, hydrogen-bonding interactions or outside random bindingmT). DNA denaturation is usually measured spectrophotometrically at the temperature-dependent excitation point of 260 nm. In this work, mT of ctDNA was found to be 68 °C whereas in the presence of 7-MEOTA-THA thioureas 12–16 slightly increased (ΔmT was 1–2 °C). In the presence of 7-MEOTA-THA ureas 18–22, no increase of mT of ctDNA was observed, except for compound 22 which exhibited a profound increase of mT of more than 10 °C. Thermal characterization of the studied compounds 12–22 and the first derivative of ctDNA denaturation curves in the presence of the studied compounds are shown in Supplementary Figure S7. Usually, ligands which could bind to DNA through intercalation significantly increase mT of DNA, and Δ mT is typically around 5–8 °C, due to stabilization of dsDNA. Groove binding and electrostatic binding ligands produce mostly negligible effects on mT of DNAmT (0.9 °C) in melting temperature analysis with ctDNA for resmethrin characterizes this compound as a groove binder. Results from our study indicated that only compound 22 could possibly bind strongly to ctDNA in intercalative mode, but it is not excluded that 7-MEOTA thio-/ureas 12–21 are able to insert between the planar bases of ctDNA.DsDNA tertiary structure is stabilized via hydrogen bonds and base stacking interactions. When a solution of dsDNA is exposed to extreme heat, the double helix dissociates to the single-stranded form (known as denaturated DNA). This leads to disruption of intermolecular forces and hydrogen bonding interactions between the DNA base pairs12–22 with ctDNA was accomplished using steady-state fluorescence measurements. Fluorescence spectra of studied heterodimers at fixed concentration with increasing concentration of ctDNA showed a progressive quenching in the fluorescence intensity which was directly proportional to the ctDNA concentration. Representative results are displayed in 17 and 22) and suggest interaction between the compounds and ctDNA. Data from these fluorescence quenching experiments were also used for determination of SVKthat provides a measure of the quenching ability of ctDNA. The inserted graphs in 0/F versus [Q]. These plots are characterized by an intercept of one on the y-axis and the slopes are equal to SVK, as summarized in A more sensitive evaluation of the interaction of studied 7-MEOTA-THA thio-/ureas SVK (by increasing the temperature random collision is more probable). On the other hand, increased temperature goes hand in hand with decreased complex stability and thus the static quenching constant is lower,SVK values it is clear that ctDNA can quench fluorescence of the studied compounds as a static quencher. To further confirm the quenching process qk was calculated; this indicates the accessibility of a fluorophore to a quencher, or the efficiency of quenching. Calculated qk are listed in 11 M−1 s−1 at different temperatures. The limiting diffusion rate constant of biomolecules in the case of dynamic quenching was found to be 2.00 × 1010 M−1 s−1. However, there is no limit for static quenching,qk were higher than the limiting diffusion rate constant, it is suggested that the quenching process in this study was static rather than dynamic.Fluorescence quenching is observed in two processes, as dynamic or static quenching. Static quenching is based on the fact that the fluorophore and added quencher form a stable non-fluorescent complex in the ground state which promptly goes back to the ground state without photon emission after light absorption. For dynamic  quenching, quencher diffusion to the fluorophore is typical during the lifetime of an excited state and the rate of this quenching is dependent upon temperature, viscosity and diffusion. The decrease of fluorescence in the case of dynamic quenching is due to random collision between the fluorophore and quencherbK of the forming complex between 7-MEOTA-THA thio-/urea hybrids and ctDNA. The calculated bK and n are recorded in ΔH, ΔS and ΔG. Values of all thermodynamic parameters are summarized in ΔG are characteristic of a favorable and spontaneous binding reaction. Positive values of both ΔS and ΔH are typical for hydrophobic interaction, while negative values of both can be regarded as indicating van der Waals forces and hydrogen bonds. However, positive values of ΔH and negative values of ΔS describe electrostatic interaction between ionic species in an aqueous solution,12–22 and ctDNA was spontaneous due to negative ΔG values at all three temperatures, and the positive values of ΔH and ΔS showed that hydrophobic interactions play a central role in the binding of the studied 7-MEOTA-THA thio-/ureas 12–22 with ctDNA. Based on the negative ΔH and ΔS for 16 and 20, we can assume that interaction in these ligand-ctDNA complexes is mediated by van der Waals forces  can be deduced structure information about the relative conformation between the DNA axis and the ligand. Circular dichroism (CD) is another powerful tool to gain insight into the structural determinants characterizing such complexes. Whereas LD can be used to monitor the angles between DNA-base transition states relative to the axis orientation which are identical with the helix axis in flow or electric field-oriented DNA, the induced CD of the same transition could provide information about molecular orientation relative to the surrounding nucleobases//−A⊥, where A//and A⊥ are absorbance values measured with the light plane polarized parallel and perpendicular, respectively, to the oriented ctDNA in this study. The orientation of measured ctDNA in the absence and in the presence of the studied 7-MEOTA-THA thio-/ureas 12–22 was obtained by flow using a cuvette cell. Representative spectra are shown in 12–22 interact with DNA and become oriented after anchoring to DNA. The negative values of these LD signals are consistent with perpendicular orientations of the chromophoric molecules 12–22 relative to the DNA helix axis as expected for intercalative binding mode12–22 indicating either lengthening and/or an increase in rigidity of double-helical DNA, which can usually be related to unwinding due to intercalation,LD is defined as LD = ASupplementary data .All chemicals and reagents purchased were of reagent grade and used without further purification. The applied material and cell lines are listed in −1 cm−1. The purity of ctDNA was identified by monitoring the value A260/A280. Stock solutions of tested 7-MEOTA-THA thio-/ureas 12–22 were prepared in DMSO (stock concentration of samples was 30 mM). Further dilutions were prepared in the appropriate aqueous buffer.CtDNA was dissolved in TE buffer (Tris–EDTA) overnight at 4 °C to completely solubilize. The concentration of ctDNA was determined from its absorbance at 260 nm using extinction coefficient ε = 6600 M6 per well and changes in the total cell number, metabolic activity, MMP, cell cycle distribution and cell death were analyzed at 24, 48 and 72 h after treatment with the studied 7-MEOTA-THA thio-/ureas 12–22, as described previously,4) were seeded into a 12-well plate  and treated with the studied 7-MEOTA-THA thio-/ureas 12–22 for 72 h, and changes in MMP were analyzed.For the flow cytometry analysis, HL-60 cells were seeded in six-well plates  at 1 × 10Immunophenotype characterization of human fibroblasts. Isolated dermal fibroblasts were characterized using flow cytometry. After dissociation with trypsin–EDTA , the cells were washed twice with PBS  supplemented with 2% (v/v) FBS. Aliquots of 2.0 × 105 cells were incubated with mouse anti-human CD90/FITC, CD105/PE, CD73/APC, CD26/PE, and with a cocktail of hematopoietic markers: CD14-/CD20-/CD34-/CD45/PerCP  for 10 min, washed with 1–2 mL of PBS and centrifuged at 3000g for 10 min. The resuspended cell pellet was analyzed in a Becton Dickinson FACSCalibur using CellQuest software (Becton Dickinson).Cell cycle distribution. HL-60 cells were treated with various concentrations of the studied seven MEOTA-THA thio-/ureas  for 24, 48 and 72 h. Untreated cells were also included in this test for comparison. The method used to perform the experiment has been published previouslym).Analysis of MMP  for 24, 48 and 72 h (for fibroblasts only 72 h). The method used to perform the experiment has been published according to a previously described protocol,Analysis of metabolic activity and viability. Analysis of metabolic activity and viability of HL-60 cells was performed using fluorescein diacetate  and PI double-staining. HL-60 cells were treated with various concentrations of the studied 7-MEOTA-THA thio-/ureas  for 24, 48 and 72 h and subsequently harvested by centrifugation, washed with HBSS and stained with FDA (100 ng mL−1) in HBSS buffer for 20 min in darkness at RT. Prior to measurements, cells were stained with PI (1 µg cm−3) and analyzed using a BD FACSCalibur flow cytometer. Fluorescence of FDA was detected via a 530/30 nm band pass filter (FL-1) and PI via a 670 nm long-pass filter (FL-3). Results were analyzed using FlowJo software (TreeStar Inc.).t-test and results were considered significant if p<.05*, p<.01** and p<.001***.Results were presented as the mean ± SD (standard deviation) of at least three independent experiments. Statistical significance was determined by Student’s 3 per well) were seeded onto microscopy glass slides with mounted 12-well silicone chamber  and left to settle for 24 h. They were then treated with the studied 7-MEOTA-THA thio-/ureas 12–22 (5 µM) or with the medium alone (control group) for 24 h and washed with pre-warmed PBS in order to remove the unbound fraction. Intracellular accumulation of studied heterodimers in A549 cells was evaluated by fluorescence microscope Leica DMI6000B  using two channels as stated in Supplementary Table S1. Every sample was captured using the same settings for HCX PL APO CS 10.0 × 0.40 DRY UV objective. Results were analyzed using Leica Application Suite Lite (LAS AF Lite) software. Each presented image represents a single sample and consists of separated images from a channel and their mutual overlap.A549 cells (6 × 104 per well) were seeded into a 12-well plate, left to settle for 24 h and treated with the studied 7-MEOTA-THA thio-/ureas 12–22  or with the medium alone (control group). After 24 h of incubation with the studied derivatives, the cells were analyzed with an inverted fluorescence microscope Leica DMi3000B (Leica Microsystems).Fibroblasts . The gel was photographed under UV and gel images were obtained using a photogel documentation system .The influence of the studied 7-MEOTA-THA thio-/urea hybrids on the catalytic activity of Topo I was identified according to a previously described protocol12–22: 5, 50 and 100 µM) was incubated for 45 min at 37 °C. The method used to perform the experiment has been published previously,Freshly prepared 5× complete assay buffer for Topo II decatenation containing 0.16 µg catenated kinetoplast DNA (kDNA) and 2 U of human Topo IIα in the absence or presence of the studied 7-MEOTA-THA thio-/ureas (12–22: 10 and 100 µM) at 37 °C for 30 min. The method used to perform the experiment has been published previously,DNA cleavage reactions were carried out in a 20 µL final volume containing freshly prepared 5× complete assay buffer, 0.2 µg supercoiled pHOT-1 and 5U human Topo IIα. Reactions were incubated in the absence or in the presence of the studied 7-MEOTA-THA thio-/ureas (12–17: 49 µM) and ureas  in the absence or in the presence of an increasing concentration of ctDNA (0–1.24 µM) were recorded in the wavelength range 230–450 nm.UV–vis spectroscopic analyses were realized in 10 mM Tris–HCl buffer . UV–vis absorption titration spectra were measured on a Varian Cary 100 spectrophotometer in a 100-QS quartz cuvette with optic length 1 cm. UV–Vis spectra of newly developed 7-MEOTA-THA thioureas (b) types:f+Cb) and εf and εb are the appropriate extinction coefficients C, where CDNA is the total DNA concentrationK of the derivative-ctDNA complexes using a Scatchard plot in the form r/Cf , the McGhee and von Hippel equation (Equation (3)The absorbance A measured at any wavelength indicates both the free (Af) and DNA-bound . The temperature was increased at a rate of 1 °C min−1 over the range 40–90 °C. The absorbance was measured at 260 nm for ctDNA alone (concentration of ctDNA in measurements with 12–17: 93.2 µM and with 18–22: 0.32 µM) and for ctDNA in complex with the studied derivatives (concentration of 12–17: 37.5 µM and 18–22: 50 µM). The thermal melting points  were determined as the maxima of the first derivative plots of the melting curves.DNA melting temperature measurements were performed in a quartz cuvette (1 cm path length) on a Varian Cary 100 spectrophotometer equipped with a heating multiple cell block apparatus. Measurements were realized in BPE buffer  containing a constant concentration of the studied derivative (12–22: 5 µM), and to which were added equivalent increased amounts of ctDNA (0–280 µM). The steady state fluorescence measurements were performed at three different temperatures  for the evaluation of various thermodynamic parameters important in the detection of complex formation between DNA and 7-MEOTA-THA heterodimers.Fluorescence spectra of the studied 7-MEOTA-THA thio-/ureas were recorded in a quartz cuvette (1 cm path length) on a Varian Cary Eclipse spectrofluorimeter. The widths of both the excitation and emission slit were set at 10 nm. The excitation wavelengths were 340 nm for compounds SVK) by applying the equation (Equation (4)0 is the fluorescence intensity of the studied compound alone, F is the fluorescence intensity of the studied 7-MEOTA-THA thio-/urea in the presence of ctDNA as a quencher and [Q] is the molar concentration of ctDNA. To further confirm the quenching process were calculated biomolecular quenching constants kq using the equation  of the forming complex between 7-MEOTA-THA thio-/urea hybrids and ctDNA:b and n were calculated at three different temperatures from double logarithm regression curves of log(F0−F)/F versus log[Q] and they were obtained from intercept and slope, respectively.The modified Stern–Volmer equation (Equation (6)−1 mol−1. ΔH and ΔS were determined as the slope and intercept respectively of the van’t Hoff plot (log Kb against 1/T). The ΔG values at three different concentrations were calculated using equation (The van’t Hoff equation  was usedequation 96:(8)ΔG12–22: 0.3 mM) were recorded in 10 mM Tris–HCl buffer  in the wavelength range of 200–500 nm. The results are presented as the mean of at least three repeated measurements and obtained data were transferred to Grafit 7.0  for analysis.Spectral measurements of CD were realized using a J-810 Jasco spectropolarimeter in a quartz cuvette with optic length 1 mm. CD spectra of ctDNA (7.46 µM) in the absence or in the presence of the newly developed 7-MEOTA-THA thio-/urea hybrids (12–22: 0–0.1 mM) were recorded in 10 mM Tris–HCl buffer  in the wavelength range of 200–500 nm. The baseline (10 mM Tris–HCl) was also recorded for each measurement. The results are presented as the mean of at least three independent measurements and obtained data were transferred to Grafit 7.0 (Erithacus Software) for analysis.Flow LD spectra were measured using a Jasco J-720 spectropolarimeter in a flow Couette cell adapted for LD measurements. The flow cell includes a fixed outer cylinder and a solid rotating quartz inner cylinder. These two cylinders are separated with a 0.5 mm gap . LD spectra of ctDNA (310 µM) in the absence or in the presence of the newly synthesized 7-MEOTA-THA thio-/urea derivatives . Column chromatography was performed at normal pressure on silica gel 100 . Elemental analysis was measured on a Perkin-Elmer CHN Analyzer 2400 Series II apparatus . Mass spectra were recorded using combined high-performance liquid chromatography (HPLC) and mass spectrometry (MS). The HP1100 HPLC system was obtained from Agilent Technologies . It consisted of vacuum degasser G1322A, quaternary pump G1311A, autosampler G1313A and quadrupole mass spectrometer MSD1456 VL equipped with electrospray ionization source. Nitrogen for MS was supplied by a Whatman 75–720 nitrogen generator. Data were collected in positive ion mode with an ESI probe voltage of 4000 V. The pressure of nebulizer gas was set to 35 psig. Drying gas temperature was operated at 335 °C and flow at 13 L/min. 1H NMR and 13C NMR spectra were recorded on a Varian Mercury Plus 400 spectrometer operating at 400 and 100 MHz, respectively, in deuterochloroform  or hexadeuterodimethylsulfoxide  using tetramethylsilane (TMS) as internal reference (=0 ppm for both nuclei). Chemical shifts are reported in parts per million  relative to TMS. The assignment of chemical shifts is based on standard NMR experiments . Uncalibrated purity was ascertained by LC − UV using a reverse phase C18 chromatographic column. All of the biologically tested compounds exhibited purity 96 − 99% at wavelength 254 nm. Melting points were measured on a micro heating stage PHMK 05  and were uncorrected.7-MEOTA was prepared at the University of Defense  by the previously described method3–8 (1.7 mmol) and 11  were added to 20 ml of dichloromethane and stirred for 48 h at room temperature (RT). The mixture was concentrated under reduced pressure to give the crude product. Purification was performed by flash chromatography  to give 12–17 as a yellow solid. In a further step, the appropriate 7-MEOTA-THA product containing the thiourea moiety  was stirred in dry dichloromethane (25 ml) with 2,4,6-trimethylbenzonitrile N-oxide  for 48 h. The resulting mixture was subsequently evaporated under reduced pressure and purified by flash chromatography using CHCl3/MeOH (9:1) as eluent to yield 18–23 as white solids.Intermediates 1-ethyl)-3-thiourea12. Yellow solid : m.p.=142.1–143.2 °C; 1H NMR (CDCl3) δ 1.83 , 2.63 and 2.88 , 2.95 and 3.05 , 3.87 , 3.60 and 3.66 , 7.33 , 7.20 , 7.27 , 7.56 , 7.74 , 7.82 , 7.90 , 6.80 ; 13C NMR (CDCl3) δ 21.8, 22.0, 22.2, 22.4 , 24.6 and 25.3 , 32.2 and 33.6 , 42.5 and 46.8 , 55.7 (OCH3), 101.9 (C-8), 116.2 (C-9a), 119.6 (C-8a), 121.6 (C-6), 122.6 (C-8′′), 124.2 (C-8a′′), 126.5 (C-7′′), 128.8 , 129.1 (C-9a′′), 129.3 (C-6′′), 139.8 (C-9′′), 143.2 (C-10a), 147.2 (C-10a′′), 150.2 (C-9), 156.2 , 160.5 (C-4a′′), 181.5 (C = S); ESI-MS: m/z 512.2 [M]+ ; Anal. Calcd. for C30H33N5OS: C, 70.42; H, 6.50; N, 13.69; S, 6.27. Found: C, 70.37; H, 6.81; N, 13.78; S, 6.05.1-propyl)-3-thiourea13. Yellow solid : m.p.=106.3–107.5 °C; 1H NMR (CDCl3) δ 1.81 , 2.50 and 2.70 , 2.94 , 3.24 and 3.48 , 3.85 , 5.65 , 7.17 , 7.33 , 7.51 , 7.81 , 7.83 , 7.94 ; 13C NMR (CDCl3) δ 22.0, 22.2, 22.4, 22.6 , 24.8 and 25.3 , 30.6 (C-2′), 33.6 , 43.3 and 42.3 , 55.7 (OCH3), 101.5 (C-8), 116.4 (C-9a), 120.6 (C-8a), 121.6 (C-6), 122.6 (C-8′′), 124.4 (C-8a′′), 126.3 (C-7′′), 128.5 , 129.1 , 139.0 (C-9′′), 143.0 (C-10a), 147.0 (C-10a′′), 150.5 (C-9), 156.3 , 160.3 (C-4a′′), 181.5 (C = S); ESI-MS: m/z 526.2 [M]+ ; Anal. Calcd. for C31H35N5OS: C, 70.82; H, 6.71; N, 13.32; S, 6.10. Found: C, 70.65; H, 6.67; N, 13.45; S, 6.01.1-butyl)-3-thiourea14. Yellow solid : m.p.=95.6–96.4 °C; 1H NMR (CDCl3) δ 1.64 , 1.85 , 2.64 and 2.87 , 2.98 and 3.04 , 3.43 and 3.64 , 3.87 , 5.46 , 7.17 , 7.20 , 7.40 , 7.60 , 7.81 , 7.83 , 7.94 ; 13C NMR (CDCl3) δ 22.2, 22.4, 22.6, 22.8 , 24.7 and 25.3 , 26.5 and 28.6 , 32.8 and 33.8 , 44.8 and 48.1 , 55.6 (OCH3), 101.7 (C-8), 117.0 (C-9a), 120.7 (C-8a), 120.9 (C-6), 122.5 (C-8′′), 124.1 (C-8a′′), 126.7 (C-7′′), 128.8 , 129.1 (C-9a′′), 129.4 (C-6′′), 139.0 (C-9′′), 142.5 (C-10a), 147.3 (C-10a′′), 150.3 (C-9), 156.2 , 160.6 (C-4a′′), 181.4 (C=S); ESI-MS: m/z 540.2 [M]+ ; Anal. Calcd. for C32H37N5OS: C, 71.21; H, 6.91; N, 12.98; S, 5.94. Found: C, 71.19; H, 6.55; N, 13.26; S, 5.80.1-pentyl)-3-thiourea15. Yellow solid : m.p.=93.2–94.1 °C; 1H NMR (CDCl3) δ 1.43 , 1.59 and 1.71 , 1.83 , 2.63 , 2.95 , 3.57 , 3.87 , 7.18 , 7.27 , 7.33 , 7.54 , 7.82 , 7.90 ; 13C NMR (CDCl3) δ 21.8, 22.2, 22.5, , 24.0 (C-3′), 24.6 and 25.3 , 28.6 and 30.9 , 31.4 and 33.8 , 44.8 and 48.3 , 55.7 (OCH3), 102.3 (C-8), 117.0 (C-9a), 119.6 (C-8a), 121.6 (C-6), 122.6 (C-8′′), 124.3 (C-8a′′), 126.4 (C-7′′), 128.6 and 128.7 , 129.2 , 139.8 (C-9′′), 143.5 (C-10a), 147.1 (C-10a′′), 151.8 (C-9), 156.3 , 160.4 (C-4a′′), 181.5 (C = S); ESI-MS: m/z 554.3 [M]+ ; Anal. Calcd. for C33H39N5OS: C, 71.57; H, 7.10; N, 12.65; S, 5.79. Found: C, 71.23; H, 6.95; N, 12.88; S, 5.93.1-hexyl)-3-thiourea16. Yellow solid : m.p.=74.2–74.8 °C; 1H NMR (CDCl3) δ 1.25 and 1.36 , 1.50 and 1.61 , 1.89 , 2.65 and 2.88 , 2.97 and 3.07 , 3.44 and 3.58 , 3.88 , 7.20 , 7.27 , 7.41 , 7.59 , 7.82 , 7.94 ; 13C NMR (CDCl3) δ 22.2, 22.3, 22.5, 22.7 , 24.6 and 25.3 , 26.3 and 26.4 , 28.8 and 31.4 , 32.6 and 33.8 , 45.1 and 48.6 , 55.5 (OCH3), 102.0 (C-8), 116.1 (C-9a), 120.4 (C-8a), 120.8 (C-6), 122.5 (C-8′′),124.2 (C-8a′′), 126.5 (C-7′′), 128.3 (C-5), 128.7 (C-5′′), 129.0 (C-9a′′), 129.3 (C-6′′), 138.2 (C-9′′), 142.0 (C-10a), 147.3 (C-10a′′), 150.7 (C-9), 156.0 (C-7), 154.8 (C-4a), 160.5 (C-4a′′), 181.2 (C = S); ESI-MS: m/z 568.3 [M]+ ; Anal. Calcd. for C33H39N5OS: C, 71.57; H, 7.10; N, 12.65; S, 5.79. Found: C, 71.23; H, 6.95; N, 12.88; S, 5.93.1-heptyl)-3-thiourea17. Yellow solid : m.p.=80.8–81.9 °C; 1H NMR (CDCl3) δ 1.32 , 1.43 and 1.63 , 1.85 , 2.64 and 2.90 , 2.97 and 3.10 , 3.52 , 3.88 , 7.20 , 7.27 , 7.40 , 7.58 , 7.84 , 7.94 ; 13C NMR (CDCl3) δ 22.0, 22.2, 22.3, 22.6 , 24.5 and 25.3 , 26.5 (C-4′), 28.6 and 28.7 , 30.3 and 31.3 , 31.7 and 33.9 , 45.2 and 48.6 , 55.6 (OCH3), 102.3 (C-8), 115.5 (C-9a), 120.0 (C-8a), 121.4 (C-6), 122.6 (C-8′′), 124.8 (C-8a′′), 126.4 (C-7′′), 128.7 , 129.2 , 139.7 (C-9′′), 145.2 (C-10a), 147.3 (C-10a′′), 151.3 (C-9), 156.2 , 160.5 (C-4a′′), 181.3 (C = S); ESI-MS: m/z 582.3 [M]+ ; Anal. Calcd. for C35H43N5OS: C, 72.25; H, 7.45; N, 12.04; S, 5.51. Found: C, 72.43; H, 7.58; N, 12.12; S, 5.43.1-ethyl)-3-urea18. White solid : m.p.=103.7–104.8 °C; 1H NMR (CDCl3) δ1.70 , 2.66 and 2.81 , 3.0 , 3.66 and 3.93 , 3.85 , 7.20 , 7.26 , 7.31 , 7.42 , 7.83 , 7.91 , 8.0 , 9.05 ; 13C NMR (CDCl3) δ 22.2, 22.4, 22.5, 22.6 , 25.4 , 28.4 and 33.8 , 40.1 and 51.5 , 55.2 (OCH3), 102.1 (C-8), 116.5 (C-9a), 119.8 (C-8a), 121.1 (C-6), 123.2 (C-8′′), 125.3 (C-7′′), 124.5 (C-8a′′), 127.2 (C-5), 128.0 (C-5′′), 128.3 (C-6′′), 128.7 (C-9a′′), 139.9 (C-9′′), 146.6 , 150.0 (C-9), 155.4 (C-4a), 156.3 (C-7), 157.1 (C=O), 159.5 (C-4a′′); ESI-MS: m/z 496.2 [M]+ ; Anal. Calcd. for C30H33N5O2: C, 72.70; H, 6.71; N, 14.13. Found: C, 72.43; H, 6.35; N, 13.69.1-propyl)-3-urea19. White solid : m.p.=104.5–105.4 °C; 1H NMR (CDCl3) δ 1.72 , 2.56 and 2.77 , 2.84 and 2.95 , 3.34 and 3.50 , 3.85 , 6.01 , 7.20 , 7.26 , 7.30 , 7.43 , 7.70 , 7.82 , 8.35 ; 13C NMR (CDCl3) δ 22.4, 22.6 , 25.3 , 31.2 (C-2′), 32.1 and 33.8 , 36.7 and 44.0 , 55.4 (OCH3), 101.5 (C-8), 114.6 (C-9a), 119.9 (C-8a), 122.0 (C-6), 122.9 (C-8′′), 124.6 (C-8a′′), 125.3 (C-7′′), 127.3 (C-5), 128.2 (C-5′′), 128.4 , 139.8 (C-9′′), 146.8 , 152.1 (C-9), 153.3 (C-4a), 156.1 (C-7), 157.6 (C=O), 159.7 (C-4a′′); ESI-MS: m/z 510.2 [M]+ ; Anal. Calcd. for C31H35N5O2: C, 73.06; H, 6.92; N, 13.74. Found: C, 73.42; H, 6.86; N, 13.95.1-butyl)-3-urea20. White solid : m.p.=69.2–69.8 °C; 1H NMR (CDCl3) δ 1.70 , 1.80 , 2.57 and 2.80 , 2.90 and 3.0 , 3.40 and 3.50 , 3.85 , 4.50 , 6.15 , 7.20 , 7.26 , 7.30 , 7.43 , 7.75 , 7.84 ; 13C NMR (CDCl3) δ 22.1, 22.4, 22.5, 22.6 , 24.6 and 25.3 , 27.5 and 28.5 , 32.0 and 33.8 , 39.5 and 48.1 , 55.5 (OCH3), 102.1 (C-8), 115.6 (C-9a), 120.1 (C-8a), 121.1 (C-6), 122.7 (C-8′′), 124.4 (C-8a′′), 125.4 (C-7′′), 127.4 (C-9a′′), 128.2 (C-5), 128.5 , 139.5 (C-9′′), 146.8 , 151.1 (C-9), 154.2 (C-4a), 156.1 (C-7), 156.4 (C=O), 159.7 (C-4a′′); ESI-MS: m/z 524.3 [M]+ ; Anal. Calcd for C32H37N5O2: C, 73.39; H, 7.12; N, 13.37. Found: C, 73.68; H, 7.34; N, 13.48.1-pentyl)-3-urea21. White solid : m.p.=114.2–114.9 °C; 1H NMR (CDCl3) δ 1.43 , 1.55 and 1.66 , 1.80 , 2.56 and 2.73 , 2.98 and 3.02 , 3.30 , 3.85 , 5.62 , 7.20 , 7.26 , 7.27 , 7.43 , 7.67 , 7.77 , 7.82 , 7.84 ; 13C NMR (CDCl3) δ 22.9, 23.1 , 24.8 (C-3′), 24.7 and 25.4 , 29.0 and 33.4 , 30.7 and 31.2 , 40.4 and 48.5 , 56.4 (OCH3), 102.2 (C-8), 115.7 (C-9a), 120.3 (C-8a), 121.0 (C-6), 122.8 (C-8′′), 124.5 (C-8a′′), 125.3 (C-7′′), 127.3 (C-5), 127.8 (C-9a′′), 128.2 (C-5′′), 128.5 (C-6′′), 139.2 (C-9′′), 143.5 (C-10a), 145.8 (C-10a′′), 150.5 (C-9), 154.9 (C-4a), 156.1 (C-7), 156.3 (C=O), 160.1 (C-4a′′); ESI-MS: m/z 538.3 [M]+ ; Anal. Calcd for C33H39N5O2: C, 73.71; H, 7.31; N, 13.02. Found: C, 73.39; H, 7.35; N, 13.33.1-hexyl)-3-urea22. White solid : m.p.=91.4–92.1 °C; 1H NMR (CDCl3) δ 1.32 , 1.57 , 1.80 , 2.57 and 2.77 , 2.90 and 2.97 , 3.10 and 3.40 , 3.85 , 4.52 , 5.82 , 7.15 , 7.23 , 7.43 , 7.68 , 7.74 , 7.82 ; 13C-NMR (CDCl3) δ 22.1, 22.4, 22.7 , 24.6 and 25.3 , 26.2 , 29.9 and 31.3 , 32.1 and 33.8 , 39.8 and 48.2 , 55.6 (OCH3), 102.2 (C-8), 115.5 (C-9a), 120.1 (C-8a), 121.1 (C-6), 122.8 (C-8′′), 124.5 (C-8a′′), 125.4 (C-7′′), 127.4 (C-9a′′), 127.5 (C-5), 128.2 (C-5′′), 128.5 (C-6′′), 139.6 (C-9′′), 140.5 (C-10a), 146.8 (C-10a′′), 151.2 (C-9), 154.2 (C-4a), 156.1 (C-7), 156.4 (C=O), 159.7 (C-4a′′); ESI-MS: m/z 552.3 [M]+ ; Anal. Calcd for C34H41N5O2: C, 74.02; H, 7.49; N, 12.69. Found: C, 74.48; H, 7.15; N, 12.88.1-heptyl)-3-urea23. White solid : m.p.=66.4–67.4 °C; 1H NMR (CDCl3) δ 1.25 , 1.58 and 1.74 , 1.80 , 2.62 and 2.77 , 2.93 and 3.0 , 3.08 , 3.41 , 3.86 , 5.62 , 7.18 , 7.26 , 7.46 , 7.57 , 7.77 , 7.83 ; 13C NMR (CDCl3) δ 22.3, 22.4, 22.6, 22.7 , 24.5 and 25.3 , 26.6 (C-4′), 28.6 and 28.8 , 30.3 and 31.3 , 32.4 and 33.8 , 40.0 and 48.6 , 55.5 (OCH3), 102.1 (C-8), 115.8 (C-9a), 120.3 (C-8a), 120.9 (C-6), 122.8 (C-8′′), 124.5 (C-8a′′), 125.4 (C-7′′), 127.4 (C-5), 128.1 (C-9a′′), 128.5 , 139.5 (C-9′′), 141.1 (C-10a), 146.8 (C-10a′′), 150.9 (C-9), 154.6 (C-4a), 156.0 (C-7), 156.3 (C=O), 159.7 (C-4a′′); ESI-MS: m/z 566.3 [M]+ ; Anal. Calcd for C35H43N5O2: C, 74.30; H, 7.66; N, 12.38. Found: C, 74.55; H, 7.95; N, 12.36.12–22 combining the two fragments via a linker of varying length and containing either urea or thiourea moieties. In conclusion, extensive comparative biochemical, biophysical and biological measurements consistently indicated that 12–22 acted as DNA-binders and inhibitors of Topo I/II, and were able to affect the MMP and viability of HL-60 cells and suppress selectively cancer cell proliferation.In this study, we developed novel 7-MEOTA-THA heterodimers 6 M−1. The obtained K for the heterodimers complexed with ctDNA were higher than K for THA (K = 3.8 × 104 M−1) and 7-MEOTA (K = 7.9 × 104 M−1) complexed with ctDNA, and moreover were comparable with K for the acridine–ctDNA complex (K = 4.5 × 106 M−1). Data from fluorescence quenching experiments clearly demonstrated that ctDNA can quench fluorescence of the studied heterodimers, by static quenching. The latter correlates well with decreasing KSV values and increasing temperatures and was further confirmed by obtained values of kq (4.77–10.69 × 1011 M−1 s−1) which were higher than the limiting diffusion rate constant for dynamic quenching. Thermodynamic parameters  indicated that hydrophobic interactions play the key role in the binding of novel compounds to ctDNA, except in the case of heterodimers 16 and 20 which revealed a different pattern of negative ΔH and ΔS, pointing to van der Waals associations with ctDNA. The existence of LD signals also demonstrated that 12–22 interact with DNA and become oriented after attachment to DNA.In summary, spectroscopic data proved that linking of THA and 7-MEOTA with an alkyl linker containing thio-/urea moieties positively influenced the DNA interaction power. From UV–vis absorption spectroscopic titration were calculated DNA-binding constants K = 0.5–8.0 × 1017 and urea hybrid 22 can be highlighted as the most potent. The studied heterodimers 14–17 and 21, 22 were able to evoke MMP dissipation after 24 h treatment and caused cell death with an efficiency between 80 and 100%. In addition, thioureas 14–17 significantly altered cell cycle progression and induced block of the cell cycle in S phase, and ureas 21, 22 demonstrated an increase of cells in G1 phase.Based on all the obtained biological positive outcomes, 7-MEOTA-THA thio-/ureas can be considered as interesting and potential drug candidates as anticancer agents. This assertion is supported by the fact that they are able to affect cancer cell lines, whereas they remained unattached in healthy non-cancer fibroblasts at the tested concentration. The more effective heterodimers are those which contain longer alkyl chains. The higher antiproliferative effect of the more lipophilic agents containing either pentyl or hexyl chains could be ascribed to better cell membrane penetration. In this regard, thiourea hybrid We believe that our study can contribute towards further development of novel, more potent and highly selective antiproliferative agents with intercalating properties.


Adult-onset Still’s disease (AOSD) is a rare inflammatory condition characterized by fever, rash, and arthritis. Because of its rarity, clinical trials are inherently small and often uncontrolled. Our objective was to develop recommendations for the use of interleukin (IL)-1 inhibitors in the management of patients with AOSD, based on the best evidence and expert opinion.A panel of 10 experts (9 rheumatologists and 1 pediatrician) was established. The first step was dedicated to a comprehensive literature review and development of statements. Two separate literature searches were performed on the MEDLINE (Pubmed), EMBASE, and BIOSIS databases through April 2018 to identify (1) differences and similarities between AOSD and pediatric Still’s disease (systemic juvenile idiopathic arthritis [SJIA]) and (2) the efficacy and safety of IL-1 inhibitors in AOSD treatment. In the second step, the statements were submitted in a Delphi process to a panel of 67 rheumatologists. Consensus threshold was set at 66%: positive, > 66% of voters selected scores 3 to 5; negative, > 66% of voters selected scores 1 or 2. In the third step, the voting results were analyzed, and the statements were finalized.Eleven statements were developed. Forty-six of 67 rheumatologists (72%) participated in the Delphi process. A positive consensus was reached after the first round of voting and was full (> 95%) on the majority of statements. A large consensus was achieved in considering AOSD and SJIA as the same disease. The use of anti-IL-1 therapies in refractory patients was considered quite safe and effective both as the first and as a subsequent line of biologic treatment, especially in systemic patients. Because of the lack of head-to-head comparisons, a different profile of efficacy among IL-1 inhibitors could not be established. There was a large consensus that failure of the first IL-1 inhibitor does not preclude response to another one. The lack of studies comparing early versus late treatment did not allow to draw conclusions; however, data from SJIA suggest a better response in early treatment.The Delphi method was used to develop recommendations that we hope will help clinicians in the management of patients with AOSD refractory to conventional therapies. Still’s disease is a rare systemic inflammatory disorder that can develop both in adults and children and is characterized by a wide spectrum of manifestations, including a typical triad of symptoms: daily spiking fever, polyarthritis, and a characteristic salmon-colored skin rash . Still’sThe pathophysiology of Still’s disease is largely unknown, but recent advances have identified interleukin (IL)-1β as a crucial inflammatory mediator , 11. BotTo date, there are no guidelines for the management of Still’s disease, and the treatment of this condition is mostly empirical and challenging for many clinicians. Because of the rarity of the disease, very few clinical studies are available. Specifically, there is only one randomized controlled trial (RCT) on the use of anakinra in patients with AOSD , and datA panel composed of 9 Italian rheumatologists and 1 pediatrician expert in pediatric rheumatology (scientific board of the project) were chosen based on their clinical experience and expertise in the treatment of Still’s disease  comprehensive literature review and writing of statements; (ii) evaluation of statements by a panel of rheumatologists, who were asked to express their agreement or disagreement on each statement; and (iii) assessment of voting results and finalization of the statements.The first step involved two meetings. The first meeting was held in Milan on April 3, 2018, and was attended by the scientific board and by 4 additional rheumatologists , who were responsible for the search and selection of the literature (bibliographic board). The aim of this meeting was to design the strategy of the literature search. During the second meeting, held on September 10, 2018, the bibliographic board presented the results of the literature search. Based on the results of the literature review, 11 statements were drafted. The level of evidence (range 1–5) and the grade of recommendation (range A–D) were assigned to each statement according to the Oxford Centre for Evidence-Based Medicine .In the second step of the consensus process (the Delphi portion), the statements were circulated electronically to a panel of 67 rheumatologists from 31 Italian rheumatology centers; since AOSD is a rare disease requiring specialist management, these rheumatologists were all members of the Working Group of Systemic Autoinflammatory Diseases of the Società Italiana Reumatologia  and were from all areas of Italy . No start date for the literature search was defined, but the end date was April 20, 2018.First, a systematic literature review was performed to identify reports describing the similarities and differences of SJIA and AOSD. A search strategy was developed using Mesh (or Emtree) terms and free-text words identifying AOSD and SJIA  efficacy and safety of IL-1 inhibitors in general, (2) comparative efficacy and safety of different IL-1 inhibitors, (3) comparison of early versus late treatment with IL-1 inhibitors, and (4) relative efficacy of IL-1 inhibitors in patients with a systemic versus chronic articular pattern of AOSD. Each clinical question was phrased according to the Patient, Intervention, Control, Outcomes (PICO) strategy for study selection. The main database searches were complemented by a manual search of proceedings from the European League Against Rheumatism (EULAR) and American College of Rheumatology (ACR) congresses of the previous 2 years (2016 and 2017) and also by scrutinizing the reference lists of included articles. A search strategy was developed using Mesh (or Emtree) terms and free-text words identifying AOSD, along with potential terms for anti-IL-1 agents  are shown in Table A total of 49 rheumatologists out of the 67 invited by the scientific board participated in the Delphi process (72% participation rate). The threshold for positive consensus (> 66% agreement) was reached on each statement during the first round of voting, and no additional Delphi rounds were required. Consensus exceeded 95% for the majority of statements.Statement 1.1. Adult Onset Still’s Disease (AOSD) and Systemic Juvenile Idiopathic Arthritis (SJIA) show substantial similarities in terms of clinical manifestations [3b], laboratory features [3b], response to treatment [3b] and, possibly, genetic background . . [Grade B]Statement 2.2. In AOSD, treatment with IL-1 Inhibitors is effective on different clinical and laboratory parameters and displays a significant steroid-sparing effect in most patients [2a]. Therapeutic response is rapid and sustained over time. [Grade B]Statement 2.3. IL-1 inhibitors are effective in the treatment of AOSD-related Macrophage Activation Syndrome (MAS) , althougStatement 2.4. IL-1 Inhibitors have an overall satisfactory safety profile in AOSD [2b]. Among adverse events, infections have been reported [2b]. Treatment with anakinra has been associated with frequent injection-site reactions [2b] and occasionally severe cases of hepatotoxicity, reversible after treatment withdrawal . . [Grade B]Early use (soon after disease onset) of IL-1 inhibitors in AOSD has not been explored yet. Direct comparisons of early versus late initiation of treatment with IL-1 inhibitors are also not available. The evidence from studies in SJIA suggests that early treatment with anti-IL-1 therapy is associated with a better response and that IL-1 inhibitors may be a valuable option in the first-line disease setting , 143–145Statement 5.1. Some data suggest that IL-1 inhibitors may be more effective on systemic rather than chronic articular manifestations of AOSD [2b]. [Grade C]n = 15) compared to 38.4% of patients with the chronic articular pattern (n = 13) [n = 12) compared to 37.5% of patients with articular disease (n = 8) [To date, there is no study specifically aimed at comparing the effects of anti-IL-1 therapy on systemic versus articular manifestations of AOSD. However, indirect evidence suggests that anti-IL-1 agents may be more effective at relieving systemic disease , 70, 77.(n = 13) . A much  (n = 8) , 146–148A large body of evidence supports the similarity between AOSD and SJIA. Furthermore, both conditions respond to agents that inhibit IL-1β by distinct mechanisms, which confirm the pivotal role of IL-1β in the pathogenesis of these conditions.The likely differences in pharmacokinetics and pharmacogenetics between AOSD and SJIA do not allow us to extrapolate information on the effectiveness and safety profile of IL-1 inhibitors from SJIA to AOSD. This is the main reason why we decided to perform this systematic review of the literature specifically on AOSD. The aim of this study was to clearly collect and summarize all of the available evidence supporting the use of IL-1 inhibitors specifically in patients with AOSD, for which information is scarce compared with SJIA. While studies on the pharmacokinetics of anakinra  or canakMost of the data on the efficacy and safety of IL-1 blockers in AOSD describe the use of anakinra, and the evidence from RCTs is limited to one small study investigating this medication . NeverthWe used a modified Delphi process to develop the reported consensus statements. This technique has the advantage of being able to systematically synthesize the knowledge and opinions of a large group of individuals with diverse expertise and from different geographic locations, thereby limiting the potential for a small group of experts from one area to dominate  Appendi. By collmaking the best use of available information [Our study is not without limitations. We set a relatively low threshold for consensus (> 66%) , althougormation . Howeverormation , while dormation , 76, 77.ormation .Although the evidence of the overall efficacy of anti-IL1 agents in patients with AOSD, especially in those with the systemic form of the disease, is quite convincing, the effectiveness of these medications on articular involvement should be further explored. For this reason, the results of the ongoing RCT with canakinumab (NCT02204293) in patients with AOSD and active joint involvement are eagerly awaited.Data on anakinra in patients with AOSD are mainly based on retrospective observational studies. Following the only existing RCT of anakinra in patients with AOSD, the results of which were published in 2012 ; a new RAlthough adequately powered RCTs on the use of IL-1 inhibitors in AOSD are lacking, the literature is consistent in demonstrating a beneficial effect of these agents, with a high proportion of patients achieving rapid and sustained remission of systemic symptoms and normalization of inflammatory markers. Given the absence of a strong evidence base for the use of IL-1 inhibitors in AOSD, a robust and careful Delphi process was undertaken to develop consensus recommendations on the use of these agents, which we hope will assist physicians in the rational prescribing of these agents for patients affected by this condition.Additional file 1: Table S1. Search strategy† to identify articles describing similarities and differences between SJIA and AOSD. Table S2. Search strategy† to identify articles describing the efficacy and safety of IL-1 blockade in AOSD. Table S3. Case reports (with ≤5 patients) describing the use of anti-IL-1 therapy in patients with AOSD.


Fecal incontinence is a socially and emotionally destructive condition that has a negative impact on personal image, self-confidence, and quality of life. Acupuncture is commonly used to treat chronic conditions, including fecal incontinence. However, no relevant systematic review or meta-analysis has been designed to evaluate the effects of acupuncture on fecal incontinence.We will identify relevant randomized controlled trials (RCTs) from the Cochrane Library, Medline, Embase, PubMed, Springer, Web of Science, China National Knowledge Infrastructure, VIP Chinese Science and Technology Journals Database, Wanfang database, and clinical trial registration center from their inception to February 28, 2019. The primary outcome measures will be clinical effective rate, functional outcomes, and quality of life. Data that meets the inclusion criteria will be extracted and analyzed using RevMan V.5.3 software. Two reviewers will evaluate the studies using the Cochrane Collaboration risk of bias tool. Publication bias will be assessed by funnel plots, Egger test, and Begg test using the Stata software. Acupoints characteristics will be analyzed by Traditional Chinese Medicine inheritance support system.This study will analyze the clinical effective rate, functional outcomes, quality of life, daily average number of fecal incontinence, and effective prescriptions of acupuncture for patients with fecal incontinence.Our findings will provide evidence for the effectiveness and potential treatment prescriptions of acupuncture for patients with fecal incontinence.PROSPERO CRD42019119680. It has been confirmed in numerous studies that fecal incontinence affects personal image and self-confidence, and even leads to social isolation, resulting in a significant impact on quality of life.,4 Previous studies have also shown that obstetric trauma and irritable bowel syndrome are common risk factors for fecal incontinence.,6 Currently, management or treatment measures for fecal incontinence include bowel routines, changing dietary habits, biofeedback training, electrostimulation, and surgical interventions, as well as supportive care measures such as pads, deodorants, and protective ointments.,8 However, only 46% of patients who underwent surgery for prolapse reported continence improvement at 38.9 months of follow-up, while those who underwent an operative procedure other than ventral rectopexy were similar to nonoperated patients. Therefore, a growing number of people are seeking conservative therapies, including dietary changes, antidiarrheal medications, bulking agents, enemas, and biofeedback,,11 as well as complementary and alternative therapy.Fecal incontinence is characterized by the impaired ability to control stool or gas to allow evacuation at a socially acceptable time and location, Compared with sham acupuncture, acupuncture at the lumbosacral region is effective for women suffering from stress urinary incontinence; the similar but slightly different clinical outcomes can be seen in nerve stimulation, which may also have an impact on functional outcomes or quality of life of patients with fecal incontinence. In recent years, the number of clinical trials involving acupuncture treatment of fecal incontinence has increased significantly,17; however, the clinical efficacy and potential treatment prescriptions of acupuncture for fecal incontinence remain unclear, prompting us to further explore its efficacy and effective prescription. In this study, we will investigate current evidence associated with the effectiveness and safety of acupuncture for fecal incontinence, which will help clinicians use it in clinical practice better.As a kind of complementary and alternative therapy, acupuncture is often used to treat chronic functional diseases, and its application in fecal incontinence has gradually spread in recent years.22.1We will collect articles of randomized controlled trials (RCTs) that evaluated the clinical effective rate, functional outcomes, quality of life, daily average number of fecal incontinence, or side effects of acupuncture on fecal incontinence for systematic review and meta-analysis. RCTs, observational studies, case series, and case–control studies will be included for data mining. Observational studies, case series, case–control studies, animal experiments, qualitative studies, proceedings, conferences, comments, and review articles will be excluded in the meta-analysis. There will be no restrictions regarding the race, region of the studies, gender, and age of patients.2.2 Participants with urinary and fecal incontinence will be excluded.Participants who are diagnosed with fecal incontinence will be included. Fecal incontinence should be confirmed according to the 1999 consensus standard of the American Association of Colorectal Surgeons.2.3Participants in the intervention group are those undergoing acupuncture, including electroacupuncture therapy, acupuncture alone, laser acupuncture, and warm acupuncture. There will not be any restrictions on age and original countries of the participants. Control group intervention could be no treatment, sham or placebo acupuncture, electrical stimulation, etc. The other treatments between intervention group and control group should be the same.2.4 The criteria of quality of life according to the Fecal Incontinence Quality of Life Scale was developed by Rockwood et al, including thoughts about lifestyle, depression/self-perception, coping/behavior, and embarrassment. The Fecal Incontinence Quality of Life Scale measures impairment caused by fecal incontinence or fear of fecal incontinence, ranging from 1 (most of the time) to 4 (none of the time).The primary outcomes will include clinical effective rate, functional outcomes, quality of life. Functional outcomes will be calculated on the basis of the Wexner score.The secondary outcomes will be daily average number of fecal incontinence, adverse events, and discontinuations due to adverse events.2.5www.controlled-trials.com; Clinical Trials: www.ClinicalTrials.gov; and Chinese Clinical Trial Registry: www.chictr.org.cn/index.aspx.An electronic search will be conducted. We will identify relevant studies from The Cochrane Library, Medline (Ovid), Embase, PubMed, Springer, Web of Science, China National Knowledge Infrastructure, VIP Chinese Science and Technology Journals Database, Wanfang database from their inception to February 28, 2019. The search term will consist of 3 parts: intervention method, disease, and study type: (“electroacupuncture therapy” or “acupuncture” or “laser acupuncture” or “warm acupuncture” or “electroacupuncture” or “acupuncture therapy” or “laser acupuncture therapy” or “warm acupuncture therapy”) and  and  and (“blind”). The details of the PubMed and Wanfang database search strategies are provided in Tables 2.6http://www.inoteexpress.com/aegean/). Duplicate citations from different databases will be identified and excluded. Two reviewers (Lin HX and Hu GJ) will independently screen the titles and abstracts of citations to delete irrelevant studies, and then examine the full-text reports, identify potential studies, and assess eligibility of studies for inclusion. Any disagreement will be resolved on the eligibility of studies through discussion and consensus or, if necessary, with the help of a third partner (Chen YJ). We will record details of the excluded studies and the reasons for exclusion. The selection process will be showed in a PRISMA flow chart (http://www.prisma-statement.org/)  with experience in the field will guide the search. All retrieved results will be imported into NoteExpress 3.2.0 (Available at: g/) Fig. . Two rev2.7We will obtain the missing data or additional information by contacting the study authors via email or telephone if possible. Otherwise, we will analyze the available information and conduct sensitivity analysis to explore the potential impact of insufficient information on the results of the meta-analysis.2.8Two review authors  will independently evaluate each included study and will follow the domain-based evaluation as developed by the Cochrane Handbook for Systematic Reviews of Interventions. They will assess the following domains: Randomization sequence generation, allocation concealment, blinding of outcome assessors, incomplete outcome data, selective outcome reporting, other sources of bias. Each domain will be divided into 3 categories: low, unclear, and high.2.9https://community.cochrane.org/help/tools-and-software/revman-5) provided by The Cochrane Collaboration. A meta-analysis using random or fixed effects models will be conducted to summarize the data. Continuous data will be pooled and presented as mean differences or standardized mean difference with their 95% CI. Dichotomous data will be pooled and expressed as risk ratio with their 95% CI. Statistical heterogeneity will be investigated using the Chi-square test and I2 values. We will interpret it using the following criteria: I2 values of 25% are considered low levels of heterogeneity, 50% indicated moderate levels, and 75% indicated high levels. As low or moderate heterogeneity suggests little variability among these studies, the data will be analyzed in a fixed-effects model. When significant heterogeneity occurs among the studies , a random-effect model will be performed to analyze the data, as well as meta-regression will be adopted to investigate the potential sources of heterogeneity.Effect sizes with their 95% confidence intervals (95% CIs) will be extracted. We will analyze the data using RevMan software (Version 5.3) .3 It is a socially and emotionally destructive disease that has a negative impact on quality of life of suffers. In the United States, the 4-year incidence of fecal incontinence among the community residents over 65 years old is 17%. And the prevalence increases with age, affecting 16.16% of adults over the age of 70. At the same time, previous studies have confirmed that advancing age, pelvic floor dysfunction, parity, stool form disturbance, urinary incontinence, and multiple chronic diseases are potential pathophysiological mechanisms of fecal incontinence.–32 At present, the treatment of fecal incontinence vary from medical methods, including biofeedback to various surgical interventions, but the results are still unsatisfactory.,34 A number of patients with fecal incontinence attempt to use complementary and alternative therapy, including acupuncture.Fecal incontinence is an embarrassing condition involving unintentional loss of solid or liquid feces.,35 It is confirmed that acupuncture could relieve pain and regulate peripheral circulation, which is beneficial for bladder dysfunction and even gastrointestinal dysfunction. Besides, electroacupuncture stimulation combined with bone marrow mesenchymal stem cell transplantation can effectively repair the impaired anal sphincters. Therefore, acupuncture is gradually being applied to the treatment of fecal incontinence. To the best of our knowledge, even though acupuncture is often used for fecal incontinence, there is no planned or published systematic review of the effectiveness and safety of acupuncture for fecal incontinence. The purpose of this study is to evaluate the effect of acupuncture on clinical effective rate, functional outcomes, quality of life, daily average number of fecal incontinence, adverse events, and discontinuations events in patients with fecal incontinence. In particular, we will analyze specific acupuncture prescriptions that are used in fecal incontinence by Traditional Chinese Medicine Inheritance Support System. Herein, this study will be the first to assess the clinical efficacy and effective prescriptions of acupuncture for patients with fecal incontinence, and may benefit practitioners in the fields of complementary and alternative therapies.Adequate anorectal sensation combined with pelvic floor muscle strength helps maintain fecal continence.4Ethics approval is not required due to this work is carried out on published data. We aimed to explore the clinical effective rate, functional outcomes, quality of life, daily average number of fecal incontinence, as well as effective prescriptions of acupuncture for patients with fecal incontinence. In the end, the results will be submitted to a peer-reviewed journal.Lin HX and Wang XT had the original idea of this work and drafted the protocol. Hu GJ and Zhang ZQ designed the search strategies. Chen YJ proposed some advice for the design and revision. Lin CN designed the flow chart. All authors critically revised the draft and approved the final manuscript.Conceptualization: Haixiong Lin, Xiaotong Wang.Data curation: Zhiqing Zhang, Guijuan Hu, Xiaotong Wang.Formal analysis: Haixiong Lin, Xiaotong Wang.Funding acquisition: Yongjun Chen.Methodology: Guijuan Hu, Chunni Lin.Project administration: Xiaotong Wang, Yongjun Chen.Resources: Haixiong Lin, Zhiqing Zhang, Guijuan Hu, Xiaotong Wang.Software: Xiaotong Wang, Chunni Lin.Supervision: Yongjun Chen.Validation: Haixiong Lin, Xiaotong Wang.Visualization: Yongjun Chen.Writing – original draft: Haixiong Lin, Xiaotong Wang.Writing – review & editing: Zhiqing Zhang, Guijuan Hu, Yongjun Chen.Haixiong Lin orcid: 0000-0002-9939-7698.


The higher MCLR dose increased expression of genes involved in fibrosis and inflammation in the control and HFHC groups. These data suggest MCLR toxicity in the context of preexisting NASH may drive the liver to a more severe phenotype that resembles burnt-out NASH.Microcystin-LR (MCLR) is a hepatotoxic cyanotoxin reported to cause a phenotype similar to nonalcoholic steatohepatitis (NASH). NASH is a common progressive liver disease that advances in severity due to exogenous stressors such as poor diet and toxicant exposure. Our objective was to determine how sub-chronic MCLR toxicity affects preexisting diet-induced NASH. Sprague-Dawley rats were fed one of three diets for 10 weeks: control, methionine and choline deficient (MCD), or high fat/high cholesterol (HFHC). After six weeks of diet, animals received vehicle, 10 µg/kg, or 30 µg/kg MCLR via intraperitoneal injection every other day for the final 4 weeks. Incidence and severity scoring of histopathology endpoints suggested that MCLR toxicity drove NASH to a less fatty and more fibrotic state. In general, expression of genes involved in  Photoautotrophic cyanobacteria are increasingly prevalent and persistent in the environment as a result of anthropogenic activity and global warming ,2. CyanoMultiple epidemiological and exposure studies have linked MCLR with liver damage. For example, an association between MCLR exposure and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was reported in a Chinese fisherman population exposed to TDI levels of MCLR (2.2–3.9 µg/day) . In addiThe most common chronic liver disease in the United States is nonalcoholic fatty liver disease (NAFLD), and the worldwide prevalence of NAFLD is estimated to be ~25% . NALFD iA potential role of microcystins in NALFD development and progression has been previously suggested. Recently, a correlation between algal blooms and county level incidences of NAFLD in the United States was reported using a novel satellite imaging technique . In addiThe interaction between preexisting NASH with MCLR toxicity was determined using common dietary models of NASH and a common sub-chronic MCLR treatment design  ,37,38,39Final body, liver, and spleen weights are shown in Plasma ALT is a potential biomarker for MCLR-induced liver toxicity and NALFD ,46,47,48An analysis of plasma insulin and glucose levels revealed similarities between the control and HFHC groups in response to MCLR exposure  . It has Slco1b2 mRNA expression were observed  and inhibiting its activity ,66. In oPP2A/C-bound MCLR can be measured by western blot using an MCLR specific antibody . As expede novo lipogenesis, fatty acid oxidation, and export of lipids [The progression of NASH to a more severe pathophysiological phenotype is characterized by the loss of steatosis and an increase in inflammation and fibrosis . In our f lipids .de novo lipogenesis are shown in Cd36 in rodent models can exacerbate steatosis potentially due to increased de novo lipogenesis and decreased very low lipoprotein (VLDL) secretion [Cd36 expression in the HFHC group  catalyzes the rate-limiting step in the conversion of saturated fatty acids to mono-unsaturated fatty acids. Total or liver specific knockout of fibrosis ,75. Scd ol group D, suggesde novo lipogenesis, and ACC1 inhibition is currently being explored as a target for NAFLD treatment by reducing steatosis [Acc1 in the control group, and 30 µg/kg MCLR decreased Acc1 gene expression in the MCD group (Acc1 may play a role in the modulation of steatosis after MCLR exposure. Acetyl CoA carboxylase-1 (ACC1) is another integral enzyme in teatosis . Both MCCD group E, suggesde novo lipogenesis [Srebp1 and there was a significant difference between the control 30 µg/kg group and the HFHC 30 µg/kg group  and carbohydrate regulatory element-binding protein (ChREBP) are two key transcription factors involved in regulating the genes involved in ogenesis ,77,78. Tkg group B. In cont groups C. Liver-exposure . CollectAcadl) and acetyl-CoA acyltransferase-2 (Acaa2) (Acadm), hydroxyacyl-CoA dehydrogenase (Hadh), acyl-CoA oxidase-1 (Acox1), carnitine palmitoyltransferase-1A (Cpt1), and carnitine palmitoyltransferase-2 (Cpt2)  A,G. Acco2 (Cpt2) B–G. MCLRol group B,C. ThesGpat) is the rate-limiting enzyme, and diaclyglycerol O-acyltransferase 2 (Dgat2) catalyzes the final step [Gpat and Dgat2 expression (Gpat or Dgat2 prevents steatosis in mouse models [Mttp) gene is involved in producing lipoproteins responsible for lipid export from hepatocytes. Mttp expression increased after MCLR exposure  after MCLR exposure, whereas the HFHC group exhibited changes in interleukin-6 (IL-6) interleukin-1β (IL-1β), interleukin-10 (IL-10), and chemokine C-X-C motif ligand 2 (CxCl2)  . In cont to NASH . The reaTgfβ), collagen type 1 alpha 1 chain (Col1a1), cellular communication network factor 2 (Ccn2), and smooth muscle actin alpha 2 (Acta2) gene expression in the control and HFHC groups  ,50,93,94Three diets were utilized in this study: a control diet , an MCD diet (Dyets Inc. Cat. 518810), and a HFHC diet . These diets were selected based on their strengths and weaknesses in recapitulating features of clinical NASH. The control diet produces a healthy phenotype, while the MCD and HFHC diets are established dietary models used to produce a NASH phenotype. The two NASH diets have their respective strengths and weaknesses. The MCD diet replicates NASH liver pathology and is reported as a good model to study NAFLD progression because it causes steatosis, inflammation, oxidative stress, and fibrosis in a short period of time (less than 6 weeks) ,97,98,99The MCLR exposure scenario  is commonly utilized to study sub-chronic MCLR toxicity and was selected to isolate hepatic exposure and toxicity mechanisms from those occurring in the gastrointestinal tract ,39,40,41g for 5 min at 4 °C and plasma was removed and stored at −80 °C until further analysis. Animals were housed in metabolic cages starting 24 h prior to, and 24 h after, the first and last MCLR doses (48-h housing duration each time). Twenty-four hours after the final MCLR injection, animals were euthanized by carbon-dioxide asphyxiation and tissues were collected. Portions of tissues were formalin fixed for histopathological scoring or snap frozen for total RNA isolation or protein isolation.Animals were divided into three groups (n = 18 per group) and fed one of the diets listed above for 6 weeks, at which point each diet group was further divided into three treatment groups (n = 6 per group): vehicle , 10 µg/kg MCLR, or 30 µg/kg MCLR . The aniChrebp), Sterol regulatory element-binding protein 1 (Srebp1), Acetyl CoA Carboxylase 1 (Acc1), stearoyl CoA desaturase (Scd), Cluster of differentiation 36 (Cd36), Transforming growth factor β (Tgfβ), Collagen type 1 alpha 1 (Col1a1), Cellular communication network-2 (Ccn2), Actin, alpha 2, smooth muscle, aorta (Acta2), C-X-C motif chemokine ligand 2 (Cxcl2), Tumor necrosis factor-α (Tnf-α), Interleukin -6 (IL6), Interleukin 1-Beta (IL1B), Interleukin 10 (IL10), C-X-C motif chemokine ligand 1 (Cxcl1), acyl-CoA dehydrogenase long chain (Acadl), acyl-CoA oxidase-1 (Acox1), carnitine palmitoyltransferase-1A (Cpt1) and carnitine palmitoyltransferase-2 (Cpt2) and acetyl-CoA acyltransferase-2 (Acaa2), Acyl-CoA dehydrogenase medium chain (Acadm) and hydroxyacyl-CoA dehydrogenase (Hadh), Glycerol-3-phosphate acyltransferase (Gpat), Diaclyglycerol O-acyltransferase 2 (Dgat2), Microsomal triglyceride transfer protein (Mttp), Ubiquitin C (Ubc), Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), and Beta-2 microglobulin (β2M). Primers for Slco1b2 was purchased from Integrated DNA Technologies (IDT) . The expression for the genes of interest were normalized to the average expression of three housekeeping genes . Total RNA was extracted from rat liver using TRIzol reagent  according to the manufacturer’s protocol. RNA concentrations were determined using a nano-drop, and RNA integrity was confirmed by agarose gel electrophoresis. iScript cDNA synthesis kit (Bio-Rad) was used for cDNA synthesis from total RNA, and SYBR green master mix  was used for real time quantitative PCR analysis as per the manufacturer’s protocol. Primers  were purFormalin fixed liver tissues were paraffin embedded, stained with hematoxylin and eosin (H&E), and analyzed by a board-certified veterinary pathologist. Stained sections were incidence and severity scored using an established rodent NASH system  with endg at 4 °C for 1 h. Supernatants were collected, avoiding the lipid layer. Protein concentrations were determined using the Pierce BCA Protein Quantification Assay kit  according to the manufacturer’s protocol. Approximately 50 mg of liver tissue was homogenized with a handheld homogenizer in 1 mL of NP40 lysis buffer with protease inhibitors. The homogenized samples were agitated at 4 °C for 2 h followed by centrifugation at 10,000× Tissue lysates (20 µg/well) were prepared in Laemmli sample buffer with 2.5% BME and heated at 37 °C for 30 min. Protein was transferred from the gel to polyvinylidene fluoride (PVDF) membrane using the Trans-Blot Turbo Transfer System at 25 V and 1.0 A for 30 min. Following transfer, the membranes were imaged under UV to capture Stain-Free image used for protein normalization. The blots were then blocked with 5% non-fat dry milk in Tris-base buffered saline-Tween 20 (TBS-T) for 1 h at room temperature, then incubated with primary antibody overnight at 4 °C. Membranes were probed for OATP1B2  and MCLR . The blots were incubated with secondary antibody in 5% non-fat dry milk in TBS-T for 1 h at room temperature. Densitometry was performed using Image Lab . Proteins of interest were normalized to total protein as captured by Stain-Free image. Total protein normalization is a commonly accepted technique for protein densitometry analysis instead of the single-protein loading control .All results are represented as mean ± SEM. All data, except the histopathological data, were analyzed by two-way ANOVA statistical analysis with diet and dose constituting the main two factors. A Dunnett multiple comparison post-test was performed to determine statistical differences between different treatment groups. Histopathological data were rank ordered before statistical analysis. All analyses were carried out using GraphPad Prism software. .