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+{"text": "This study was carried out to assess the reliability of core biopsy in predicting the grade and type of cancer accurately as obtaining this information can influence initial therapeutic decisions.Core biopsy is a method of choice for the triple assessment of breast disease as it can reliably distinguish between benign and malignant tumours, between kappa statastics.A total of 105 patients who had invasive breast carcinoma diagnosed by core biopsy in year 2001 and who subsequently underwent surgical management were included. The core biopsy results were compared with final histology with the help of A moderate level of agreement between the predicted grades and final grades was noted (kappa = 0.585). The agreement was good between predicted and final type of tumour (kappa = 0.639).Core biopsy as a predictor of grade and type has limited use at present. We suggest that initial clinical decisions should not be based on the results of core biopsy. Collins et al have shown that majority (83%) of core biopsies and excisional procedures demonstrate exact histological agreement [Core biopsy is rapidly replacing fine needle aspiration cytology (FNAC) as a procedure of choice for the triple assessment of the breast problems. Where there is access to an experienced cytopathologist, the FNAC can provide a rapid and cost effective means of triage of patients who would benefit from more expensive core biopsy . Core bigreement . Core bigreement .As the range of options for the treatment of the breast cancer widens, it has become increasingly important that clinicians are provided with accurate prognostic information to base the initial therapeutic decisions on. Prognostic factors for breast cancer have been extensively studied. Histological grade and type can be used to predict biological behaviour as has been assessed by overall survival and local recurrence for women with primary breast carcinoma -9. HistoThe aim of this study, therefore, was to see how reliable core biopsy is in predicting the grade and the type of cancers, as that could influence the further management of breast cancer.in-situ diagnosed by core biopsy and patients who underwent neo-adjuvant therapy were excluded from the study. Level of agreement between core and excision biopsy was assessed using kappa statistics.All patients with invasive breast cancer diagnosed by the core biopsy and treated subsequently by surgical excision in the year 2001, at a district general hospital were included in the study. Of the 105 patients whose records were studied retrospectively, 47 lesions were palpable and 58 lesions were screen detected. The core biopsies were performed under ultrasound guidance as a part of triple assessment and at least four cores were obtained from palpable lesions and six or more from screen detected lesions with a 22 mm automated core biopsy gun. Two dedicated breast pathologists had authorised all the reports. Age of patients ranged from 35 to 84 with a median age of 62 years. The histology reports for the core biopsy and final histology were extracted and compared. Carcinoma Of 105 patients there was no prediction of grade in 2 patients and in 19, a prediction of grade 1 or 2 or grade 2 or 3 was made. This left 84 where a clear prediction was made. On final histology 35 (33.3%) were categorised as grade I, 45 (42.8%) were grade II and 25 (23.8%) were grade III. The predicted grades versus final grade results are detailed in table Of the 84 cores in which clear prediction of grade was made 63 (75%) were correct. All 21 of grade 1's were predicted correctly, 35 (71%) of grade 2's were predicted correctly but only 7 (50%) or grade 3's were predicted correctly on core biopsy. Of the predicted grade 2's which were reclassified, 5 (10%) were downgraded and 9 (18%) were upgraded. Of the reclassified grade 3's, 6 (43%) were downgraded to grade 2 and 1 (7%) was downgraded to grade 1.Of 105 patients, 101 patients had a prediction of type made. Of 84 cases predicted to be ductal, 81 (96%) were correct and one case was reclassified as mixed histology. Of the 14 predicted to be lobular 9 (64%) were correct and one reclassified as mixed Table . Of the In general the level of agreement between the predicted grades and final grades was moderate (kappa = 0.585) and between predicted and final types was slightly better (kappa = 0.639).et al reported percutaneous, image guided biopsy to be an accurate diagnostic alternative to surgical biopsy in women with mammographically detected suspicious breast lesions [Fajardo  lesions . The fal lesions . However lesions  or amoun lesions  has not  lesions .Histological grade and type, tumour size and presence or absence of axillary node metastases is well-recognised prognostic factors of breast cancer. Tumour grade, size and nodal involvement are three factors considered in Nottingham Prognostic Index . HistoloGreen Hough 1925) was the first to categorise the breast tumours into three grades according to its differentiation. He also assessed the association of grades with \"cure\" though the term cure was not clearly defined . Since t was the Of the two major histological types, lobular is known for its multifocality and multicentricity and its diffusely infiltrating nature . It is iOur results suggest that the prediction of grade and type of breast cancer from core biopsy has only limited use at present. The group of patients we would like to be predicted most accurately would have been those with a high-grade and lobular type, for the reasons stated above. Our results suggest that these patients are most difficult to predict in practice. However, present study being retrospective has its own drawbacks. A prospective study specifically aimed at Kappa statistics between core biopsy and final histopathology may be able to answer this question better. Further refinements are needed in technique of core biopsy and these technical innovations will ultimately improve the results of core biopsy.The author(s) declare that they have no competing interests.AD: Original idea, planning of study, background search, data compilation and drafting the manuscript.TG: Data collection, data compilation, help with the manuscript drafting.SH: Overall supervision and guidance with the study, helped in the analysis and helped with manuscript drafting and revisions.All authors read and approved the final versionNone"}
+{"text": "Six hundred and eighty-four primary breast cancers from the International (Ludwig) Breast Cancer Study Group (IBCSG) were studied for Helix pomatia lectin (HPA) binding. There was a weak correlation between lymph node-positive and HPA positive (P = 0.04). In our series there was a large advantage in disease-free survival (DFS) and overall survival (OS) for node-negative patients (P < 0.0001 DFS and OS). However, there was no such advantage for HPA-negative patients (P = 0.23 DFS and P = 0.32 OS). We conclude that in this randomised patient group HPA is of no clinical predictive value."}
+{"text": "This special issue on Cell Stress and Cell Death is aimed at bringing together recent developments in the fields of cellular stress and cell death and, in particular, the interplay between cell stress responses and cell death. The special issue opens with a review by S. Fulda et al. which provides an overview of how cells can respond to stress in a variety of ways ranging from the activation of survival pathways to the initiation of cell death that eventually eliminates damaged cells. Whether cells mount a protective response or succumb to death depends to a large extent on the nature and duration of the stress as well as the cell type. For example, milder stresses can lead to protection through activation of the heat shock response or the unfolded protein response (UPR). This review also describes several types of cell death  and the mechanism by which a cell dies often depends on various exogenous factors as well as the cell's ability to handle the stress to which it is exposed. The implications of cellular stress responses for human physiology and disease are multifold and are discussed in this review in the context of some major world health issues such as diabetes, Parkinson's disease, myocardial infarction, and cancer. 2+, address this topic. In recent years there has been a significant increase in the number of papers in the field dealing with ER stress and ER stress-induced cell death. This reflects the growing recognition of the importance of the ER in cell stress and in different modes of cell death. The ER is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the ER initiate a complex intracellular signal transduction pathway, known as the UPR. This response is an attempt to reestablish ER homeostasis, although it can also lead to cell death. The review by A. Samali et al. provides a comprehensive overview of current methodologies for monitoring the UPR and ER stress, and it puts together a set of criteria to assess this response, which will be useful for researchers who wish to examine these phenomena in different model systems. The UPR is essentially tailored to reestablish ER homeostasis and it can also signal through other adaptive mechanisms involving the stimulation of autophagy. However, when ER stress is persistent, the cytoprotective functions of the UPR and autophagy can switch to cell death-promoting mechanisms. A review by T. Verfaillie et al. discusses the relationship between ER stress and autophagy and the implications for cancer therapy. Recently, a variety of anticancer therapies have been linked to the induction of ER stress in cancer cells, envisaging strategies that stimulate prodeath function or block its prosurvival function, to improve tumoricidial action. A better understanding of the molecular mechanisms that determine the final outcome of UPR and autophagy activation by chemotherapeutic agents will offer new opportunities to improve existing cancer therapies as well as revealing novel targets for cancer treatment. Prolonged or severe ER stress has been linked to induction of apoptosis. Caspase activation is one of the key steps in commitment of cells to apoptosis and is dependent on mitochondrial outer membrane permeabilization (MOMP) and the release of cytochrome c from the mitochondria. The research article by S. Gupta et al. investigates the mechanism of ER stress-induced MOMP using thapsigargin as an inducer of ER stress. They genetically dissected the role of caspase-9, -3, and -2 in the induction of MOMP by ER stress using embryonic fibroblasts derived from knockout mice and also treated cells with chemical and molecular inhibitors of the mitochondrial permeability transition. Their results suggest that caspase-9 and -2, Bcl-2 family members, and the mitochondrial permeability transition pore all play a role in MOMP during ER stress-induced apoptosis. There are many molecules and cellular processes that play critical roles in normal cell signaling and survival responses, while also having a dual role in inducing cell death. A number of papers in this special issue, covering endoplasmic reticulum (ER) stress and Ca2+ is an important second messenger which is also poised at the intersection between cell survival and cell death. The review by C. Cerella et al. examines the events that occur during Ca2+ toxicity and how reparative or death pathways can be activated. They also discuss the observations that while Ca2+ can elicit these opposing responses, it also plays a role as a second messenger in signal transduction associated with cell death and survival. CaKnowledge of cell stress and cell death pathways, and the interplay between these, beneficially provides us with new ways to tackle diseases, particularly cancers and degenerative disease. Resistance to apoptosis is a feature of many cancer cells and this is the subject of the review by S. Fulda which summarizes the main mechanisms by which cancer cells evade the intrinsic and extrinsic apoptosis pathways. Generally, altered ratios of key pro- and antiapoptotic proteins are responsible for resistance to cell death; for example, altered ratios of Bcl-2 family proteins as well as increased expression of caspase inhibitors (IAPs) regulate the intrinsic pathway, while reduced sensitivity of extrinsic pathways occurs due to decreased expression of death receptors on the plasma membrane and increased expression of intracellular decoy proteins. Given this increased resistance to cell death pathways, the search for novel molecules to induce cell death in cancer cells is an important goal. Histone deacetylase inhibitors have become a promising new avenue for cancer therapy, and many are currently in clinical trials for various tumor types. The research article by N. Rivera-Del Valle et al. demonstrates that a novel hydroxamic acid histone deacetylase inhibitor, PCI-24781, exerts cytotoxicity and histone alterations in leukemia cells, via a mechanism that is dependent on caspase-8 and Fas-associated death domain. Another avenue for identifying novel therapeutic targets for cancers is study of inflammation, since many human malignancies have been strongly linked with chronic inflammation. The review by C. Sobolewski et al. describes the role of cyclooxygenase-2 in these diseases. This enzyme is a member of a family, which catalyzes the rate-limiting step of prostaglandin biosynthesis. Cyclooxygenase-2 is upregulated during both inflammation and cancer, and has been described to modulate cell proliferation and apoptosis mainly in solid tumors and more recently in hematological malignancies. Thus, the use of cyclooxygenase-2 inhibitors, together with other therapeutic strategies, may further improve the efficiency of anticancer treatments in the clinic. inhibit cell death. One such disease is Parkinson's disease, where there are currently no therapies which halt the degeneration of dopaminergic neurons. In the research article by K. Mnich et al. the ability of the endogenous cannabinoid, anandamide, to inhibit apoptotic cell death by the Parkinson mimetic, 6-hydroxydopamine, was shown. The protection provided by anandamide involved activation of phosphatidylinositol 3-kinase and prevention of 6-hydroxydopamine induced activation of c-Jun-NH2-terminal kinase (JNK). These data add to the growing body of literature concerning cannabinoids and Parkinson's disease. Certain other neurodegenerative diseases, termed tauopathies, feature filamentous tau-positive protein inclusions in neurons and glia, which are characterized by the expression of stress response proteins, particularly heat shock proteins (Hsps), in these inclusions. The article by L. Schwarz et al. investigated the contribution of small Hsps, Hsp27, and \u03b1B-crystallin, to neurodegenerative diseases by analyzing the association of Hsp27 with pathological lesions of tauopathies. Their results suggest distinct mechanisms for Hsp27 action in glial and neuronal cells, with prominent expression in unstressed astrocytes but with low expression observed in neurons even after stress situations.In contrast to cancers, an important aim of research into degenerative diseases is to discover novel ways to We hope that this special issue will alert researchers to some new developments in the fields of cell stress and cell death, particularly the interplay between prosurvival and prodeath responses, and how our knowledge of these can direct our efforts in discovering new therapeutic strategies for the treatment of cancers and degenerative diseases.Afshin SamaliAfshin SamaliSimone FuldaSimone FuldaAdrienne M. GormanAdrienne M. GormanOsamu HoriOsamu HoriSrinivasa M. SrinivasulaSrinivasa M. Srinivasula"}
+{"text": "Cervical cancer is the most common cancer experienced by women worldwide; however, screening techniques are very effective for reducing the risk of death. The national cervical cancer screening program was implemented in Taiwan in 1995. The objective of this study was to examine and provide evidence of the cervical cancer mortality trends for the periods before and after the screening program was implemented.Data from 1981 to 2010 of the causes of death registered were obtained from the Department of Health, Taiwan. Age-standardized mortality rates, age-specific rates, and age-period-cohort models that employed the sequential method were used to assess temporal changes that occurred between 1981 and 2010, with 1995 used as the separating year.The results showed that for both time periods of 1981 to 1995 and 1996 to 2010, age and period had significant effects, whereas the birth cohort effects were insignificant. For patients between 80 and 84 years of age, the mortality rate for 1981 to 1995 and 1996 to 2010 was 48.34 and 68.08. The cervical cancer mortality rate for 1996 to 2010 was 1.0 for patients between 75 and 79 years of age and 1.4 for patients between 80 and 84 years of age compared to that for 1981 to 1995. Regarding the period effect, the mortality trend decreased 2-fold from 1996 to 2010.The results of this study indicate a decline in cervical cancer mortality trends after the screening program involving Papanicolaou tests was implemented in 1995. However, the positive effects of the screening program were not observed in elderly women because of treatment delays during the initial implementation of the screening program. Cervical cancer is the most common and serious gynecologic malignancy worldwide. This finding highlights the urgent need to significantly increase cancer survival rates and reduce mortality through screening programs. Cervical screening methods that involve cytology tests, the Papanicolaou technique (PT), human papillomavirus (HPV) tests, and visual inspections of the cervix with 5% acetic acid (VIA) have been established to reduce the cervical cancer mortality rate by almost 60% in numerous countries ,2. NatioAccording to the World Health Organization (WHO)\u2019s cervical cancer screening guidelines, screening is defined as testing all women at risk of cervical cancer, most of whom will not exhibit symptoms. The objective of screening is to detect precancerous changes that, if left untreated, can lead to cancer. However, screening is only effective if a well-organized follow-up and treatment system is also provided. Women who are found to have abnormalities during screening require follow-up consultations, diagnosis, and possibly treatment to prevent the development of cancer or to treat cancer in the initial stage. Several tests can be used to screen for cervical cancer. However, the Papanicolaou smear (Pap cytology) is the only test that has been applied to large populations and been shown to reduce the incidence and mortality rate of cervical cancer. Although VIA, and HPV test have shown potential, to date, no comparable evidence of their effectiveness exists. Large-scale studies are still being conducted. Regardless of the test used, the key to an effective program is to target the largest proportion of women at risk with quality screening and treatment. Organized screening programs that are designed to reach most women at risk and managed at a central level are preferable to opportunistic screening.The Bureau of Health Promotion of the Department of Health implemented the screening program in Taiwan in 1995. Under the screening policy, free annual medical examinations are provided to all Taiwanese women aged 30 years or older. Women who are sexually active are strongly advised to undergo a smear test at least once every 3 years. Women who have been diagnosed with a sexually transmitted disease, the human immunodeficiency virus, the human papillomavirus, or cervical dysplasia, and who have numerous sexual partners, should undergo testing once every year. The results of a smear test provide a reference for whether further examinations are necessary. If the test results are unclear regarding pathological changes, women are recommended to undergo a biopsy. If the biopsy indicates carcinoma in situ, women will only require a simple operation to fully cure the disease.Through a time trend analysis of mortality rates, we can evidence the efficacy of cervical screening programs. The age-period-cohort (APC) model is one of the most common statistical methods. In a previous study , a numbeth for 180.0, and ICD-10th for C53.0), exocervix , other specified sites of the cervix , and unspecified sites of the cervix uteri .Data from 1981 to 2010 of the causes of death registered were obtained from the Department of Health, Taiwan. We selected three variables for sequentially analysis, that is, age of death, period of death, and the coding of International Classification of Diseases (ICD). To determine the three factors  for time trend analysis, we first excluded patients who were aged 19 and under and patients who were aged 85 and over before separating the participants into 13 groups of 5 age bands each. Second, we separated 6 aggregations by 5 years of death for each period group. Finally, cohort groups were calculated according to age groups and period groups. Cervical cancer was defined as malignancies of the endocervix  exhibited an increasing trend before 1995, but not among younger women. i.e. the mortality risks for women aged 70 years or more increased annually before 1995; whereas no significant change in mortality trends was observed among women under 70. Present studies cannot provide a suitable explanation for this finding. The reason for these results may be a particular risk factor directly or indirectly related to age that remains unknown. However, we still propose some possible reasons causing this phenomenon for subsequent study validation. Regarding the phenomenon of multiple births among women of the post-war generation, a number of previous studies have suggested that pregnancy at an excessively early age and excessive pregnancies may be risk factors for cervical cancer . Taiwan However, the results of numerous previous studies also support the limited effectiveness of cervical cancer screening among elderly women -27; one In 2006, Lloroa  contendeth to 10th) and 5 versions (ICD-6th to 10th) of ICD codes. To the best of our knowledge, the different versions of ICD codes were developed through diagnosis and treatment. Thus, we can infer that the old version may be the primary cause of the assumption. Based on this condition, we calculated the cervical cancer mortality trends for 1981 to 2010, but we only included two versions of ICD codes. However, the death registration system in Taiwan cited ICD-9th for death certificates for 1981 to 2008, and ICD-10th for 2008 to the present. Therefore, the effects of various versions of ICD codes are the lowest in this study.However, this assumption and concept do not need to be considered in this study. Furthermore, the above conditions indicate that the study design included analysis of long-term trends and more than one version of ICD codes. In the studies conducted by Lloroa and Levi, the periods analyzed were 1955 to 1995 and 1960 to 1998, and they included 4 versions . The percentage of death-certificate-only cases (DCO%) between 1980 and 2009 show steady improvements in the quality of Taiwan Cancer Registry. The MV% increased from 82.4% between 1980 and 1984 to 89.05% between 2005 and 2009. The DCO% declined from 28.8% between 1985 and 1989 to 1.4% between 2005 and 2009.The results of this study support the effectiveness of the screening program through analysis of the results of the APC model and other standard statistic techniques. For both older and younger women in Taiwan, the screening program provided positive effects by reducing mortality rates; however, it also provided a delayed effect among elderly women. The reliability of supplementary measures following screening using Papanicolaou tests may be significant contributors. However, additional related studies with demographic analyses of the initial screening program implementation delay pattern are required for clarification in future research.PT: Papanicolaou technique; HPV: Human papillomavirus; VIA: Visual inspection of the cervix with 5% acetic acid; APC: Age-period-cohort; ICD: International classification of diseases; AIC: Akaile information criterion; WHO: World health organization; ASMR: Age-standardized mortality rate; UNOS: Cancer of the uterus at an unspecified site.The authors declare that they have no competing interests related to this manuscript.SSY participated in the design, data analysis, research implementation, results interpretation, and manuscript writing. LYP participated in the design, research implementation, and manuscript drafting and editing. JYH participated in the data analysis. DPJ and CCH contributed to the searching and management of the reference list. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/13/prepub"}
+{"text": "Apoptosis is a tightly regulated process: cellular survive-or-die decisions cannot be accidental and must be unambiguous. Since the suicide program may be initiated in response to numerous stress stimuli, signals transmitted through a number of checkpoints have to be eventually integrated.In order to analyze possible mechanisms of the integration of multiple pro-apoptotic signals, we constructed a simple model of the Bcl-2 family regulatory module. The module collects upstream signals and processes them into life-or-death decisions by employing interactions between proteins from three subgroups of the Bcl-2 family: pro-apoptotic multidomain effectors, pro-survival multidomain restrainers, and pro-apoptotic single domain BH3-only proteins. Although the model is based on ordinary differential equations (ODEs), it demonstrates that the Bcl-2 family module behaves akin to a Boolean logic gate of the type dependent on levels of BH3-only proteins (represented by Bad) and restrainers (represented by Bcl-xL). A low level of pro-apoptotic Bad or a high level of pro-survival Bcl-xL implies gate AND, which allows for the initiation of apoptosis only when two stress stimuli are simultaneously present: the rise of the p53 killer level and dephosphorylation of kinase Akt. In turn, a high level of Bad or a low level of Bcl-xL implies gate OR, for which any of these stimuli suffices for apoptosis.analogous inputs into a digital survive-or-die decision.Our study sheds light on possible signal integration mechanisms in cells, and spans a bridge between modeling approaches based on ODEs and on Boolean logic. In the proposed scheme, logic gates switching results from the change of relative abundances of interacting proteins in response to signals and involves system bistability. Consequently, the regulatory system may process two  Apoptosis is a programmed cell death required for removal of infected, damaged or unwanted cells . It assic from mitochondria results in the formation of apoptosome  and activation of caspase-9 [c, which ensures that the apoptotic decision is irreversible [In mammalian cells, apoptosis can be induced via two classes of apoptotic pathways: extrinsic and intrinsic ,7. In boaspase-9 , which aaspase-9 ,12 respoaspase-9 . Caspaseversible ,15.\u03b1, TWEAL and Trail) [\u03baB-mediated) signals are induced. Caspase-8 and -10 recruited to death receptors autoactivate themselves and activate further effector caspases (caspase-3 and -7) either by direct processing or by engaging the intrinsic death pathway [The extrinsic apoptotic pathways, also known as \u2018death receptor pathways\u2019, are initiated through the activation of membrane death receptors, including Fas, TNFR, DR3 and DR4/DR5, by their respective ligands  ,17. Enga pathway .c. This process is regulated by the Bcl-2 family proteins, which share up to four BH (Bcl-2 homology) domains. The proteins can be classified as: pro-apoptotic multidomain effectors (Bax and Bak), pro-survival multidomain restrainers  and pro-apoptotic single domain BH3-only upstream sentinels  [The intrinsic apoptotic pathway is also called the mitochondrial pathway because it is associated with the disruption of mitochondrial outer membranes and consequent release of cytochrome  others) ,19.bax or bak affects apoptosis only slightly, but deletion of both these genes dramatically impairs apoptosis in many tissues [bim\u2013/\u2013bid\u2013/\u2013 mice develop rather normally [L:Bad heterodimers, no Bax:Bcl-xL heterodimers are present [\u03b8) instead of Bcl-2 family pro-survival restrainers [Bax and Bak are effectors directly responsible for the mitochondrial outer membrane permeabilization via either channel formation  or openi tissues ,25. Desp tissues ,27) can  tissues -30 . Oth tissues ,27,34,35normally . Additionormally , and thenormally ,39. The normally ,41. This present ,42. In ntrainers -45. Althtrainers .\u03b3 and UV irradiation, nutrient deprivation, viral infection, hypoxia and increased intracellular calcium concentration [Bcl-2 family proteins are activated or inhibited in response to numerous stress factors including heat shock, ntration ,48. In tntration ,50 and 2ntration -53.In not onco-transformed cells, p53 protein remains inactive . In respIn healthy cells but also frequently in cancer cells, Akt, in contrast to p53, maintains its  activity and suppresses apoptotic signals by phosphorylating and thereby inhibiting pro-apoptotic Bad . Akt actInterestingly, phosphorylation of p53 at Ser46 enables it to activate expression of phosphatase PTEN ,73, whicHere we review mathematical models of the apoptotic pathway, which are relevant to our study.et al. [c\u2013Apaf dimers) is a key element ensuring bistability in survival-or-death decisions. They examined the influence of Bax and Bcl-2 synthesis and degradation rates, as well as the number of mitochondrial permeability transition pores (MPTPs) on the cell response to apoptotic stimuli. The model predicts that above some critical Bax degradation rate the system is monostable and cells survive. Bistability arises for Bax degradation smaller than critical, while the cell fate depends on the initial level of caspase-3. When the number of MPTPs is large, the bistability vanishes and the apoptosis is initiated regardless of the initial condition.Stucki and Simon  have focet al.  describeet al. [L . In the model the pro-survival action of Akt relies on Bad phosphorylation which primes it for degradation. The authors demonstrated that repeated oscillations of the p53 level lead to the depletion of Bcl-2 and Bcl-xL, which is considered as a marker of apoptosis. Antagonism between pro-apoptotic signals of p53 and pro-survival signals of Akt can also lead to bistability, which, if DNA repair is not accomplished in time, allows for termination of p53 oscillations [et al. [et al. [Wee et al.  analyzedllations -81, signllations ,83. In cllations ,82,83, L [et al.  construc [et al.  proposedet al. [et al. [c and caspase-3. In correspondence to the model proposed here, the study of Wee et al. [et al. [In summary, the above models revealed that apoptosis is possible based on the system bistability, which makes the apoptotic decision irreversible. Bagci et al.  showed t [et al.  bistabile et al.  indicate [et al.  presentewhether the interaction network of Bcl-2 family proteins allows for the integration of apoptotic signals, and if yes, whether this signal integration is analogous to Boolean logic gates. Boolean networks are used to model various regulatory pathways, including apoptotic ones [et al. [L.In this study we aim to address the question, tic ones , however [et al.  we focusode23tb solver implemented in MATLAB . The source code is available as the Additional file The system of ODEs following the proposed model was solved using the bax is well-established [L was reported between Bax and Bad, with the latter having higher affinity [L is selected to represent the group of restrainers. Neutralization of Bcl-xL is the primary pro-apoptotic function of Bad [L binding [Our model of the apoptotic decision module involves the following components: p53, Akt, scaffolding protein 14-3-3, caspases, and the Bcl-2 family proteins. The evidence that p53 serves as a transcription factor for ablished . Contrarablished . The comaffinity . For then of Bad ,88, and  binding . Thus, Bc from the mitochondrial intermembrane space [c enables the formation of the apoptosome, which in turn initiates the downstream caspase program [c creating positive feedback loop, which introduces bistability and makes the apoptotic decision irreversible [Active Bax, when freed from its antagonists, releases cytochrome ne space . Release program . In the versible ,15,83. Ip) and p53 in its killer form  will serve as inputs. Surviving cells are characterized by high level of Aktp and lack (or very low level) of p53killer. In these cells, most of Bad remains in the phosphorylated form (denoted Badp) bound to Scaffold14-3-3, while Bax is inhibited (sequestered) by Bcl-xL[u) may bind to Bcl-xL, which limits the amount of Bcl-xL protein available for inhibiting Bax. The steady state is controlled by the total levels of Bad (Badtot) and Bcl-xL (Bcl-xL tot), which will be considered as parameters in the model. The fraction of Bcl-xL bound to Bad increases with Badtot rendering cells of high Bad level more prone to apoptosis. In the model Figure\u00a0, levels by Bcl-xL. UnphospWe consider two pro-apoptotic stimuli, which may lead to the accumulation of free Bax and consequent apoptosis: killer, which triggers Bax transcription and Bax protein accumulation;1) increase of the level of p53Aktp and corresponding increase of the level of unphosphorylated Akt (Aktu).2) dephosphorylation of Akt, i.e. decrease of 14-3-3. Dephosphorylated Bad captures Bcl-xL which eventually releases Bax.Akt dephosphorylation is followed by the dephosphorylation of Bad and its release from ScaffoldImportant notice: We assume that the total level of Akt (Akttot=Aktp+Aktu) remains constant. Under this assumption, the appearance of unphosphosphorylated Akt (Aktu) will be considered as a pro-apoptotic stimulus. The strength of both pro-apoptotic stimuli will be thus measured by p53killer and Aktu.mRNA, Bax, Bcl-xL, Bax:Bcl-xL, Bcl-xL:Badu, Badu, Badp, Badp:Scaffold14-3-3, Scaffold14-3-3, pro-caspases and caspases. The mathematical representation of the apoptotic module consists of 11 ordinary differential equations for levels of molecular species or their complexes: BaxmRNA transcription, induced by p53 killer, is modeled according to the sigmoidal kinetics  [Baxequal 2) . The cubequal 2) . Model pCasp with Bax considered as a bifurcation parameter. Casp undergoes the saddle-node bifurcation at Bax=Baxbif\u22435000 (with Casp\u22431000). Below the bifurcation point there exist three steady states: one unstable and two stable corresponding to low (<1000) and high (>9.9 \u00d7 104) caspase levels. Above the bifurcation point there exists the unique stable steady state characterized by high Casp. The structure of the bifurcation diagram ensures that the apoptotic switch is irreversible, i.e. once the system switches to the apoptotic state, it may not switch back even if the level of free effectors, Bax, drops to zero. The apoptotic switch occurs when Bax exceeds Baxbif for sufficiently long time; as we will show later, short excursions of Bax over Baxbif do not induce apoptosis. For the steady state analysis, however, the states with Bax>Baxbif will be interpreted as apoptotic.The last two equations exhibit bistability. In Figure\u00a0p53killer and Aktu. In this section, we consider digital steady-state inputs: the signal from p53killer is assumed to be logic 0 for p53killer=0, and is assumed logic 1 for 0 for Aktu=0  and is considered logic 1 when Aktu=Akttot=2 \u00d7 105. Intermediate input values of Aktu\u2208 will be analyzed in a forthcoming section.We demonstrate that apoptosis can be controlled in a manner similar to logic gates OR and AND with inputs defined as Badtot=2 \u00d7 105, while gate AND is achieved for Badtot=0.6 \u00d7 105; here, for both gates we assume the same Bcl-xL tot=1 \u00d7 105. Surviving cell steady state is associated with conditions in which both apoptotic signals are equal zero. In these cells  Bad is bound to Bcl-xL. Rest of Bcl-xL sequesters Bax or remains free. Free Bcl-xL may be considered as the anti-apoptotic buffer, which may potentially capture the excess of Bax. This buffer of free Bcl-xL is small (about 104 molecules) for gate OR  for gate AND  is released.Accordingly, to trigger apoptosis, gate OR Figure\u00a0 requiresL is larger  and eventually triggers apoptosis  level of (free) Bcl-xr Figure\u00a0, Table\u00a02L Figure\u00a0B. The siL Figure\u00a0C. HoweveL , dephosphorylated Bad is not able to displace Bax from its complex with Bcl-xL: high abundance of Bcl-xL allows it to restrain whole Badu and whole Bax at the same time.Alternatively, gate OR may be transformed into gate AND (denoted AND* to avoid confusion) by a significant increase of the level of pro-survival Bcl-xL Figure\u00a0, Table\u00a02In the next section we summarize the above numerical analysis introducing approximate formulas defining OR and AND gates.An important property of the model is that, as long as steady-state solutions are considered, a change of any of its 20 parameters can be compensated by a proper change of other (conjugate) parameter(s). Therefore, steady-state concentrations of proteins depend on clusters of parameters rather than on individual parameter values. We identified 7 such parameter clusters, which define \u2018macro-parameters\u2019 Table\u00a0. When co1/m3, 1/m4, and 1/m5 are larger than the characteristic numbers of molecules, all allowed complexes are formed. The conditionm3\u226am4 implies that Bcl-xL binds preferentially to Badu and therefore free Bax is released only whenBased on the macro-parameters one can obtain approximate algebraic formulas which must be satisfied for a given gate type. First, let us notice that since all  dissociation constants Baxbif, the cell sets in the apoptotic state; in the opposite case the cell survives. The amount of Badu is (1\u2212m6)Badtot. In this way we obtain simple inequalities defining logic gates. These inequalities, given in Tables\u00a0When the level of free Bax exceedsp53killer andAktu both in the range , assuming levels ofBadtot andBcl-xL tot adequate to the gate type (Table\u00a0Bax = Baxbif=5000 isoline (white line) separates inputs {p53killer,Aktu} leading either to apoptosis or to survival. In gate OR -plane for different levels ofBadtot. For {p53killer,Aktu} \u201cabove\u201d each isoline value the cell undergoes apoptosis, whenever itsBadtot is greater or equalBadtot value for that isoline. Gate AND arises forBadtot\u22720.9 \u00d7 105, while gate OR arises forBadtot\u2208 the full dephosphorylation of Akt (Aktu= Akttot) leads to apoptosis (for an arbitraryp53killer) but the increase ofp53killer to its highest assumed value  needed to trigger apoptosis. We assume that before and after the stimulation phasep53killer=0 andAktu=0. The transient stimulation by p53killer and/or Aktu may cause that the caspase level passes a threshold above which the apoptotic decision is irreversible. This threshold caspase level (\u22482 \u00d7 103) is determined by the value of the unstable steady state forBax = 0, see the bifurcation diagram  that such stimulation lasting sufficiently long leads to apoptosis. Here we estimated that the minimal duration of the stimulation phase is 10.5 hours. In Figure\u00a0Bax reaches 1.3 \u00d7 104>Baxbif and Casp reaches 1.6 \u00d7 103>Caspbif, the system returns to the initial steady state of the low caspase level. In Figure\u00a0Bax returns to its initial steady state. In the case of gate AND*, the minimal duration of the stimulation phase (for the same stimulation) is 20.5 hours.For the gate AND we consider stimulation during whichp53killer signal  data, but allows also for a more precise description. Wittman and colleagues  introduckiller and pro-survival signals from Akt. The final output of the module is the level of caspase-3, which (in the model) is activated by Bax (or Bak), but also auto-catalytically, which renders the apoptotic decision irreversible. After the caspase level surpassed unstable steady state, the presence of Bax is no longer needed, since caspase autoactivation suffices for their further build-up and execution of apoptosis. Caspase activation requires that free Bax surpasses a threshold of about 5000 molecules per cell, and remains over this level for sufficiently long time. The regulation of Bcl-2 family module can be summarized as follows: in resting cells, Bax (chosen as a representative for pro-apoptotic multidomain effectors) remains in the inactive form bound to Bcl-xL (chosen as a representative for anti-apoptotic restrainers), while most of Bad (chosen as a representative for BH3-only pro-apoptotic proteins) is in the phosphorylated form bound to Scaffold14-3-3. The signal coming from p53 leads to the accumulation of Bax, while the signal coming from Akt (Akt dephosphorylation) results in Bad dephosphorylation. Dephosphorylated Bad may release Bax from Bcl-xL. In this way, both mechanisms contribute to the appearance of free Bax.The considered mitochondrial apoptotic module integrates pro-apoptotic signals from p53L, have relatively small reservoir of free Bcl-xL (required to inhibit/sequester Bax) and thus are more prone to apoptosis. In these cells apoptosis follows from p53killer accumulation or Akt dephosphorylation (gate OR); a BH3-domain mimetic, ABT-737, which mirrors binding capacities of Bad and engages pro-survival proteins (mostly Bcl-2), was shown to induce Bax/Bak-dependent killing [L have much larger reservoir of free Bcl-xL and in these cells apoptosis requires both signals simultaneously (gate AND); this is consistent with experimental results showing that the overexpression of Bcl-xL blocks apoptosis [L level, which confirms that levels of these proteins are important in regulating cell sensitivity to apoptosis.Cells characterized by high Bad level or/and low level of Bcl-x killing . Cells cpoptosis . We demo14-3-3, decreased caspase activity, and consequently enhanced cell viability [It is known that Bad modifications, which interfere with Bad phosphorylation, can make cells more or less sensitive to apoptosis. Bad phosphorylation is limited by PRMT1-mediated methylation of two Bad arginine residues (Arg94 and Arg96), which prevents Akt-mediated phosphorylation of Bad at Ser99. Respectively, decreased methylation of Bad increases the fraction of phosphorylated Bad leading to its enhanced sequestration to Scaffoldiability ,96.L level) apoptosis can result only from the simultaneous presence of both pro-apoptotic signals, i.e. elevated p53killer level and Akt dephosphorylation . Radiotherapy against these cells which leads to the increase of p53killer level (via DNA damage) should be accompanied by the inhibition of pro-survival Akt pathway in order to be effective. In turn, it suggests that cells characterized by high Bad level, or low Bcl-xL level are very prone to apoptosis, which can follow even from growth factor withdrawal.In the therapeutic context, our findings suggest that in some (cancerous) cell lines or cell mutants . Such regulation is based on stoichiometry of interacting proteins, and introduces dynamical thresholds for all protein levels, which, when surpassed, change output from NO to YES. Specifically, apoptosis arises when the level of anti-apoptotic restrainers is surpassed by the combined levels of pro-apoptotic unphosphorylated BH3-only proteins (controlled by Akt) and pro-apoptotic effectors (controlled by p53). We demonstrated that the transition between OR and AND gates is accomplished by the change of a level of single component , without any modification of topology of the network or even kinetic rate parameters.We demonstrated that the mitochondrial apoptotic module may process signals in the way similar to logic gates OR and AND, which suggests a mechanism of the integration of apoptotic and survival signals before the death-or-survival decision is reached. The correspondence between ODEs and Boolean logic arises due to the high affinity, competitive binding of two subfamilies of pro-apoptotic proteins, multidomain effectors (represented by Bax) and BH3-only proteins (represented by Bad) to anti-apoptotic restrainers (represented by Bcl-xThe authors declare that they have no conflict of interest.TL and MK conceived of the study. MNB, BH and MK analyzed the model and performed simulations. All authors wrote and approved the manuscript.Matlab source code.Click here for file"}
+{"text": "Fluocinolone acetonide slow release implant (Iluvien\u00ae) was approved in December 2013 in UK for treatment of eyes which are pseudophakic with DMO that is unresponsive to other available therapies. This approval was based on evidence from FAME trials which were conducted at a time when ranibizumab was not available. There is a paucity of data on implementation of guidance on selecting patients for this treatment modality and also on the real world outcome of fluocinolone therapy especially in those patients that have been unresponsive to ranibizumab therapy.Retrospective study of consecutive patients treated with fluocinolone between January and August 2014 at three sites were included to evaluate selection criteria used, baseline characteristics and clinical outcomes at 3-month time point.Twenty two pseudophakic eyes of 22 consecutive patients were included. Majority of patients had prior therapy with multiple intravitreal anti-VEGF injections. Four eyes had controlled glaucoma. At baseline mean VA and CRT were 50.7 letters and 631\u00a0\u03bcm respectively. After 3\u00a0months, 18 patients had improved CRT of which 15 of them also had improved VA. No adverse effects were noted. One additional patient required IOP lowering medication. Despite being unresponsive to multiple prior therapies including laser and anti-VEGF injections, switching to fluocinolone achieved treatment benefit.The patient level selection criteria proposed by NICE guidance on fluocinolone appeared to be implemented. This data from this study provides new evidence on early outcomes following fluocinolone therapy in eyes with DMO which had not responded to laser and other intravitreal agents. Diabetic Macular Oedema (DMO) is one of the leading causes of blindness in the working-age population , 2 In thThere are currently three corticosteroid agents that have been used for DMO; triamcinolone , fluocinThis retrospective study reports the initial real world experience of implementation of TA301 in the clinical setting, in particular, the patient level criteria used for selection of patients for intravitreal fluocinolone implant, the initial treatment response and the short term safety profile.Consecutive patients treated at each of three hospital departments using fluocinolone implant for DMO according to NICE guidance (TA301) between January 2014 and August 2014 were identified at each site using their respective patient database systems for tracking patients on intravitreal therapies. The study involved collection of data retrospectively from case notes and OCT scans performed as part of patients\u2019 standard care pathways at each institution, approval by Ethics Committee was not required according to the institutional review policies at each site. Case notes review was performed and data were collected at baseline, which was at the time of fluocinolone implant and at the visit nearest to the 3\u00a0month time-point of follow-up. At the baseline time-point, data were collected on demographics, visual acuity (treated and fellow eyes), OCT parameters, IOP, IOP lowering medication use, presence of co-existing ocular pathology and prior laser for DMO and DR or intravitreal therapy and duration of DMO. At the 3\u00a0month time-point, data were collected on visual acuity (treated eyes), IOP, IOP lowering medication use and OCT parameters. Snellen visual acuity was converted using a standard estimation method so that all acuity data was presented in terms of number of ETDRS letters. The type of conversion method we used would convert 6/60 to 35 letters and 6/6 to 85 letters as obtained when an ETDRS acuity is used at 2 meters for acuity scoring. , 13. DesA total of 22 patients (14\u00a0M:8\u00a0F) aged between 42 and 85\u00a0years (mean 67.2\u00a0years) were included. Nineteen patients had Type-2 and 3 patients had Type-1 diabetes mellitus; mean duration of diabetes was 17.9\u00a0years (range: 3\u201360). All patients had unilateral fluocinolone implants ie; 11 right and 11 left eyes treated.Visual acuity conversion for Snellen fraction to ETDRS was required for five patients. In all other patients, acuity scores were captured directly using ETDRS chart. Treated eyes had mean baseline visual acuity of 50.7 letters and mean CRT of 631\u00a0\u03bcm. Twenty two eyes had foveal eversion and 13 eyes had pre-existing vitreo-retinal interface abnormalities. Majority of eyes had prior laser of either macular or pan retinal photocoagulation. All 22 eyes had prior intravitreal therapy. Thirteen eyes had prior ranibizumab or bevacizumab, 3 eyes had both ranibizumab and bevacizumab and 6 eyes had prior intravitreal triamcinolone for DMO. Mean duration of DMO was 26.2\u00a0months (range: 9-47). All patients had a minimum of 8\u00a0weeks wash out period after using Anti-VEGF before having the fluocinolone implant.Fellow eyes had visual acuities ranging from NPL to 85 letters. In 11 patients, the fellow eye had visual acuities equal to or worse than treated eyes. Baseline characteristics of 22 treated eyes are shown in Table\u00a0At the 3\u00a0month time-point, mean change in BCVA for all patients was +6.4 ETDRS letters . A gain of >0 letters was seen in 21 eyes (95.4\u00a0%); 0-4 letters in 4 eyes (18.1\u00a0%), 5-9 letters in 7 eyes (31.8\u00a0%) and \u226510 letters in 6 eyes (27.2\u00a0%), CRT after three months reduced by 148.9\u00a0\u03bcm . A total of 18 out of 22 eyes (81.8\u00a0%) showed some reduction in CRT at 3-months. In the 4 eyes without CRT improvement, the mean increase in CRT was 134.75\u00a0\u03bcm .Overall, 15 patients (68.2\u00a0%) showed some improvement in both visual acuity and CRT, 3 patients (13.6\u00a0%) showed some visual gain without any reduction in CRT, another 3 patients (13.6\u00a0%) achieved a reduction in CRT without any corresponding visual gain and 1 patient (4.5\u00a0%) did not show any improvement in visual acuity or CRT.Of the 5 eyes which had undergone prior vitrectomy, visual acuity was improved by +7.2 letters (range: 0 to +14) and CRT reduced by 176.8 (range:-714 to +385). Four of 5 eyes showed both a reduction in CRT with improved visual acuity but one patient had reduced CRT (-345) with no gain in visual acuity.One patient worsened by 11 letters and had increase in CRT of 385\u00a0\u03bcm; this particular patient had received prior treatment with macular laser and four ranibizumab and one triamcinolone intravitreal injections.Mean baseline IOP was 16.9\u00a0mmHg , with the mean change of 0.3\u00a0mmHg  at month 3. No cases with substantial elevation in IOP were documented in the short follow up period. Of 22 eyes, four eyes, were receiving IOP-lowering drops (timolol and/or latanoprost) prior to fluocinolone implant. Following implant an additional one eye needed IOP lowering medication.Our study set out to evaluate two important aspects in the use of fluocinolone acetonide in the real world clinical setting for treatment of DMO; firstly, the characteristics of patients selected for treatment by clinicians and secondly, the early treatment response seen in their patients.In terms of selection, we found that fluocinolone implant was used infrequently in patients with DMO. In all three sites, there were only 22 procedures performed in three sites in the 8-month period following approval of fluocinolone by NICE. Given the large number of patients needing repeated injections of ranibizumab for DMO at these sites , this inIn terms of early outcome, we found promising results with 18 out of 22 eyes reported to have visual gain and 18 out of 22 eyes with at least a reduction in CRT at month 3 time-point. Figure\u00a0There were limitations in the design of this real world study. The selection of patients for therapy is a complex process involving careful explanation and the confidence expressed by the treating clinician at counselling and a final decision made with the patient preference. It was possible that many other patients who were eligible for therapy but declined due to the risk factors involved. We could not capture those patients who declined treatment with our retrospective design and this study may represent the highest threshold for using fluocinolone at this early stage of implementing NICE guidance. We also captured basic baseline data on patient selection. We were unable in the scope of this study to capture the timescale and progression of DMO in these patients. We recognise that other factors, including the recent worsening of oedema or visual acuity and the frequency of previous visits do have an impact on tendency to use fluocinolone and these factors were not be captured uniformly in this retrospective design.The strength of this study was the accuracy of the representation of those patients who were treated with fluocinolone. The sites selected were medium sized district general hospital settings as well as large teaching hospital centres which enables translation of our results to the majority of hospital eye departments. The inclusion of all consecutively treated patients in an 8-month period ensured a representative cohort of patients for reporting of a potentially wide spectrum of selection criteria used or variable response to treatment.Numerous review articles have been published mentioning fluocinolone therapy for DMO but there is a paucity of published data on use of fluocinolone 0.2\u00a0\u03bcg/day or iluvien\u00ae outside the experience of the FAME trial , 17. We"}
+{"text": "Recently, the shape and design of the panel on the official ball used in the FIFA World Cup was considerably different from that of a conventional soccer ball . Depending on the number of different panels and their orientation, the aerodynamic force experienced by a ball is believed to change, which in turn changes the ball trajectory. However, not much is known about the impact of the surface forms of a ball on its aerodynamics. Therefore, in the present study, 10 different types of soccer balls were produced and their aerodynamic properties were studied by wind tunnel experiments. The results confirmed that the aerodynamic force acting on the ball varied considerably depending on the existence of dimples on the ball surface. In addition, the 4 types of soccer balls, which had different kinds of roughness, revealed that even balls having the same number and shapes of panels experienced greatly varying aerodynamic forces depending on the surface form of the balls. In particular, with the FIFA World Cup tournament in mind, significant changes have been made to the shape and design of the panel of its official soccer ball. The Teamgeist, which was the official ball of the 2006 FIFA World Cup held in Germany, was made of 14 panels, and its form was quite different from the typical form of a conventional soccer ball, which had 32 hexagonal and pentagonal panels. Subsequently, the 8 panel Jabulani (Adidas) was used in the 2010 FIFA World Cup held in South Africa. Later, in the 2013 FIFA Confederations Cup held in Brazil, the 32 panel Cafusa (Adidas) was used as the official ball. The Cafusa had the same number of panels as a conventional ball. However, the panel arrangement of the Cafusa varied depending on the direction of the panel and was quite different from the simple hexagonal and pentagonal panel arrangement of the conventional ball. Then, a ball  consisting of 6 panels was used as the official ball in the 2014 FIFA World Cup held in Brazil. The trajectory of this ball was reported to be stable compared to those of other official balls , 8, 9.The 6 panel Beau Jeu (Adidas), whose panel shape is similar to that of the 6 panel Brazuca but has a slightly different surface form, has been selected as the official ball of the 2016 EURO Cup. The surface of the Beau Jeu has small uneven uniformly arranged square projections; this feature differentiates it from the Brazuca, which has square projections that spread out giving a wave-like appearance . ConsideIt is known that a major aerodynamic characteristic of a sports ball is the rapid decrease in the aerodynamic drag when the boundary layer around the ball transitions from laminar flow to turbulent flow during the flight of the ball In this study, the pattern of the surface of a soccer ball (surface form including the presence or absence of dimples) is investigated using wind tunnel experiments to determine its impact on the aerodynamic characteristics of the ball. The results confirm that the aerodynamic force acting on the ball vary greatly depending on the impediments on the surface of the soccer ball.22.1\u22121, the nozzle size was 1.5 m \u00d7 1.5 m, the wind speed distribution was within \u00b10.5%, the degree of turbulence was less than 0.1%, and the blockage of the measured soccer ball was within 5% of the nozzle size. For example, when the wind speed was set to 25 m s\u22121, the measured mean wind speed was 25.28 m s\u22121, with a standard deviation from the measurement position in the range \u22120.46 to 0.45 and a wind speed distribution within \u00b10.5%. Similarly, the degree of turbulence downstream of the nozzle when the wind speed was 25 m s\u22121, was 0.05\u20130.06, within approximately \u00b10.1%, so the error from the ball position was believed to have little effect on the wind speed. Furthermore, in the measuring system of this study, the dynamic pressure can be measured automatically with 0.1 kg m\u22121 s\u22122 intervals by means of the Pitot-static tube placed above the measuring portion of the soccer ball. In addition, because the position of the ball during the measurement procedure is set almost at the center of the nozzle cross section to adjust the distance between the nozzle and the ball to zero, the flow generated from the Pitot tube may not have a direct effect on the flow around the ball. Furthermore, the length of the sting used in this study was 0.8 m and its width was 0.02 m. This wind tunnel was used to perform experiments on the same orientation of six type balls. Moreover, because the effect of sting vibrations is reduced to a small value by placing the six-component force detector behind the soccer ball, the magnitude of the sting forces was ignored in this study. We performed experiments on soccer balls made using the same material (leather) in the thread (seam) between the panels. Three types of soccer balls, with 32, 12, and 6 panels, were fabricated, and in each type, balls with and without dimples  were made. The diameter of the soccer ball used in this study was uniformly 0.22 m, its weight was 0.436 \u00b1 0.004 kg, and its internal pressure was set to 9.0 psi. In total, six types of soccer balls (\u22121 (approximately Re\u00a0=\u00a01.0\u00a0\u00d7\u00a0105) to 35\u00a0m\u00a0s\u22121 (approximately Re\u00a0=\u00a05.5\u00a0\u00d7\u00a0105) at intervals of 1\u00a0m/s.Re) is a dimensionless number defined as the ratio of inertial force to viscous force, \u03bd is wind speed (m\u00a0s\u22121), D is ball diameter (m), and v is the kinematic viscosity (m2/s), as shown in Eq. The circulating-type low-speed low-turbulence wind tunnel located at the University of Tsukuba   was useder balls  were stuIn the present research, however, a comparative study has been performed by keeping focus on drag coefficient involving simulation of the force acting on the ball due to its surface characteristics.SD is standard deviation, \u2211 means sum up, xi is a value in the data set, \u03bc is the mean of the data set, and N is the number of data points.Also, as wind speed increased, the changes in the side and lift forces on each soccer ball and the corresponding standard deviations were shown for each wind speed and compared, as shown in Eq. Furthermore, to examine the characteristics of the surface form of soccer balls in greater detail, four types of soccer balls with different surface forms  were invThe width and depth were measured using a high-speed 2D laser scanner  To measure these parameters, all seams of the football were covered using clay with the height of the imprint representing the panel joint depth and the width representing the panel joint width . The depdC), lift coefficient (Cl), and side coefficient (Cs), as shown in Eqs. \u03c1 is the air density (\u03c1 = 1.2 kg m\u22123), U is the wind speed, and A is the projected area of the football (A = \u03c0 \u00d7 (0.11)2 = 0.038 m2).The force acting on the soccer ball was measured by using a sting-type 6-component force detector . The measured aerodynamic force was converted into the drag coefficient (2.2Re = 4.6 \u00d7 105) and the 32 panel dimple-type ball (32_D) and the 32 panel dimple-less ball (32_S) in the critical regime (Re = 2.3 \u00d7 105) on their flight distance and flight trajectory We conducted a simple 2D flight simulation to compare the effects of the drag coefficients of the 12 panel dimple-type ball (12_D) and the 12 panel dimple-less ball (12_S) in the super-critical regime  by selecting conditions such that the difference between the flight distance of the two balls with and without dimples is a maximum. Thus, by setting various conditions, the difference between the flight distances of various balls can be explained with more clarity.In flight simulation, by using the 33.1Cd value for balls without dimples showed a trend of being smaller than that for balls with dimples in the supercritical range ; therefore, the difference caused by practice could not be seen. This is believed to be due to the fact that in the critical Reynolds number range (Re = 1.5 \u00d7 105\u20133.0 \u00d7 105), the dimple-type soccer ball experiences a smaller drag (resistance) than the dimple-less ball. From the results, in the case of dimple-type balls, in the critical Reynolds number range, the shape of the dimples causes transition of the boundary layer on the ball. Therefore, the dimple-type balls are perceived to be faster than the dimple-less balls. In contrast, in the supercritical Reynolds number range (Re = 3.8 \u00d7 105\u20135.0 \u00d7 105), the dimple-less ball had a smaller drag value. Furthermore, it can be inferred that frictional drag for dimple-less balls are lower than that for the dimple type ball in the supercritical Reynolds number range. Therefore, we found that the drag acting on the ball changes depending on the Reynolds number interval. The 32_D soccer ball had the smallest supercritical drag coefficient of approximately 0.10 (Re = 2.3 \u00d7 105), and the 12_D ball had a corresponding value of approximately 0.15 (Re = 2.1 \u00d7 105). The 6_D soccer ball had a corresponding value of approximately 0.17 (Re = 2.1 \u00d7 105). Further, in the case of the dimple-less balls, the value for the 32 panel ball was approximately 0.13 (Re = 3.5 \u00d7 105) and that for the 12 panel ball was approximately 0.12 (Re = 3.5 \u00d7 105). The 6 panel ball had a value of approximately 0.12 (Re = 3.4 \u00d7 105). In the case of the dimple-type balls, the supercritical drag coefficient increased as the number of panels reduced, but no such difference attributable to the number of panels was observed in the case of the dimple-less balls. For soccer balls with dimples, the supercritical drag coefficient was observed to increase as the number of panels decreased, so it is believed that the supercritical drag coefficient will decrease as the total length of the seams on the surface of the soccer balls with dimples increases . Furthermore, we observed the same trend as reported in prior research: that the length of the seams has a large effect on the supercritical drag coefficient The aerodynamic coefficients of the balls indicate that the aerodynamic drag  tends toal range B and C. ype ball A. This CFrom these results, the dimple shape in the soccer ball surface is also expected to have the effect of maintaining a constant boundary layer around the ball. However, because the air flow around the ball was not studied in this experiment, it is necessary to study from multiple angles how friction resistance, due to the presence or absence of dimples, affects boundary layer separation, so in the future, it will be necessary to study the ball surface air flow in further detail by using visualization methods such as PIV.\u22121, attack angle: 30\u00b0), we compared the flight distance of the 12_S ball , and the 12_D ball (a large one). The flight distances for the 12_S ball and 12_D ball were approximately 55.1 and 49.0 m respectively , comparing the flight distance of the 32_D ball and the flight distance of the 32_S ball, the 32_D ball distance (approximately 17.9 m) was approximately 0.5 m longer than that of 32_S ball (approximately 17.4 m)  was approximately 0.4 m higher than that of 12_D ball (approximately 1.7 m). However, in the present study, investigation of the unsteady aerodynamic force acting on the soccer ball in air has not been performed (such as knuckle effect); therefore, in future research it is necessary to investigate the difference in the knuckle effects of soccer balls in-flight by studying the vortex structure of the wake flow of the soccer ball in flight.Using a ball trajectory simulation in the supercritical regime  B. FurtheSD)) due to the increase in the flow velocity for the six types of soccer balls divided according to the presence or absence of dimples and the number of panels. First, looking at the entire graph, the lift coefficient (red line) and side coefficient (blue line) acting on the ball are seen to vary greatly depending on the types of ball. Further, although the force variation is different for the ball types, no significant difference were observed in the tendency for force variation in the lift and side coefficients in the same type of balls. In terms of the force variation (SD) in the side and lift coefficients due to the presence or absence of the dimples, the dimple-less balls (\u22121 (Re = 4.6 \u00d7 105), the variation is seen to exceed 2 in terms of both the lift and side coefficients, showing a greater variation than that for the other types of balls with an increase the flow velocity. However, for the 32_D ball, the magnitude of the force variation is seen to be less than 2, showing a smaller variation than that for the dimple-less ball (\u22121 (Re = 4.6 \u00d7 105), the variation in the lift and side coefficients of the 32_D ball was approximately 1.2 and 0.9 (respectively). However, the 32_S ball was approximately 2.0 and 1.9 (respectively). Therefore, the dimple-type balls are observed to have less force variation than the dimple-less balls. These results indicate that the shape of dimples on the surface of the soccer ball may have the effect of constantly maintaining a boundary layer around the ball. Moreover, it has been made clear that the dimple structure on the surface of the soccer ball significantly affects the force variation on the flight distance and force variation on lift force and side force according to the Reynolds number intervals. Therefore, we found that the drag acting on the ball changes depending on the Reynolds number interval. Furthermore, it is now understood that the aerodynamics of the ball varies not only because of the structure of the dimples, but also because of the shape of the surface protrusions. Also, the surface of the ball involves complicated factors such as shape or number of panels, their width and depth and therefore, a detailed study of the flow of air at the surface of the ball by using a visualization method such as PIV is essential in future studies.ss balls A\u2013C with ss balls D, at floess ball A. For ex3.2Cd values in the four types of soccer balls with different surface shapes. Each of these four types of soccer balls consists of 32 same panels. Also, the measured ball orientation was set identically to that shown in Re = 1.5 \u00d7 105\u20133.0 \u00d7 105), the dimple-type balls have a smaller drag (resistance) than the balls with other types of panels. In addition, the 32_T1 ball experiences a smaller drag than the other balls in the supercritical Reynolds number range used during a long kick or a powerful shot (Re = 3.8 \u00d7 105\u20135.0 \u00d7 105). Further, the 32_T2 soccer balls with dimples have the smallest supercritical drag coefficient of approximately 0.11 (Re = 2.3 \u00d7 105), whereas the 32_T4 balls with hexagonal surface forms have the largest value of approximately 0.14 (Re = 2.7 \u00d7 105). Moreover, the 32_T1 balls made of square projections have a value of approximately 0.12 (Re = 2.7 \u00d7 105), whereas the 32_T3 balls with triangular projections have a value of approximately 0.12 (Re = 2.7 \u00d7 105). Further, we compared the flight distance of these four different types using a ball trajectory simulation in the supercritical regime . The flight distances for the 32_T1 ball and 32_T2 ball were approximately 27.0 and 25.9 m respectively (ectively . TherefoRe = 4.2 \u00d7 105), the 32_T4 ball has values of approximately 1.45 and 1.50 (respectively), which are smaller than the corresponding values for the other types . It is seen that dimples (surface texture) have a greater impact on the aerodynamic characteristics of the balls. Further, creating forms on the surface of the soccer ball makes it possible to control the irregular movement of the ball in the up and down and left and right directions to some extent. Thus, the fact that the aerodynamic force acting on the ball varies depending on the surface form of the soccer ball suggests that surface roughness is also one of the important factors that determine the aerodynamic characteristics of the ball in addition to the shape and number of panels.4Recent studies on the fluid dynamics of soccer balls have reported that the number of panels on the ball, their orientation, and changes in panel shapes have a major impact on the aerodynamic characteristics of the ball. The present researches also show results similar to those of previous studies. In particular, this study has established the fact that changing the texture of the ball surface changes the aerodynamic characteristics of the ball. This suggests that by considering the changes in the aerodynamic characteristics obtained in response to variations in the number and shape of the panels and modifications to the surface form, soccer balls with even more diverse aerodynamic characteristics can be developed. However, it is necessary to examine dimples of various sizes and patterns other than the ones used in this study. Therefore, in the future, we intend to study the surface roughness in more detail by examining the air flow around the dimple on the ball surface through visualization experiments such as particle image velocimetry.Sungchan Hong and Takeshi Asai: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paperThe authors declare no conflict of interest.Ministry of Education, Culture, Sports, Science and Technology of the Japanese government.This work was supported by JSPS KAKENHI Grant Number 24240084, 25750283, 15K16442 of the No additional information is available for this paper."}
+{"text": "With the advances in next-generation sequencing (NGS) technology and significant reductions in sequencing costs, it is now possible to sequence large collections of germplasm in crops for detecting genome-scale genetic variations and to apply the knowledge towards improvements in traits. To efficiently facilitate large-scale NGS resequencing data analysis of genomic variations, we have developed \u201cPGen\u201d, an integrated and optimized workflow using the Extreme Science and Engineering Discovery Environment (XSEDE) high-performance computing (HPC) virtual system, iPlant cloud data storage resources and Pegasus workflow management system (Pegasus-WMS). The workflow allows users to identify single nucleotide polymorphisms (SNPs) and insertion-deletions (indels), perform SNP annotations and conduct copy number variation analyses on multiple resequencing datasets in a user-friendly and seamless way.https://github.com/pegasus-isi/PGen-GenomicVariations-Workflow) and a web-based implementation of the PGen workflow integrated within the Soybean Knowledge Base (SoyKB), (http://soykb.org/Pegasus/index.php). Using PGen, we identified 10,218,140 single-nucleotide polymorphisms (SNPs) and 1,398,982 indels from analysis of 106 soybean lines sequenced at 15X coverage. 297,245 non-synonymous SNPs and 3330 copy number variation (CNV) regions were identified from this analysis. SNPs identified using PGen from additional soybean resequencing projects adding to 500+ soybean germplasm lines in total have been integrated. These SNPs are being utilized for trait improvement using genotype to phenotype prediction approaches developed in-house. In order to browse and access NGS data easily, we have also developed an NGS resequencing data browser (http://soykb.org/NGS_Resequence/NGS_index.php) within SoyKB to provide easy access to SNP and downstream analysis results for soybean researchers.We have developed both a Linux version in GitHub  contains supplementary material, which is available to authorized users. In-depth informatics analysis of genotypic data can provide a better understanding of genotype-phenotype correlations with applications designed to assist in the work toward improvement of traits. In order to achieve this, many research institutions are generating large-scale sequencing datasets for crop germplasm , 2 for aG. Max) and wild soybean genomes (G. Soja) [http://soykb.org/NGS_Resequence/NGS_index.php. PGen workflow can also be utilized for other organisms and crops by easy customization and serves as a good template for reproducible workflow for bioinformatics analysis with different types of NGS data.Soybean is an important economic crop and is no exception to the computational barriers associated with a lack of access to advanced HPC and other NGS resources just mentioned. Soybean is a great source of dietary protein and oil for human and animal consumption. The soybean community has invested a great deal of efforts in both sequencing germplasm and creating phenotypic datasets, which has resulted in hundreds of resequencing datasets for both cultivated (G. Soja) . Here weG. Soja)  for aligG. Soja)  for SNP G. Soja)  environmG. Soja) , 8. We h500+ soybean germplasm lines were sequenced at different coverages (15X and 40X) from multiple datasets and utilized for SNP and indel identification. Table\u00a0G. Max Williams 82 Wm82.a2.v1 version [ version  availabl version  was used version  workflow version . Only th version . Picard  version  is also  version . Filteri version  and the  version . CNVs am version . The SNP version  tool, deThe PGen workflow has been optimized to obtain the most efficient and large-scale analysis of the sequenced lines using iDS for cloud storage, Extreme Science and Engineering Discovery Environment (XSEDE) . XSEDE ipegasus-run, keep track of the workflows status using pegasus-status, generate statistics using pegasus-statistics, identify problems in case of failed workflows using pegasus-analyzer, resume the workflows after resolving the problems and remove them using pegasus-remove. With this optimization we can conduct analysis on all datasets that have more than hundreds of soybean lines with multiple PGen workflows submitted simultaneously on TACC resources, generating results within a 5- to 10-day timeframe. Each workflow consists of a batch of up to 50 soybean lines. This timeframe includes the associated wait times for individual tasks in queue before they can be executed on Stampede or Wrangler systems. For our datasets, we conducted analyses using multiple workflows submitted simultaneously for a subset of soybean lines, and later combined all results for datasets into the final variant calling using the merged GVCF file in GATK. This ensures that SNP and indel calls were made based on the entire datasets of sequenced lines. Further, the outputs were sent back to iDS and utilized in the downstream analysis as well as loaded into the SoyKB NGS data browser for sharing with biological users. The details for necessary accounts, supported input file formats and output files generated for every workflow are available in PGen documentation  tasks in the workflow as shown in Fig.\u00a0https://github.com/pegasus-isi/PGen-GenomicVariations-Workflow) and easily customize it for conducting analysis on organisms other than soybean, uploading reference genome versions of their choice and customizing SNP filtering criteria. For soybean researchers and SoyKB users, the PGen workflow is also available in a web-based implementation within SoyKB (http://soykb.org/Pegasus/index.php) to conduct analysis on their lines of interest and submitting their NGS datasets online. The PGen workflow submission within SoyKB is mainly intended for biology researchers and guides them through five easy steps for workflow creation and submissions, which allows them to access the results within SoyKB as well. The steps for using PGen workflow in SoyKB as outlined in Fig.\u00a0Introduction: We provide an introduction page, which presents the structure and computing environment of the PGen workflow. A user manual and public data for testing are also provided  using FUSE mount. Successfully uploaded data will be shown on the create workflow page when selecting inputs  of sequenced soybean lines as well as statistics related to raw datasets. It provides the total number of raw reads, mapping rates, SNPs and indels identified  of each soybean line in both searchable tabular and PDF format . The second resource comes from the Stampede and Wrangler high-performance computing cluster of TACC. The third resource is the XSEDE gateway allocation, which has been setup for SoyKB users utilizing the PGen online workflow. We are also building a fourth computing resource locally utilizing HPC resources at the University of Missouri-Columbia to provide PGen execution. More computing resources can be added as they become available to users in future.PGen, together with its source code, is freely available to academic users via GitHub. It outlines best practices for efficient utilization of distinct and unique cyberinfrastructure (CI) resources available through multiple providers, with an emphasis on creating extensible and scalable workflows that can be easily modified and deployed. A similar approach can be utilized for designing many other bioinformatics analysis pipelines using the Pegasus workflow management system (Pegasus-WMS)."}
+{"text": "One way of expediting materials development is to decrease the need for new experiments by making greater use of published literature. Here, we use data mining and automated image analysis to gather new insights on nanoporous gold (NPG) without conducting additional experiments or simulations. NPG is a three-dimensional porous network that has found applications in catalysis, sensing, and actuation. We assemble and analyze published images from among thousands of publications on NPG. These images allow us to infer a quantitative description of NPG coarsening as a function of time and temperature, including the coarsening exponent and activation energy. They also demonstrate that relative density and ligament size in NPG are not correlated, indicating that these microstructure features are independently tunable. Our investigation leads us to propose improved reporting guidelines that will enhance the utility of future publications in the field of dealloyed materials. However, it may be possible to accelerate materials development by systematically extracting new insights from already published literature using advanced image analysis tools. We apply this strategy to nanoporous gold (NPG): a material that has been studied extensively due its potential uses in catalysis3, sensing4, actuation5, and energy storage6. NPG consists of a network of interconnected nanometer-scale pores and ligaments, as shown in Fig.\u00a08. By mining and analyzing published, peer-reviewed, images such as those shown in Fig.\u00a0Materials research and development is often frustratingly slow due to the time and resources needed to conduct new experiments9, to extract microstructure characteristics \u2013 such as NPG ligament and pore dimensions \u2013 in a consistent and reproducible manner. Combining this information with reported processing histories, we obtain a quantitative numerical description of NPG coarsening as a function of time and temperature. Our analysis confirms that coarsening in NPG is a thermally activated process, with an Arrhenius dependence on temperature and an activation energy consistent with surface self-diffusion of Au. However, our analysis finds a coarsening exponent that is lower than classical predictions developed for idealized systems, indicating a need for revised models to capture the coarsening behavior of dealloyed materials.Our method relies on novel image-analysis software, developed and discussed in a previous publicationIn addition, we find new insights concerning the relative density of NPG, approximated as the area fraction of the solid phase in published NPG images. Relative density is found not to correlate with other physical characteristics of NPG, such as ligament diameter, parent alloy composition, or processing conditions such as dealloying time or temperature. This finding suggests the existence of unreported, \u201chidden\u201d processing parameters that may enable NPG relative density and ligament size to be independently tuned. Based on our investigation, we propose new publication guidelines that will facilitate the discovery of such unanticipated, hidden parameters from data mining studies on future publications in the field of dealloyed materials.10. It is an ideal target material for a data mining study such as ours because it has inspired a large volume of literature to analyze. Indeed, keyword searches on \u201cdealloying\u201d and \u201cnanoporous\u201d using the Web of Science database yield more than 1,500 and 22,000 hits, respectively. However, only a small fraction of these publications refers to work that uses de-alloying to synthesize NPG. An even smaller fraction contains the information needed for our study, such as high-quality images and a comprehensive description of material processing conditions. Since most widely used search engines do not have the capability to sort publications according to the type of data they contain, we confine our work to manuscripts citing the seminal paper on dealloyed NPG by Erlebacher et al.11. At the time of our search (03/01/2017), there were a total of 1293 citations for this paper listed on Web of Science. Out of these, only 145 contained sufficient information for our analysis. Of these 145 manuscripts, only 28 were focused on NPG37.NPG is a prototypical dealloyed material, formed by selectively dissolving Ag or Cu out of a parent Ag/Cu-Au alloy using an acid solvent9, applies advanced algorithms to extract accurate microstructural information even with significant additions of noise, blurring, and magnification errors  to light blue at the largest (1630\u2009\u00b1\u2009770\u2009nm), where\u2009\u00b1\u2009is not an uncertainty but instead one standard deviation. Figure\u00a0\u03bb, indicating that coarsening for long times at low temperatures may yield similar ligament diameters as short anneals at elevated temperatures. For example, room temperature coarsening for 864,000\u2009seconds gives rise to a ligament diameter of 56\u2009\u00b1\u200920\u2009nm, comparable to the ligament diameter produced by coarsening at 473\u2009K for 600\u2009sec, 63\u2009\u00b1\u200916\u2009nm.Figure\u00a0The literature used in our study reports ligament diameters for approximately 70% of the data points shown in Fig.\u00a038 and only three manuscripts employed computer-aided software to measure the ligament diameter37. Some authors reported using fast Fourier transform (FFT) image analysis to measure the ligament diameter under the assumption that ligament and pore diameters are equivalent21. However, this assumption does not accurately represent most NPG samples. Many manuscripts, moreover, failed to report the number of manual measurements used to determine the ligament diameter and often reported values without quantifying uncertainty 37. By contrast, AQUAMI analyzes all the ligaments in each image using automated image analysis to determine \u03bb as well as its variance.We attribute these differences to approximations made in the publications mined for our study. For example, some authors measured only a handful of representative ligament diameters to estimate the ligament diameter of their samples38. It is likely that similar choices were also made by other groups. AQUAMI avoids such approximations by determining ligament diameters over the entire ligament length, and allows us to examine the literature data in a consistent manner.In addition to random scatter, some systematic discrepancies are also evident in Fig.\u00a039. The data we mined for our study allow us to determine quantitative descriptions of this coarsening behavior. We expect that coarsening of NPG depends on one dominant mass transport mechanism, so\u2014 following Herring\u2019s analysis40 \u2013 we fit a general power-law expression for the ligament diameter:t is coarsening time excluding the time spent in dealloying , Ds is surface self-diffusivity, n is a coarsening exponent, and k is a proportionality constant. Ds has the usual Arrhenius form, Ea is the activation energy for rate-limiting process of coarsening. Thus, Eq.\u00a0NPG is known to undergo thermally-driven coarsening, manifested as a continuous increase in ligament diameter with time at rates that rise with temperature42. Note that, according to Eq.\u00a0nEa and not Ea.The data shown in Fig.\u00a0nEa, we determine via free corrosion  in nitric acid. However, some of the data are obtained from NPG fabricated using electrochemical corrosion  or other electrolytes. Data from all processing conditions were included when fitting the exponent.The second data group includes ligament diameters at a single temperature (room temperature) from a wide range of times. Plotting this data in Fig.\u00a042. It should be noted that our activation energy calculation is not for the surface self-diffusion of Au in electrolytes, which is typically lower at ~0.6\u2009eV43. Finally, using the average coarsening time of the samples in Fig.\u00a0Combining the power-law exponent obtained from Fig.\u00a044. Although analysis of 2D images cannot give a direct measurement of relative density, we may nevertheless use the area fraction of the solid (gold) phase in NPG images as a convenient proxy for relative density. To determine the factors that control relative density, we sought to correlate solid phase area fractions for all the images mined in our study with NPG processing parameters, such as coarsening time and temperature, free vs. potentiostatic dealloying conditions, dilute  vs. concentrated acid solvent (greater than 0.1\u2009M), as well as the composition of the parent alloy. As shown in Fig.\u00a0via data mining.Relative density is a key characteristic for predicting the properties of porous materials47.To gain further insight into the factors controlling NPG relative density, we search for correlations between solid phase area fractions and other descriptors of NPG morphology, as shown in Fig.\u00a0In this study, we demonstrated that mining and analysis of published images is an effective way to gain new insight into processing-structure-property relations in materials. Applying this approach to NPG, we confirmed that coarsening is thermally activated in this material and calculated activation energies consistent with surface self-diffusion of Au being the rate-limiting process for coarsening. We also determined the coarsening exponent to be ~1/8. The strong correlation of ligament diameter\u00a0with time and temperature demonstrates that they\u00a0are\u00a0the primary factors\u00a0influencing coarsening, but our analysis may become more precise if we are able to include secondary and tertiary factors such as average grain size and defect densities. In addition, we find that NPG relative density, represented by solid phase area fraction, is not correlated with any of the processing conditions reported in the literature mined for our study. Furthermore, while solid area fraction is not correlated to ligament length or diameter, it shows a distinct correlation with ligament aspect ratio, supporting the notion that all the NPG images we investigated are topologically equivalent.48 and there has been one in-depth study regarding the dealloying potential, volume shrinkage, and remaining Ag content49. Unfortunately, we were unable to extract data from ref.48 due to poor image contrast while ref.49 provided no images corresponding to the 36 reported processing conditions.Our findings have important consequences for future investigations of NPG. First, Fig.\u00a050. However, several key assumptions \u2013 particularly that surface diffusion is isotropic and the surface profile remains sinusoidal as it decays \u2013 are not expected to hold during coarsening in nanoporous gold. Departures from classical behavior have been observed in materials with finite terrace widths below the roughening transition temperature52. In addition, kinetic Monte Carlo coarsening studies of NPG by Erlebacher showed that a power-law exponent of n\u2009=\u20091/4 is only observed at long times, when the morphology approaches that of a sphere. Our findings indicate that the NPG samples investigated in the literature mined for our study are still far from this limiting condition. Had we used an exponent of n\u2009=\u20091/4 in our analysis, we would have obtained estimates of 0.64\u2009\u00b1\u20090.04\u2009eV for the activation energy of Au surface self-diffusion, which is out of the range, 0.73\u20131.73\u2009eV, of reported activation energies in the literature42.The coarsening exponent of n\u2009\u2248\u20091/8 obtained in our study stands in contrast with the classical surface diffusion exponent of 1/4, which was derived for the idealized case of a sinusoidal surface profile decaying by surface diffusion56. Reported coarsening power-law exponents have ranged from 0.1354 to 0.3256. One coarsening study55 did not directly report a coarsening relationship, but showed that their data was poorly captured by power-law exponents of 1/3 and 1/4. Only ref.54 measured ligament diameters directly from images, yielding a value close to that determined in our present study. The other manuscripts estimated the ligament diameter from scattering peaks corresponding to a characteristic length scale in the material under the assumption that ligament and pore diameters are identical. This assumption does not always hold, and there are appreciable differences in our calculated values and those reported in ref.12. Regarding the activation energy for coarsening, ref.12 reported a value of ~0.65\u2009eV in an electrolyte, and is thus not comparable to our study, while ref.53 reported an unphysically low value of ~0.35\u2009eV in air. Ref.56 did not directly report an activation energy, but showed that the data was better fit by a value of 0.64\u2009eV than 2.2\u2009eV. Of these three studies, we can only draw direct comparison with ref.53, but the value reported in that study corresponds to nEa and not Ea.There are\u00a0a few studies on coarsening of\u00a0NPG\u00a0reported in the literature, but the results are inconclusiveA in the coarsening law for NPG  the remaining Ag content in the parent alloy upon completion of dealloying and b) the extent of sample shrinkage (and consequently reduction in number of lattice sites) during dealloying. Unfortunately, neither Ag dissolution rate, nor final Ag content, nor sample shrinkage are consistently reported in the literature, even though individual studies show volume shrinkage may be as large as 30%, in some casesThe conclusions of the study presented here depend on the availability of data in the open\u00a0literature, and our investigation reveals serious challenges in extracting this\u00a0data. A surprisingly large number of the manuscripts we considered, ~89%, did not meet the minimum criteria to be used in our study due to poor image contrast, low image resolution, or lack of detailed processing history. Even if a manuscript met the minimum criteria, reported ligament diameters frequently had no corresponding images: a significant concern in light of the discrepancies between the reported and calculated ligament diameters shown in Fig.\u00a058. It would be useful to quantify this relationship in nanoporous metals and other complex structures due to the popularity of 2D analysis in metallic foams and other cellular materials59.To enhance the utility of future publications, we propose that the following data be included in every publication on NPG : high quality images with minimum resolution of 300 DPI and at least 10 pixels per ligament diameter; representative cross-section images ; dealloying and coarsening times; dealloying and coarsening temperatures; electrolyte solution and concentration; applied potential and current relative density; composition of the parent and final dealloyed material  and percent volume change of the sample ; and finally, include all images as supplemental material whenever possible. Meeting these criteria does not require significant additional effort, given access to standard materials research equipment, such as a scanning electron microscope with elemental analysis capabilities. Although we are unaware of published data in the field of nanoporous metals demonstrating significant inaccuracies in using 2D over 3D images to gather quantitative structural information, there is evidence that 2D measurements are accurate in comparison to 3D measurements in metallic foamsThe data mining approach used in this manuscript is not confined to NPG. As noted in the Data Mining section, 116 additional manuscripts contained sufficient information for image analysis, but were not used in our study because they focused on dealloyed materials other than Au, such as Cu or Pt. Our approach is directly applicable to those materials, given sufficient data. More broadly, data mining and image analysis may be applied to study numerous materials-related phenomena, such as solidification, precipitation, and grain growth. To accelerate investigations such as ours, it would be helpful to develop techniques for the automatic acquisition and screening of images and data from the published literature.9. The segmentation procedure consists of two steps: first, bilateral filtering to remove noise from the micrographs while preserving edges; second, Local Otsu\u2019s Method to assign pixels to the solid or void phase, generating a binary image. The measurement procedure consists of three steps: first, a distance map is generated where pixels belonging to the solid phase are replaced with a value equal to the pixel\u2019s Euclidean distance to the nearest pixel belonging to the void phase; next, a binary array is generated comprised of one pixel-thick lines along the center of the solid phase in the distance map; finally, a radius map is generated by element-wise multiplication of the distance map and binary array. This procedure is able to output the area fraction and full ligament diameter distribution. A similar approach was used to measure the ligament length, where nodes were removed from the binary array and a connected-components labelling algorithm was used to determine the number of pixels in each ligament. Image analysis details may be found in ref.9.The NPG images used in this study were exported in TIFF format from manuscripts using Adobe Illustrator without any reduction in image resolution. The images were analyzed using a custom segmentation and measurement procedure implemented in the AQUAMI softwareAll data generated or analyzed during this study are included in Supplementary Information files.Supplementary InformationSupplementary Dataset 1"}
+{"text": "N-demethylation of N-methylated (both mono- and di-methylated) arginine residues of histones and other proteins, including HSP70 (heat-shock protein 70), estrogen receptor \u03b1, and RNA helicase A. Here, we report MS- and NMR-based kinetic assays employing purified JMJD6 and multiple substrate fragment sequences, the results of which support the assignment of purified JMJD6 as a lysyl hydroxylase. By contrast, we did not observe N-methyl arginyl N-demethylation with purified JMJD6. Biophysical analyses, including crystallographic analyses of JMJD6\u0394344\u2013403 in complex with iron and 2OG, supported its assignment as a lysyl hydroxylase rather than an N-methyl arginyl-demethylase. The screening results supported some, but not all, of the assigned JMJD6 substrates and identified other potential JMJD6 substrates. We envision these results will be useful in cellular and biological work on the substrates and functions of JMJD6 and in the development of selective inhibitors of human 2OG oxygenases.JmjC domain-containing protein 6 (JMJD6) is a 2-oxoglutarate (2OG)-dependent oxygenase linked to various cellular processes, including splicing regulation, histone modification, transcriptional pause release, hypoxia sensing, and cancer. JMJD6 is reported to catalyze hydroxylation of lysine residue(s) of histones, the tumor-suppressor protein p53, and splicing regulatory proteins, including u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65). JMJD6 is also reported to catalyze  A)  .N-methyl lysyl and N-methyl arginyl demethylases  (B)  (B), but the cellular relevance of this remains unclear  B 4). Jm. JmN-metaldehyde B 5). In. InN-methyde (B) B, but thC) (R hydroxylase), JMJD7 (a lysyl C-3S hydroxylase), and the ribosomal oxygenases MINA53 and NO66 (both histidine-residue C-3S hydroxylases)  . A protoC) , 9. Othexylases) \u201312. Manyylases) \u2013\u201312. Strulases) \u2013\u2013, 13, altN-methyl arginyl demethylase and lysyl C-5 hydroxylase activities (C)  with a consequently associated role in apoptosis  that is characteristic of the Fe(II) and 2OG-dependent oxygenases  , a predicted AT-hook motif (Lys283\u2013Ser326), a potential SUMOylation site (Leu316\u2013Asp319), and a C-terminal polyserine (poly-Ser) region   \u201317N-methpoptosis . Subsequpoptosis , 20, altpoptosis . Structuygenases , 20, 22.ygenases , 23. JMJygenases , 25, conFig. S1) . Human JFig. S1) .et al. (N-methyl arginyl demethylase (RDM) acting on mono- and symmetric/asymmetric di-methyl forms of arginine residues as observed in studies with isolated enzyme (B). JMJD6 is also reported to catalyze RDM reactions on N-methylated forms of heat-shock protein 70-kDa (HSP70)  , estroge (HSP70) , RNA hel (HSP70) , and the (HSP70) . Althoug (HSP70) , 31, 32.C) of the splicing regulatory (SR) proteins luc-7\u2013like 2 (LUC7L2), cisplatin resistance-associated overexpressed protein (CROP), and u2 small nuclear ribonucleoprotein auxiliary factor 65-kDa subunit (U2AF65) (S)-stereochemistry (C) (R)-product  . JMJD6-chemistry C 16), c, cC) of -product . JMJD6 i-product . More re-product .JMJD6-knockout mice dying near birth due to growth impairment of multiple organs, including heart, brain, lung, kidney, and eye , and two C-terminally truncated variants, i.e. JMJD6 comprising residues 1\u2013362 (JMJD6\u0394363\u2013403) and residues 1\u2013343 (JMJD6\u0394344\u2013403) . Circular dichroism (CD) spectroscopic analyses imply similar secondary structure content for each of the three JMJD6 variants .Because our objective was to investigate the catalytic reactions catalyzed by isolated JMJD6, the activity of which can be compromised due to oligomerization , 31, we 267\u2013278 substrate . The apparent binding constant (DKapp) of 2OG was determined using CPMG-edited NMR . mKapp values for 2OG were determined using the standard hydroxylation assay procedure and were 31, 24, and 45 \u03bcm for JMJD6FL, JMJD6\u0394363\u2013403, and JMJD6\u0394344\u2013403, respectively , consistent with Mantri et al. . In the presence of the LUC7L2267\u2013278 substrate with JMJD6\u0394344\u2013403, JMJD6\u0394363\u2013403, and JMJD6FL, these values increased to 1.8, 8.4, and 3.6 \u03bcm succinate, respectively . Thus, under both assay conditions, succinate formation for JMJD6\u0394363\u2013403 and JMJD6FL was higher than JMJD6\u0394344\u2013403, indicating a possible role of the C-terminal poly-Ser region in enhancing catalysis and/or of active protein stability. However, given the role of the poly-Ser region in oligomerization  solution being added prior to reaction, with the same outcome, i.e. with the poly-Ser region containing proteins being more active . Given that JMJD6\u0394363\u2013403 was the most active and stable variant tested, further assays were conducted with it.Hydroxylation assays using the reported LUC7L2ubstrate  revealedited NMR ; the KDai et al.  who reporization  and the \u0394363\u2013403 under standard assay conditions: H3R2(me2s)1\u201325, AR(me2s)TKQTARKSTGGKAPRKQLATKAA GKGGKGLGKGGAKRHRKVLRDNIQGIT GKGGKGLGKGGAKRHK-biotin . When treated with JMJD6, hydroxylation of K4histone3, K9histone3, K14histone3, K5histone4, and K8histone4 was observed , consistent with the MALDI results. Note, we did not accrue any evidence for JMJD6 catalyzed hydroxylation of arginine residues as reported for JMJD5 1\u201330 .Three histone peptides with symmetrically di-methylated arginine residues, all of which have been reported as JMJD6 RDM substrates , 40, werKQLATKAA ; H4R3. Furthermore, when a histone peptide monomethylated at Lys9 (H3K9me11\u201317) was tested with JMJD6\u0394363\u2013403, a +16-Da mass shift was observed, implying hydroxylation and not demethylation . This observation also suggests JMJD6 does not catalyze histone lysyl demethylation, consistent with an earlier report (9(me1) compared with Lys9 was relatively low , which suggests that under our assay conditions JMJD6 prefers to hydroxylate unmodified lysine residues and provides evidence that JMJD6-catalyzed lysyl hydroxylation has the potential to be affected by N\u03f5 lysine modifications.Evidence for JMJD6H3K9me11\u2013 was test469) is demethylated by JMJD6 as observed by MS .HSP70 has been reported as a JMJD6 substrate wherein the mono-methylated arginine residue  . Time-course assays were then performed , with peptides displaying \u226525% hydroxylation after 6 min in kinetic studies  . To inve studies .267\u2013278 were as follows: mKapp, 51.0 \u03bcm; kcat, 106 \u00d7 10\u22124 s\u22121; and kcatm/Kapp, 208 \u00d7 10\u22126 \u03bcm\u22121\u00b7s\u22121. Of the tested U2AF65 peptide fragments, U2AF6521\u201335  was found to be a better substrate than U2AF6530\u201346  and U2AF6555\u201370  . Other peptides tested, e.g. from the SR protein CROP, gave similar kinetic parameters . Of the SRSF11 fragments tested, JMJD6\u0394363\u2013403 hydroxylated SRSF11358\u2013372 with a relatively high efficiency . With RBM39 (RNA-binding protein 39), hydroxylation levels were found to vary with measured mKapp values ranging within 56.0\u201399.0 \u03bcm . Of the tested RBM39 fragments, RBM3931\u201342 was substantially more efficiently hydroxylated than the others tested, with mKapp 56.0 \u03bcm and kcatm/Kapp 455 \u00d7 10\u22126 \u03bcm\u22121\u00b7s\u22121. These results are consistent with the proposal that JMJD6 is promiscuous with respect to its lysyl hydroxylase activity in cells , suggesting possible hydroxylation of Lys171. Assays with the alanine variant pVHL166\u2013177K171A and with WT pVHL166\u2013177 in the presence of a JMJD6 inhibitor, 2,4-pyridine dicarboxylic acid ,BFig. S24). LC-MS/MS analysis of JMJD6\u0394363\u2013403-treated pVHL166\u2013177 provided further evidence for pVHL Lys171 hydroxylation .The apparent promiscuity of isolated JMJD6 with RS domains of SR proteins prompted us to test the potential of other reported JMJD6 substrates. The von Hippel-Lindau protein (pVHL) has been reported as a potential JMJD6 substrate  and was N-demethylation of methylated  Arg260 of ER\u03b1 and thereby regulates methylated ER\u03b1 function in response to estrogen (260 (MKGGIRKDRR(me2s/a)GG) were tested with JMJD6\u0394363\u2013403, and ER\u03b1 demethylation was not observed . Interestingly, a +16-Da mass shift, corresponding to hydroxylation, was observed for both peptides . In light of the results with histone peptides, we considered it possible that JMJD6 catalyzes hydroxylation of either or both of the lysine residues (Lys252 and Lys257) in the ER\u03b1 sequence. Assays using ER\u03b1 peptides with unmodified Arg260 also revealed JMJD6\u0394363\u2013403-dependent hydroxylation . Hydroxylation was ablated by alanine substitution ER\u03b1251\u2013262K252A, ER\u03b1251\u2013262K257A, or by addition of the JMJD6 inhibitor 2,4-PDCA4 . These results suggest Lys252 and Lys257 of ER\u03b1 are possible targets for JMJD6 hydroxylation and are further evidence that JMJD6-catalyzed hydroxylation is possibly regulated by other post-translational modifications.Based on Western blot analysis, it has been reported that JMJD6 catalyzes estrogen . Peptide382 .JMJD6 lysyl hydroxylase activity has also been associated with post-translational modification of the tumor-suppressor protein p53, wherein the target residue is Lys382 . Assays \u0394344\u2013403 in the presence of Fe(II) and 2OG was performed in an anaerobic glove box as reported for FIH ..\u0394344\u2013403\u0394344\u2013403\u00b7Fe\u00b72OG crystals belong to the same space group (P21) with similar unit cell dimensions to that reported by Mantri et al. (Table S1). The structure was solved by molecular replacement using PDB code 3K2O as a search model  with no Ramachandran or side-chain conformational isomer outliers ) .The JMJD6i et al.  ) . However  core fold (B) characteristic of the 2OG oxygenases, which is located within the JmjC domain and harbours the Fe- and 2OG-binding residues. Importantly, the JMJD6\u0394344\u2013403\u00b7Fe\u00b72OG structure reveals the geometry of the JMJD6 active-site coordination chemistry in the presence of its Fe cofactor and 2OG cosubstrate , a feature characteristic of most 2OG oxygenases (Asp is sometimes substituted with Glu or Gly) (C). JMJD6 His187 and Asp189 are located on the loop linking \u03b2II and \u03b2III, whereas His273 is located on DSBH \u03b2VII (B). Mutagenesis studies reveal the importance of the Fe-binding residues in JMJD6 catalysis . Although the JMJD6\u0394363\u2013404\u00b7D189A variant manifests apparent low-level formation of succinate in the absence of substrate , succinate formation was not observed in the presence of LUC7L2267\u2013278 within the limits of detection , implying the D189A variant does not support productive hydroxylation catalysis. This provides further evidence that substitution of the metal-binding residues of 2OG oxygenases does not always completely block catalysis; for example, in the case of FIH, it has been reported that substitution of the Fe binding aspartate to a glycine is insufficient to completely ablate hydroxylation activity  C. JMJD6 SBH \u03b2VII B. Mutageactivity .et al. (204 (\u03b2IV) and to make hydrogen-bonding interactions with residues Tyr131 (located immediately to the N terminus of \u03b2I), Thr184 (\u03b2II), and Asn197 (\u03b2III) (131 and Thr184 were observed in 3K2O (204 (3LDB), as reported by Hong et al. . Note in the JMJD6\u0394344\u2013403\u00b7Fe\u00b72OG structure, the 2OG C-1 carboxylate is trans to the distal His273 of the binding triad (His273JMJD6) rather than being trans to the proximal His187 (His187JMJD6), as is also observed in FIH, JMJD5, JMJD2A, and PHF8  structures  . Similar in 3K2O , whereinK2O . This is similar to other structurally characterized JmjC domain-containing proteins (B\u2013E) and likely contributes to the ability of JMJD6 to bind the positively charged basic groups of its substrate lysyl side chain as well as other lysyl-/arginyl-residues in the  basic JMJD6 substrates, e.g. histones and RS domains of SR proteins.Analysis of the electrostatic surface potential  of the Je charge A. This iproteins , B\u2013E andFig. S29), Hong et al. , with basic residues involved in these interactions, including Lys4, Lys7, Arg8, Arg48, Lys91, Arg92, Arg95, Lys111, Lys115, Lys144, Lys151, Lys154, Arg305, Lys307, and Arg310 .Following the observation of ordered sulfate molecules at conserved positions across positively-charged sites on the surface of JMJD6 , which has an additional PHD, and the JMJD2 (KDM4) JmjC KDMs . HomjC KDMs , appear e.g. due to the presence of targeting domains/proteins, knowledge of the types of reaction directly catalyzed by the 2OG oxygenase domain in vitro can assist in subsequent functional assignment work in vivo. Given its checkered history , we carried out studies on reported JMJD6 N-methyl arginyl \u201cdemethylation\u201d and lysyl \u201chydroxylation\u201d substrates . In our studies, we found no evidence that purified JMJD6 has N-methyl arginyl demethylase (RDM) activity (Table S2). Positive controls for RDM activity employing KDM4E (Table S2)  . In addig et al.  have rep . Lys382 of the tumor-suppressor protein p53 is reported to be hydroxylated by JMJD6 . Peptide fragments of two other biochemically important reported targets, i.e. pVHL and ER\u03b1 . Although we did not observe JMJD6-catalyzed RDM activity on ER\u03b1 as was reported .In addition to multiple SR domain substrates , we founby JMJD6 , and we  and ER\u03b1 , 37, werreported , hydroxypVHL is a tumor-suppressor protein in kidneys, which has a crucial role in dioxygen dependent proteasome-mediated degradation of prolyl-hydroxylated forms of \u03b1-subunits of the transcription factor HIF in many animal cells , 51, 52.6-tagged human full-length JMJD6  was inserted into the pET28a(+) expression vector. Expression plasmids for two more variants  were prepared by inserting a stop codon after the desired site using site-directed mutagenesis (see Table S3 for primer sequences). JMJD6 variants were expressed in Escherichia coli Rosetta2(DE3) cells  and stored at \u221280 \u00b0C. Cell pellets were resuspended in lysis buffer , DNase I , and 0.5% Nonidet P-40). The lysates were loaded onto a 5-ml HisTrap column , equilibrated with binding buffer  using an AKTA purifier . The column was washed with wash buffer . JMJD6 was eluted with elution buffer  using a linear gradient (0\u2013100%). Based on UV-trace (280 nm) and SDS-PAGE analyses, fractions with purified JMJD6 were pooled and concentrated to 2 ml using a membrane filter (30-kDa molecular mass cutoff membrane). Concentrated protein was loaded onto a Superdex 200 size-exclusion column , pre-equilibrated with size-exclusion buffer , for further purification. The desired fractions  were concentrated to 25\u201330 mg/ml, then flash-frozen in liquid nitrogen, and stored at \u221280 \u00b0C. Catalytic mutants of the variant JMJD6\u0394363\u2013403 were prepared by site-directed mutagenesis (see Table S3 for primer sequences) and were purified by following the same purification procedure as for WT.DNA encoding for the N-terminal His3) cells , 23. Celm phosphate buffer, pH 8.0. Spectra were acquired using a quartz cuvette with a 0.1-cm path length, between 260 and 185 nm at 0.2 nm\u00b7min\u22121 increments, a response time of 1 s, and a data pitch of 0.5 nm. Spectra were averaged over four scans and corrected for solvent background. To investigate secondary structure content, the CD secondary structure deconvolution method was applied to the data, which were accessed using the DICHROWEB server (CD spectra were acquired using a Jasco J-815 spectrometer (Jasco). Protein solutions were buffered with 10 mB server , 54.m JMJD6, 100 \u03bcm (NH4)2Fe(SO4)2\u00b76H2O (Sigma), and 400 \u03bcml-sodium ascorbate (Sigma) were mixed in deuterated Tris-d11 (Sigma), pH 7.5, in a 1.5-ml Eppendorf tube. Substrate (either only 500 \u03bcm 2OG or a mixture of 500 \u03bcm 2OG and 100 \u03bcm LUC7L2267\u2013278) was added to initiate the reaction. The mixture was immediately transferred into a 2-mm NMR tube, and the acquisition of 1H spectra was started after 3.5\u20135 min.2OG turnover assays employed a reported procedure . 10 \u03bcm Jm 2OG and 200 \u03bcm Zn(II) were mixed in deuterated Tris-d11, pH 7.5, and the 1H NMR signals for 2OG were recorded at 700 MHz (DKapp) were measured using obs represents change in an observable NMR parameter from the titration; \u0394total represents the total change of the monitored NMR parameter; [L]0 represents the titrated ligand concentration; and [P]0 represents the protein concentration. Typically, a 5\u201310% error was allowed in the curve-fitting process.20 \u03bc 700 MHz . Titratix \u2212 (\u03c4 \u2212 180\u00b0y \u2212 \u03c4 \u2212 90\u00b0y \u2212 \u03c4 \u2212 180\u00b0y \u2212 \u03c4)n \u2212 acquisition) was applied (n = 6); acquisition time 2.94 s; relaxation delay 2 s; number of transients 128. Water suppression was achieved by presaturation 2Fe(SO4)2\u00b76H2O (Sigma), 400 \u03bcml-sodium ascorbate (Sigma), and 500 \u03bcm disodium 2OG (Fluka). After 30 min, the reaction was quenched by an equal volume of 1% (v/v) aqueous formic acid (Sigma). In a 96-well MALDI plate (Waters), 0.8 \u03bcl of CHCA  (1 \u03bcg in 0.1 ml of CHCA solvent) and 0.6 \u03bcl of the quenched mixture were placed in a well. After drying, the samples were analyzed by MALDI-TOF MS using a reported procedure 2Fe(SO4)2\u00b76H2O (Sigma), 500 \u03bcml-sodium ascorbate (Sigma), and 1 mm di-sodium salt of 2OG (Fluka) for 1 h or more  and subsequent CID MS/MS spectra  of the 10 most intense peaks were acquired in the Orbitrap. CID fragmentation was performed at 35% of the normalized collision energy, and the signal intensity threshold was kept at 500 counts. ETD spectra  were acquired in the ion trap. PEAKS\u00ae 8.0  was used to analyze raw data. The raw MS files were searched against the respective protein sequences. NONE was selected as the protease. Demethylation (\u221214 and \u221228 Da) and hydroxylation  were set as variable modifications. The precursor mass tolerance was 15 ppm. The fragment mass tolerances for CID and ETD were set to 0.8 Da. All spectral assignments were manually validated.Samples were desalted using a ZipTip (Millipore) and then analyzed using a NanoAcquity-ultraperformance liquid chromatography system (Waters) connected to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific), possessing an EASY-Spray nano-electrospray ion source (Thermo Fisher Scientific). Peptides were \u201ctrapped\u201d on an in-house packed guard column  using solvent A (0.1% (v/v) aqueous formic acid) at a pressure of 140 bar. Peptides were separated on an EASY-Spray Acclaim PepMap\u00ae analytical column  using a linear gradient , flow rate, 300 nl/min). The separated peptides were electro-sprayed directly into the spectrometer operating in a data-dependent mode using a collision-induced dissociation/electron transfer dissociation (CID/ETD)-based method. Full scan MS spectra  at a concentration of \u226525 mg/ml (500\u2013600 \u03bcm) was used. JMJD6\u0394344\u2013403\u00b7metal\u00b7ligand complexes were formed by incubating enzyme with the other components  on ice for \u223c1 h. A PhoenixTM RE crystallization robot (Art Robbins Instruments) and a Minstrel-HTTM platform with CrystalTrekTM  were used to prepare and monitor crystallization trials. Crystals were grown using the sitting-drop vapor-diffusion method . Crystals were cryo-protected using a mixture of reservoir solution diluted to 25% (v/v) glycerol and cryo-cooled by plunging into liquid nitrogen. Diffraction data were collected at 100 K at the Diamond Light Source synchrotron. PHASER was used for molecular replacement using PDB code 3K2O (Crystallization trials were performed using commercially available broad screens followed by optimization. JMJD6n method  either in method . The optode 3K2O  as the sode 3K2O , 63.\u0394344\u2013403\u00b7Fe\u00b72OG structure are deposited in the Protein Data Bank as code 6GDY.Coordinates and structure factors for the JMJD6M. S. I., M. A. M., R. C., and C. J. S. conceptualization; M. S. I. and M. A. M. data curation; M. S. I. and M. A. M. software; M. S. I. and M. A. M. formal analysis; M. S. I. and C. J. S. funding acquisition; M. S. I. and M. A. M. validation; M. S. I., J. G., A. K., and E. P. investigation; M. S. I. and M. A. M. visualization; M. S. I. methodology; M. S. I., M. A. M., and C. J. S. writing-original draft; M. A. M., R. C., and C. J. S. supervision; M. A. M. and C. J. S. project administration; M. S. I., M. A. M. and C. J. S. writing-review and editing; C. J. S. resources."}
+{"text": "It is known that HRmax decreases with high altitude and the duration of the stay (acclimatization). At an altitude typically chosen for training  conflicting results have been found. Whether or not this decrease exists or not is of importance since the results of previous studies assessing hypoxic training based on HR may be biased due to improper intensity. By pooling the results of 86 studies, this literature review emphasizes that HRmax decreases progressively with increasing hypoxia. The dose\u2013response is roughly linear and starts at a low altitude, but with large inter-study variabilities. Sex or age does not seem to be a major contributor in the HRmax decline with altitude. Rather, it seems that the greater the reduction in arterial oxygen saturation, the greater the reduction in HRmax, due to an over activity of the parasympathetic nervous system. Only a few studies reported HRmax at sea/low level and altitude with patients. Altogether, due to very different experimental design, it is difficult to draw firm conclusions in these different clinical categories of people. Hence, forthcoming studies in specific groups of patients are required to properly evaluate (1) the HRmax change during acute hypoxia and the contributing factors, and (2) the physiological and clinical effects of exercise training in hypoxia with adequate prescription of exercise training intensity if based on heart rate.The use of exercise intervention in hypoxia has grown in popularity amongst patients, with encouraging results compared to similar intervention in normoxia. The prescription of exercise for patients largely rely on heart rate recordings (percentage of maximal heart rate (HR Intermi2) at a specific power output is similar at sea level and altitude  exposure to a high altitude such as encountered during mountaineering  and/or inspired oxygen fraction (%). A classification was first completed to select articles reporting HRmax data were collated from 81 publications involving healthy subjects and 5 publications involving patients , a systematic assessment of hypoxic intervention was not done.2max could not be fully explained by the reduction in arterial oxygen content (CaO2)  is usually associated with lower HRmax  = \u22120.0024 \u00d7 altitude (m) + 0.73. Hence, a 1,000 m gain will reduce HRmax by 1.7 bpm. This is in agreement with, e.g., the 1.9 bpm per 1,000 m found in young subjects by Wehrlin et al. and with the 9 bpm decrease of HRmax observed at 4,300 m  = 116\u20130.0057x . Compared to our findings, this model tends to underestimate the decrease at low altitudes and overestimate the decrease at high altitudes . For a 20 years old subject, this represents a decrease of 2.5% (assuming HRmax = 200 bpm in normoxia). For a 70 years old subject, this represents 3.3% (assuming HRmax = 150 bpm in normoxia). As older subjects already have cardiac limitations, this further decrease may result in an insufficient HR to maintain performance and a tendency to reach exercise intolerance at the suggested exercise target HR.The large variability in the decrement in HRmax with altitude , the decrement in HRmax appears a little more pronounced for untrained than trained subjects  = \u22120.0026 \u00d7 altitude (m) + 1.2486 and HRmax (bpm) = \u22120.0025 \u00d7 altitude (m) + 1.4263, respectively. However, this observation is based on studies using different testing and hypoxic conditions and should be viewed with caution. Also, the definition of the \u201cuntrained\u201d and \u201ctrained\u201d states by the authors has not been harmonized as it should be , the trend is completely different with a paradoxical grseater decrease in HRmax at the lowest reported altitude  limiting the relevance of this finding. Interestingly, in one study with heart failure patients, an unexpected increase in HRmax of 8 bpm was reported at 3454 m compared to 540 m  variability exists. But this decrement should be clearly specified, as it has been previously reported in a review paper, which focused on the use of exercise training in hypoxia that hypoxia trigger an increase in both basal and maximal HRmax, which is contradictory to the current observation  . The absolute HR were identical in both normoxic and hypoxic groups in the study of Bailey et al. , an overestimation of 3 bpm (2.8%) and 5 bpm (2.8%) of target HR for moderate (40% HRmax) and vigorous (90% HRmax) intensity exercise, respectively, would have been observed for an exercise training session at 2,500 m. At 3,000 m, it would have been 4 bpm (3.5%) and 6 bpm (3.5%), respectively. Focusing only on the altitude gain ranging from 2,500 to 3,500 m  and 4 bpm (2.3%) for moderate and vigorous exercise, respectively. Also, resting HR increases under hypoxia due to increase sympathetic and decrease parasympathetic activity  and 3 bpm (2.2%) for moderate and vigorous intensity calculated from HRR. Using the data presented in Figure max group 50-59 ml/min/kg, the overestimation is roughly the same for each given example. However, using the data presented in Figure max is very close to the predicted HRmax indirectly derived from the age. But such derived HRmax could lead to large error in the prediction  for submaximal and maximal HR could be considered as high, since they have been reported to be greater than 0.9, the higher the HR, the lower the variation  and therefore also depends on the duration of hypoxic exposure. It is not known if over the course of a training program HRmax decreases in the same way, even if unlikely (a huge difference between daily exposure on one side and 3-5 sessions of 30-90 min per week exists in terms of hypoxic exposure). Hence, much remains to be done in this field and forthcoming studies with appropriate designs need to be conducted to answer questions in both the physiological and clinical area. In the first category, the effects of hypoxia on HRmax  amongst specific populations have to be better addressed, with a specific focus on sex and age. Also, it is of importance to better assess these effects in specific pathologies (in particular cardiovascular and metabolic diseases), as well as determining potential interaction with drugs. The difference between normobaric and hypobaric hypoxia should also be addressed. In all of these situations, the specific impact of SaO2 and the autonomic nervous system should be taken into consideration. In the second category, a thorough evaluation of the potential superiority of exercise training in hypoxia over normoxia on relevant outcomes need to be done with a well-controlled design that involves accounting for the placebo effect and an adequate prescription of exercise training intensity based on heart rate. Under conditions of hypoxic exercise training, the long-term effect of hypoxia exposure on HRmax and the potential need to adjust the relative HR target should also be addressed as well as the need to account for chronotropic effects of drugs. In some years, a review similar to the present one would be very instructive.A body of scientific literature exists on the evaluation of passive exposure and/or exercise training in altitude/hypoxic environment in healthy subjects. Despite this, debates still exists on the benefits and physiological effects of hypoxic training. Nevertheless, the use of exercise training in hypoxia has grown in popularity with patients. Studies have provided encouraging results, but the effectiveness and potential superiority of hypoxic training over normoxic training remains to be established. To achieve this, the exercise training programs performed by the experimental and control groups should be comparable to allow a rigorous evaluation of the isolated hypoxic stimulus. The best metric to quantify the exercise training load needs to be specified, but the prescription of exercise for patients largely rely on HR recordings. This review underlines that HRLM: Lead author, study writing. The manuscript presents clearly results, honestly, and without fabrication, falsification, or inappropriate data manipulation. Results of the present study do not constitute endorsement by ACSM.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "TBX18 to the left ventricular apex in the CAVB rats resulted in ectopic ventricular beats within days, achieving a de novo ventricular rate faster than the slow atrioventricular (AV) junctional escape rhythm observed in control CAVB animals. The model offers new opportunities to test therapeutic approaches to treat chronic and severe CAVB which have previously only been testable in large animal models.Complete atrioventricular block (CAVB) is a life-threatening arrhythmia. A small animal model of chronic CAVB that properly reflects clinical indices of bradycardia would accelerate the understanding of disease progression and pathophysiology, and the development of therapeutic strategies. We sought to develop a surgical model of CAVB in adult rats, which could recapitulate structural remodeling and arrhythmogenicity expected in chronic CAVB. Upon right thoracotomy, we delivered electrosurgical energy subepicardially via a thin needle into the atrioventricular node (AVN) region of adult rats to create complete AV block. The chronic CAVB animals developed dilated and hypertrophied ventricles with preserved systolic functions due to compensatory hemodynamic remodeling. Ventricular tachyarrhythmias, which are difficult to induce in the healthy rodent heart, could be induced upon programmed electrical stimulation in chronic CAVB rats and worsened when combined with \u03b2-adrenergic stimulation. Focal somatic gene transfer of  The subsequent slow AV junctional escape rhythm is inadequate to support CAVB patients\u2019 hemodynamic needs, requiring the implantation of an electronic pacemaker device5. If left untreated, the ventricles of CAVB patients are susceptible to spontaneous and drug-induced QT prolongation7, which often precipitates to life-threatening arrhythmias such as torsades de pointes10. An animal model of CAVB would be ideal for studies aimed at understanding mechanisms of catastrophic ventricular tachyarrhythmias11, as well as developing therapies to mitigate arrhythmogenic events in CAVB13. We and others have developed large animal models of CAVB17, but those models are resource-exhausting and not widely available for standard benchtop research. Transgenic mouse models can exhibit spontaneous AV block19, but their degree and persistence of AV block are unpredictable, as CAVB is an adverse side effect of the transgene modification rather than a direct effect. Surgical models of CAVB have been documented in rodents23. However, we found these methods difficult to reproduce and adopt independently. One approach of injecting 70% ethanol into the AVN24, is hindered by a limited number of ablation attempts, as repeated doses of alcohol lead to global heart dysfunction and systemic complications, thus reducing survival. Other approaches require the use of custom-engineered, rodent-sized radiofrequency (RF) ablation catheters23, impeding widespread implementation in conventional laboratories.Complete atrioventricular block (CAVB) is a major cardiac conduction disorder, in which the transmission of an electrical impulse from the atria to the ventricles is completely inhibited25. Here, we present a long-term survival CAVB model in rats achieved via subepicardial delivery of electrosurgical energy into, and the subsequent fibrosis of, the AVN region. CAVB was achieved immediately upon ablation of the AVN and was stable for >4 weeks. The CAVB animals exhibited electrical abnormalities, including spontaneous and inducible ventricular tachyarrhythmias, and progressive ventricular remodeling, evidenced by increased stroke volume and ventricular hypertrophy with preserved ejection fraction at week 4. Notably, our chronic CAVB model revealed striking age and sex differences in survival rates, in which all males and 3-month old females died within the first 72\u2009hours of creating CAVB, suggesting that the slow junctional escape rhythm is insufficient for supporting male and 3-month old female rats.We sought to develop a rodent model of CAVB that requires only a routine set of surgical instrumentation, yields a high success rate in 6-month old female rats, persists long-term (4 weeks), and recapitulates ventricular arrhythmias that are often manifested in CAVB patientsTBX18, a concept we have previously demonstrated for converting chamber cardiomyocytes to induced pacemaker cells27. Direct intramyocardial delivery of TBX18 into the left ventricular apex of chronic CAVB rats revealed emergence of ectopic ventricular beats at a rate higher than the slow AV junctional rhythm of the control animals. The TBX18-induced ventricular pacing is the first demonstration of the technique\u2019s feasibility and disease-modifying potential, when the biologic is delivered after chronic CAVB had been established.We also sought to reverse the severe CAVB-induced ventricular bradycardia by focal somatic gene transfer of We attempted to disrupt AVN conduction by focal ablation with electrical energy. We elected to use an electrosurgical unit over electrocautery, because electrosurgery delivers high-frequency electrical energy, traveling beyond the tissue-electrode contact site. On the other hand, electrocautery, which delivers heat only, is limited to burning tissues at the contact site. Initial trials of electrical energy delivery by direct contact of an electrosurgical pen on the AVN region caused an epicardial scar that remained largely superficial. This failed to generate reproducible CAVB. In order to deliver electrical energy subepicardially, we employed a thin needle of a 0.3\u2009mm diameter Fig.\u00a0. This neThe following steps were taken prior to delivering electrosurgical energy: The bent distal tip of the ablation needle was advanced into the epicardial surface, posterior to the fat pad and posterolateral of the aorta, and pointing toward the left ventricular apex. This maneuver often elicited skipped ventricular beats, transient AV dissociations indicative of mechanical disruption of AVN or high His bundle conduction, and/or premature ventricular complexes (PVCs) Fig.\u00a0. Parallep\u2009=\u20090.49). However, the male rats weighed significantly more at 504\u2009\u00b1\u200915\u2009g compared to the age-matched female rats weighing 372\u2009\u00b1\u200963\u2009g . We asked if the mortality in the males was strictly due to sex difference, and performed CAVB in 3-month old female rats weighing 271\u2009\u00b1\u200916\u2009g. The 3 month old female rats also expired in 72\u2009hours after CAVB induction , but slower than the 6-month old female rats . Subcutaneous injection of isoproterenol during the post-op recovery period transiently increased the slow junctional rhythm of the 3-month old animals, but did not aid in the survival of those animals beyond three days post-op. To learn if the differences in the acute survival rates CAVB animals may be due to developmental stages rather than to sex difference per se, we examined the normal compact AVN areas near the central fibrous body of 6-month old female rats, 6-month old male rats, 3-month old female rats, and 3-month old male rats (n\u2009=\u20093 for each group). The compact AVN region near the central fibrous body was distinctively smaller in the 3-month old rats of both sexes compared to that of the 6-month old rats . Likewise in male rats, the mean area of the AVN was 0.052\u2009\u00b1\u20090.028 mm2 in 3-month old, compared to 0.177\u2009\u00b1\u20090.034 mm2 in 6-month old males . No significant difference was found in AVN area between age-matched male and female rats . Immediately upon CAVB, the ventricular rate of the 6-month old male rats was 106\u2009\u00b1\u200915 bpm, statistically similar to that of the 6-month old female rats at 123\u2009\u00b1\u200939 bpm  or PVCs Fig.\u00a0 were obsTBX18, converts chamber cardiomyocytes to induced-pacemaker cells27. Focal delivery of TBX18 into the ventricular myocardium gave rise to ectopic ventricular beats in both small27 and large12 animal models, providing evidence for a transcription factor-based biological pacemaker. Although our porcine model of CAVB closely mimicked clinical CAVB31, our previous small animal model of ventricular bradycardia relied on transient AV dissociation resulting from non-specific, cholinergic suppression of AV conduction in anesthetized rodents32. Using the current rat model of chronic CAVB, we focally delivered an adenoviral vector expressing TBX18 (Adeno-TBX18) into the left ventricular apex of 6-month old female rats upon confirming stable and complete AV block at 1 week after ablation surgery  control animals  resolved two distinct peaks in the heart rates that lasted beyond 1 week post-delivery; one peak at a rate similar to the slow junctional rhythm of the control animals, and a second peak at a faster ventricular rate . Surprisingly these younger, smaller male rats showed a significantly higher mortality rate compared to their older counterparts , indicating an additional age-dependent effect on mortality. Further investigation showed a similarly increased mortality rate in 3-month female rats with CAVB . In both\u00a0sexes, 3-month old rats were shown to die of ventricular bradycardia and sudden cardiac arrest  hearts containing a smaller AV nodal area, have significantly slower ventricular rates than their older counterparts following AV ablation. As shown in the ECG data  was applied following induction of anesthesia to prevent corneal drying. Monopolar electrosurgical current was delivered to the AVN region via an active electrode connected to a high frequency electrosurgical generator in a coagulation mode . A reusable dispersive grounding pad electrode , serving as the return electrode, was placed on the back of the rat. The back of the rat was shaved to minimize electrical resistance between the animal and grounding pad. We found that a larger surface area grounding pad reduced the resistance and increased electrical current, thereby reducing the voltage necessary to achieve the same delivered power. Smaller ground pads or poor contact between the skin and the return electrode significantly reduced the effectiveness of AVN ablation and generated excessive heat at the skin-ground pad contact site, leading to severe skin burns. In order to ensure a sufficient skin-ground pad interface, we found that application of ultrasound electrode gel between the skin and ground pad significantly increased the effectiveness of electrosurgical AVN ablation. During surgery, body temperature was maintained at 37\u2009\u00b0C with a water blanket . Carprofen (5\u2009mg/kg) and Buprenorphine SR (1\u2009mg/kg) were given subcutaneously for analgesia. Atropine (0.05\u2009mg/kg) was given intramuscularly to reduce pulmonary secretion. Upon ablation of the AVN, the thoracic cage was repaired with a 4-0 polyglycolic acid absorbable suture  and the lungs were fully inflated for two seconds to avoid pneumothorax immediately before closing the thoracic cavity. External thoracic muscles were repaired layer by layer with 4-0 absorbable suture. Skin was sutured with 4-0 monofilament polypropylene non-absorbable suture . The isoflurane ventilation was reduced to zero after completion of the surgery. Once spontaneous breathing was evident, the endotracheal tube was removed. If excessive blood loss occurred (>2\u2009mL), 2\u2009mL of 0.9% sodium chloride solution was administered subcutaneously. Carprofen (5\u2009mg/kg) was administered subcutaneously as a post-surgical analgesic at 24, 48, and 72\u2009hours after surgery.Sprague Dawley rats  were anesthetized in an induction chamber with 5% isoflurane in 100% oxygen at a constant flow rate of 2\u2009L/min for 6\u2009minutes. Endotracheal intubation was performed with a 14-gauge, 57\u2009mm long intravenous catheter. The animals were ventilated with a respirator  at 85 respirations per minute with a 2.5\u2009ml stroke volume. Anesthesia was maintained in 2% isoflurane mixed with 100% oxygen throughout the surgery. Sterile lubricating eye ointment  were recorded on anesthetized animals with a BioAmp (FE135) signal amplifier, a PowerLab 8/35 digitizer and analyzed with LabChart Pro . Baseline ECGs were acquired just after intubation under anesthesia with 2% isoflurane. A partial right thoracotomy was performed in the third intercostal space to expose the right atrium and aorta. After retracting the thymus and removing pericardium, the right atrial appendage was partially ligated and the right atrium laterally retracted so as to avoid excessive tissue damage or bleeding during ablation. This maneuver exposed the epicardial aspect of atrioventricular nodal area, landmarked by the characteristic fat pad consistently located between the aortic root and the medial wall of the right atrium. The negative electrode for lead II was replaced with the electrode connected to the ablation needle for epicardial electrogram. This was achieved by attaching the electrode of a surface ECG cable  to the proximal end of a 0.3\u2009mm diameter, 5\u2009cm long ablation needle  in the abdominal cavity according to the manufacturer\u2019s instructions. Lead I was obtained by positioning the electrodes on the left and right latissimus dorsi muscles. Lead II was obtained by positioning the electrodes between the suprasternal fossi and left lower thoracic cage. The electrical signals were transmitted to SmartPad  and analyzed with LabChart Pro .Echocardiography was performed immediately before the surgery, then serially at one week and one month post-surgery with Vevo 2100 High Frequency ultrasound imaging system  and analyzed with Vevo Imaging Workstation. Inter-ventricular septal thickness (IVS), LV posterior wall thickness (LVPW), and LV internal diameter (LVID) were measured in short axis during systolic and diastolic phases on M mode imaging. From these measurements, LV end diastolic volume (LVEDV), LV end systolic volume (LVESV), LV fractional shortening (LVFS), LV ejection fraction (LVEF), and approximated LV myocardium mass were calculated.29. Pacing thresholds were determined, and stimulation was delivered at a 1\u2009ms pulse width at twice the capture threshold. An S1 drivetrain of 10 stimuli  was applied followed by an extra stimulus (S2) starting at a coupling interval of 80\u2009ms and by 1\u2009ms decrements until the effective refractory period was reached. Another extra stimulus (S3) followed S2, decremented by 1\u2009ms until the ventricular effective refractory period was reached. After this, a train of programmed stimulation with three extra stimuli after S1 (S2\u2013S4) was performed until a ventricular arrhythmia was induced. If the rat failed to develop ventricular arrhythmia with 3 extra stimuli (S1\u2013S4), the animal was deemed non-inducible.Ventricular arrhythmias were induced on five CAVB rats one month after CAVB creation. Animals were anesthetized with 5% isoflurane and placed on a mechanical ventilator after intubation. Anesthesia was maintained with 2% isoflurane during programmed electrical stimulation (PES). The right jugular vein of the anesthetized CAVB rat was cannulated with a 21-gauge polyethylene tubing which was navigated into the right ventricle. Using the polyethylene tubing as a guide sheath, a platinum wire was inserted through the lumen of the tubing into the right ventricular cavity until ventricular electrical potentials could be detected. PES was performed with current stimulus isolator  with or without isoproterenol  following our previous protocolThe heart from seventeen rats were harvested, fixed in 10% formalin for 24\u2009hours and paraffin-embedded for histopathological examination of the AVN region. The animals were grouped as follows: 3-month old normal male (n\u2009=\u20093), 3-month old normal female (n\u2009=\u20093), 6-month old normal female (n\u2009=\u20093), 6-month old normal male (n\u2009=\u20093) and 6-month old female with CAVB (n\u2009=\u20095). The paraffin-embedded tissue samples were sectioned at 5\u2009\u00b5m thickness. Masson\u2019s trichrome staining was performed to examine the native AVN structure and subsequent tissue necrosis post-electrosurgical energy delivery. Staining was performed according to manufacturer\u2019s protocol . Stained sections were scanned with Hamamatsu Nanozoomer . We measured the fibrotic area from upper margin of IVS to lower margin of IAS including the annulus ring of the mitral valve. The mitral valve leaflet was not included. Fibrotic area was measured by manually drawing a region of interest (ROI) around the circumference of blue collagen tissue. ROI area was measured using NDP.view2 software.TBX18-IRES-zsGreen1 (n\u2009=\u20094) or GFP (n\u2009=\u20094) to the heart. For each animal, 1\u2009\u00d7\u2009108 fluorescence forming units (ffu) of the adenoviral vector was injected in 100\u2009\u00b5l of volume into the left ventricular apex of the heart. A telemetry device was implanted immediately after injection of the viral vector.Upon creating CAVB, the animals were allowed to recover from the right thoracotomy and monitored for one week to confirm stable CAVB. Upon validating stable CAVB, the animals underwent a partial left thoracotomy in the fourth intercostal space to deliver an adenoviral vector expressing either All data are expressed as mean\u2009\u00b1\u2009standard deviation (SD) at a 95% confidence limit. For longitudinal comparison of continuous variables such as mean heart rates, weight, and echocardiographic parameters, the statistical significance was tested using repeated-measures ANOVA with subsequent Bonferroni multiple comparisons test. Statistical analysis was performed using SPSS version 23.0 .Supplementary figure 1Supplementary video 1Supplementary video 2"}
+{"text": "RNA binding protein (RBP) plays an important role in cellular processes. Identifying RBPs by computation and experiment are both essential. Recently, an RBP predictor, RBPPred, is proposed in our group to predict RBPs. However, RBPPred is too slow for that it needs to generate PSSM matrix as its feature. Herein, based on the protein feature of RBPPred and Convolutional Neural Network (CNN), we develop a deep learning model called Deep-RBPPred. With the balance and imbalance training set, we obtain Deep-RBPPred-balance and Deep-RBPPred-imbalance models. Deep-RBPPred has three advantages comparing to previous methods. (1) Deep-RBPPred only needs few physicochemical properties based on protein sequences. (2) Deep-RBPPred runs much faster. (3) Deep-RBPPred has a good generalization ability. In the meantime, Deep-RBPPred is still as good as the state-of-the-art method. Testing in A. thaliana, S. cerevisiae and H. sapiens proteomes, MCC values are 0.82 (0.82), 0.65 (0.69) and 0.85 (0.80) for balance model  when the score cutoff is set to 0.5, respectively. In the same testing dataset, different machine learning algorithms (CNN and SVM) are also compared. The results show that CNN-based model can identify more RBPs than SVM-based. In comparing the balance and imbalance model, both CNN-base and SVM-based tend to favor the majority class in the imbalance set. Deep-RBPPred forecasts 280  and 265  of 299 new RBP. The sensitivity of balance model is about 7% higher than the state-of-the-art method. We also apply deep-RBPPred to 30 eukaryotes and 109 bacteria proteomes downloaded from Uniprot to estimate all possible RBPs. The estimating result shows that rates of RBPs in eukaryote proteomes are much higher than bacteria proteomes. After RBPs have been identified, CLIP-related experimental technologies14 are applied to reveal the binding sites in RNAs. Also, many computational methods have been proposed to predict interaction of protein with RNA18 and RBPs25. RBP predictors can predict the RBPs, and then CLIP-related techniques can further reveal RNAs interacting with these RBPs. However, previous computational methods only considered only part features or known RNA binding domain (RBD) which plays a significant role in RBPs prediction. So, we proposed RBPPred integrating as much as features to address this problem22. Benchmarking on datasets shows that RBPPred is better than other approaches. But RBPPred runs slowly because it requires to run blast against a huge protein NR database to generate PSSM matrix. However, the prediction speed is important because a large number of RBPs are still unknown in many species. To overcome this shortcoming, we present Deep-RBPPred which is based on deep learning.RNA binding proteins (RBPs) play important functions in many cellular processes, such as post-transcriptional gene regulation, RNA subcellular localization and alternative splicing. With significant function in biology, many high-throughput experimental techniques have been developed to identify new RBPs in human, mouse, S. cerevisiae and C. elegans32. For predicting protein binding sites in RNA sequence, DeepBind32 is the first CNN-based model to predict the binding affinity. Deep-rbp29 and iDeep31 are two deep learning methods which both take RNA structure into consideration. These methods outperform the conventional approaches in term of prediction accuracy. However, deep learning algorithm is still not applied to RBPs prediction. In Deep-RBPPred, we apply a deep convolutional neural network instead of SVM. Since CNN-based method requires to input a fixed length feature vector, two solutions are handled to meet this requirement. The first solution is to pad all the sequences to fixed length sequences, and then one-hot encoding is used to encode the sequences. The second solution is to design the features by hand. It is not appropriate to predict RBPs with the padding solution because the length of RBPs varies over a wide range . Based on this consideration, we employ the hand-designed features which are proved effective to represent RBPs in RBPPred. Unlike RBPPred, we only employ physicochemical features including hydrophobicity, polarity, normalized van der Waals volume, polarizability, side chain\u2019s charge and polarity. These features are used to train the weights of 11 layers convolutional neural network with Tensorflow33. Deep-RBPPred presents comparable results to RBPPred but is significantly more efficient in the testing. And it also only needs few physicochemical features.In recently years, deep learning technology has been used in many aspects in bioinformatics and proved as a power tool22. Here we just simply describe the generating process. The positive samples are collected from the Uniprot database34, which is retrieval with GO term\u2019RNA binding\u2019 to search this protein database because the Uniprot database includes RBPs with x-ray crystal structures and RBPs identified by high-through experiment. For the negative samples, we took the approach from SPOT-stru35. The negative sequences are collected from PDB, by using PISCCES36 with sequence identity cutoff 25%, sequence length between 50 and 10,000 and resolution of X-ray better than 3.0\u2009\u00c5. The collected sequences are then mixed together so that the redundant proteins are removed with sequence identity of 25% by psi-cd-hit in the CD-HIT package37. Finally, the training set includes 2780 RBPs and 7093 non-RBPs.In order to train our deep learning model, we employed the training set used in the RBPPred. The details of generating the training set have been described in RBPPredThe training set consisting of different amount of RBPs and non-RBPs is known as an imbalance training set. The classification algorithm tends to favor the majority class when it is trained in the imbalance dataset. So, we randomly select 2780 non-RBPs to generate the balance dataset together with 2780 RBPs.22. However, only the identical sequences between the training and the testing set are removed. This may lead to a bias result caused by the redundance between training and testing set. So, we remove the homology sequences by CD-HIT. The testing and training set are mixed together to be clustered by CD-HIT with sequence identity cutoff 30%. Then all the sequences are discarded from testing set if they are in the same cluster with the training sequences. This can ensure the testing sequences are independent with training set. For one cluster, we only select the sequence provided by CD-HIT to ensure a non-redundant testing set. We finally collected 488 sequences including 239 negative samples and 249 positive samples, which are composed of 72 RBPs and 13 non-RBPs for A. thaliana, 129 RBPs and 164 non-RBPs for H. sapiens, 48 RBPs and 62 non-RBPs for S. cerevisiae. Comparing to the previous testing dataset including 2546 sequences, 2058 sequences are discarded for the redundance.For testing our deep learning model, we used the testing set from RBPPred37. But the evolutionary information and predicted secondary structure are discarded due to the computational time. The solvent accessibility is also discarded. At last, a 148-dimensional vector is encoded to represent each protein sequence including the properties of hydrophobicity, normalized van der Waals volume, polarity and polarizability, charge and polarity of side chain. We expand the dimension of feature vector to 160 with the expanded feature values assigned to 0 due to the CNN network architecture. Finally, we collect a total 160-dimensional feature vector to represent a protein sequence, as shown in Fig.\u00a022. The program encoding the feature vector is extracted from the RBPPred software package.The protein is encoded to a feature vector by the approach described in RBPPredThe performance is evaluated by sensitivity (SN), specificity (SP), accuracy (ACC) and Matthews Correlation Coefficient (MCC) which are defined as following:38 with tensorflow. In Fig.\u00a039 which randomly discards some neurons in the training phase. The dropout probability is set to 0.5. The final layer is the Softmax layer which is used to classify RBPs or not. The output of this model is a probability score which describes the probability of an RBP. All the activation functions in neurons are ReLU40. All the weights in neurons are added a L2 regularization operation. The L2 regularization losses are added to the final loss function. Adam optimizer is employed to minimize final loss consisting of cross-entropy between the label and probability score and L2 regularization loss of neurons. In this architecture, the number of trainable variable is 480,930. In training process, the learning rate is set to 0.0001.Deep-RBPPred is a Convolutional Neural Network (CNN)To avoid the overfitting and estimate an appropriate epoch of our models in the whole training sets, we perform the 10-fold cross-validation on the balance and imbalance training set. As shown in Fig.\u00a0th epoch and imbalance model of 242th epoch are selected as final models. The training loss of final balance/imbalance model is 0.15/0.17, which is almost equal to loss of the 10-folds validation process /3), which is much better than average SP of 0.62 ((0.56\u2009+\u20090.64\u2009+\u20090.65)/3) for the imbalance model. For predicting RBPs in the testing dataset, the SVM-imbalance model achieves an average of SN 0.94 ((0.90\u2009+\u20090.93\u2009+\u20091.0)/3), which is much higher than average SN of 0.82 ((0.83\u2009+\u20090.81\u2009+\u20090.83)/3) for the balance model. Indeed, the SVM models trained on the balance or imbalance model have an effect on the non-RBPs prediction. That is, the balance model has a better/worse predicting ability in RBPs/non-RBPs than the imbalance model. This effect is not significant in CNN when comparing with SVM. As shown in Table\u00a022.Deep-RBPPred is also tested and compared to other models on the testing set. Total three predictors are compared. The first approach is RBPPred, which is developed by our group previously. The second approach is RNApred which employees the amino acid composition or PSSM to predict RBPs. The third method is SONAR which integrates protein-protein interaction network and other features to predict the RBPs. The result of SPOT-Seq-RNA is not shown here because it has been compared with RBPPredAs shown in Table\u00a022. 280 and 265 of 299 new RBPs are correctly predicted by Deep-RBPPred-balance and Deep-RBPPred-imbalance, but only 260 RBPs are predicted by RBPPred22. Deep-RBPPred performs better than RBPPred. One protein (Uniprotid: P0DOC6) can\u2019t be calculated by RBPPred for that no protein sequences can be found by Blast. We also collect the 130 experimently determined human RBPs published in Wen and the co-workers\u2019 work42. RBPPred correctly predicts 24 of 130 RBPs, while Deep-RBPPred-imbalance and Deep-RBPPred-balance correctly predict 63 and 92 RBPs respectively. These results indicate that Deep-RBPPRed has better predicting ability than RBPPred.To test the predicting ability of Deep-RBPPred on new RBPs, we collect 299 new RBPs created between 2015-05-24 (consistent with RBPPred) and 2017-09-27 from Uniprot. In this section, only the RBPPred is compared because that the RBPPred have been proved to have better predicting ability than other methodsRunning time is an important metric to measure a model. We list the computational time of Deep-RBPPred in Table\u00a0E. coli. Finally, we filter out 109 bacteria and 30 eukaryote proteomes from all the bacteria and eukaryote proteomes.Deep-RBPPred is applied to estimate the RBPs in 139 reviewed proteomes for 109 bacteria and 30 eukaryote species. There are two problems in the Uniprot proteome dataset. Firstly, reviewed and un-reviewed sequences are both included in the Uniprot. The un-reviewed sequence may not be a real protein. So, the reviewed proteomes are used in the prediction. The second problem is that almost all reviewed proteomes are incomplete. For example, truepera radiovictrix (proteome id: UP000000379) only contained one reviewed sequence. These two problems can result in a bias prediction. In order to select some appropriate proteomes, we filter the proteomes with the number of sequences. For eukaryote species, the amount is set to 1/10 of S. cerevisiae. For bacteria, the number is set to 1/10 of The results of prediction are shown in Fig.\u00a02. It may lose the RBPs binding to the non-coding RNA.In this study, we develop two RBPs predicting models  based on CNN which only need hydrophobicity, normalized van der waals volume, polarity and polarizability, charge and polarity of side chain of protein sequence. Comparing with SVM models, we show that the CNN-based model performs better than SVM-based model. In comparing the balance and imbalance model, both the CNN-based and SVM-based classification show a preference for the major class. The result from the testing dataset shows that our deep learning models perform as good as RBPPred which is the best model so far. More importantly, Deep-RBPPred needs fewer features than RBPPred. Deep-RBPPred was then applied to estimate RBPs in 139 reviewed proteomes from the Uniprot dataset. The result shows that the RBPs rate in the bacteria is smaller than the eukaryote proteome. Deep-RBPPred-imbalance predicts 14,744 RBPs for the whole reviewed human proteomes. This number is almost 10-fold more than the number of the RBPs identified by high through experiments. This may be caused by these high throughput experimental limitations. For example, \u201cInteractome Capture\u201d only identifies the RBPs which bind to mRNA43 are added to avoid overfitting in the architecture of our deep learning. The process of 10-cross validation (Fig.\u00a0th epoch. The process of training (Fig.\u00a0In general, deep learning methods are applied in a large-scale data. A classic application of CNN-based methods is to classify the image. In this application, data augmentation approaches are used to enlarge the number of samples. And a large dataset may reduce the risk of deep learning model in overfitting. In Deep-RBPPred, we remove the redundant sequences as other researches have done. The process can be regarded as the opposite of data augmentation process in image recognition. In addition, L2 regularization and dropout layerSupplementary Information"}
+{"text": "Among the known triterpenes, \u03b2-d-glucopyranosyl 2\u03b1,3\u03b2,6\u03b2-trihydroxyolean-12-en-28-oate is reported for the first time in the Combretaceae, while bellericoside and asiatic acid are described for the first time in the genus Combretum. The structures of the isolated compounds have been established on the basis of spectral techniques . Their in vitro antifungal activities against standard strains of Candida albicans, C. krusei and Cryptococcus neoformans were also evaluated in this work.Two new triterpene glucosides, \u03b2- The 1H-NMR spectrum of 1 showed six aliphatic methyl singlets at \u03b4 0.85, 0.87, 0.97, 1.06, 1.18 and 1.54 and a broad singlet at \u03b4 5.38 assignable to an olefinic hydrogen. The 13C and DEPT NMR spectra exhibited signals for six methyls, eleven methylenes (two oxymethylenes), eleven methines  and eight quaternary carbons (one olefinic and one ester carbonyl carbon), of which six were attributed to a sugar unit and 30 were consistent with an triterpene aglycon moiety. The presence of a \u039412-double bond, already inferred from the broad singlet at \u03b4 5.38 ppm in the 1H-NMR spectrum was corroborated by resonances at 122.7 and 144.1 in the 13C-NMR spectrum, assigned to C-12 and C-13, respectively, of an olean-12-ene-type skeleton  assignable to diastereotopic hydroxymethylene hydrogens, which in turn showed cross-peak correlations with a carbon signal at \u03b4 65.7 in the HSQC spectrum. This information, along with an upfield shifted methyl carbon signal at \u03b4 24.1 ascribable to C-23 and a downfield quaternary carbon resonance at \u03b4 44.0 attributable to C-4 strongly suggested the location of this third hydroxyl substituent at C-24 - ; 1H-NMR: \u03b4 4.33 ; 3.54 ; 5.38 ; 3.16 ; 3.69 ; 4.42 ; 1.54 ; 0.97 ; 1.06 ; 1.18 ; 0.87 ; 0.85 ; sugar signals: \u03b4 6.29 ; 4.00 ; 4.18-4.45*  *overlapping signals; 13C-NMR: \u03b4 47.8 (C-1); 68.6 (C-2); 85.7 (C-3); 44.0 (C-4); 56.5 (C-5); 19.3 (C-6); 33.4 (C-7); 40.0 (C-8); 48.3 (C-9); 38.3 (C-10); 24.1 (C-11); 122.7 (C-12); 144.1 (C-13); 42.1 (C-14); 28.2 (C-15); 23.4 (C-16); 47.0 (C-17); 41.7 (C-18); 46.2 (C-19); 30.7 (C-20); 34.0 (C-21); 32.5 (C-22); 24.1 (C-23); 65.7 (C-24); 17.3 (C-25); 17.4 (C-26); 26.0 (C-27); 176.4 (C-28); 33.1 (C-29); 23.6 (C-30); sugar signals: \u03b4 95.8 (C-1\u2019); 74.1 (C-2\u2019); 78.9 (C-3\u2019); 71.1 (C-4\u2019); 79.3 (C-5\u2019); 62.3 (C-6\u2019).\u03b2-Arjunolic acid (2) and Asiatic acid (7) as a mixture: colorless amorphous powder; 1H- and 13C-NMR data in accordance with those of an authentic sample and with the literature [2: 13C-NMR: \u03b4 47.6 (C-1); 68.8 (C-2); 78.2 (C-3); 43.6 (C-4); 47.9 (C-5); 18.5 (C-6); 32.8 (C-7); 39.8 (C-8); 48.1 (C-9); 38.4 (C-10); 23.6 (C-11); 122.4 (C-12); 144.8 (C-13); 42.2 (C-14); 28.2 (C-15); 23.9 (C-16); 46.6 (C-17); 41.9 (C-18); 46.3 (C-19); 30.9 (C-20); 34.1 (C-21); 33.2 (C-22); 66.4 (C-23); 14.3 (C-24); 17.3 (C-25); 17.5 (C-26); 26.1 (C-27); 180.2 (C-28); 33.2 (C-29); 23.7 (C-30). Compound 7: 13C-NMR: \u03b4 47.6 (C-1); 68.8 (C-2); 78.2 (C-3); 43.6 (C-4); 48.2 (C-5); 18.5 (C-6); 32.8 (C-7); 40.0 (C-8); 47.6 (C-9); 38.3 (C-10); 23.6 (C-11); 125.5 (C-12); 139.2 (C-13); 42.2 (C-14); 28.6 (C-15); 24.8 (C-16); 48.0 (C-17); 53.5 (C-18); 39.4 (C-19); 39.3 (C-20); 31.0 (C-21); 37.4 (C-22); 66.4 (C-23); 14.3 (C-24); 17.4* (C-25); 17.4* (C-26); 23.8 (C-27); 180.6 (C-28); 17.3* (C-29); 21.3 (C-30) *interchangeable signals.terature ,17. CompArjunglucoside II (3) and Quadranoside IV (8) as a mixture: colorless amorphous powder; 1H- and 13C-NMR data in accordance with those of an authentic sample and/or with the literature [3: 1H-NMR: \u03b4 4.25* ; 4.28* ; 5.43 ; 3.19 ; 3.72 ; 4.22* ; 1.07 ; 1.10 ; 1.16 ; 0.90 ; 0.88 ; sugar signals: \u03b4 6.33 ; 4.21 ; 4.22-4.46*  *overlapping signals; 13C-NMR: \u03b4 47.6 (C-1); 68.8 (C-2); 78.1 (C-3); 43.5 (C-4); 48.0 (C-5); 18.4 (C-6); 32.7 (C-7); 39.9 (C-8); 47.7 (C-9); 38.3 (C-10); 23.8 (C-11); 122.7 (C-12); 144.0 (C-13); 42.1 (C-14); 28.1 (C-15); 23.2 (C-16); 46.9 (C-17); 41.6 (C-18); 46.0 (C-19); 30.6 (C-20); 33.8 (C-21); 32.4 (C-22); 66.3 (C-23); 14.2 (C-24); 17.5 (C-25); 17.6 (C-26); 25.9 (C-27); 176.4 (C-28); 32.9 (C-29); 23.5 (C-30); sugar signals: \u03b4 95.6 (C-1\u2019); 74.0 (C-2\u2019); 78.8 (C-3\u2019); 71.0 (C-4\u2019); 79.2 (C-5\u2019); 62.1 (C-6\u2019). Compound 8: 1H-NMR: \u03b4 4.25* ; 4.28* ; 5.43 ; 2.52 ; 3.72 ; 4.22* ; 1.07 ; 1.11 ; 1.20 ; 1.10 ; 0.92 ; 0.88 ; sugar signals: \u03b4 6.27 ; 4.21 ; 4.22-4.46*  *overlapping signals; 13C-NMR: \u03b4 47.9 (C-1); 68.8 (C-2); 78.1 (C-3); 43.5 (C-4); 48.1 (C-5); 18.4 (C-6); 33.0 (C-7); 40.1 (C-8); 47.8 (C-9); 38.2 (C-10); 23.7 (C-11); 125.9 (C-12); 138.3 (C-13); 42.4 (C-14); 28.5 (C-15); 24.5 (C-16); 47.7 (C-17); 53.2 (C-18); 39.2 (C-19); 39.0 (C-20); 30.7 (C-21); 36.6 (C-22); 66.3 (C-23); 14.2 (C-24); 17.3 (C-25); 17.5 (C-26); 23.6 (C-27); 176.1 (C-28); 17.2 (C-29); 21.1 (C-30); sugar signals: \u03b4 95.6 (C-1\u2019); 73.9 (C-2\u2019); 78.8 (C-3\u2019); 71.1 (C-4\u2019); 79.1 (C-5\u2019); 62.1 (C-6\u2019).terature ,13,21. CBellericoside (4): colorless amorphous powder; 1H- and 13C-NMR data in accordance with the literature [1H-NMR: \u03b4 4.07 ; 4.30* ; 5.42 ; 3.18 ; 4.24 ; 4.86 ; 3.97 ; 4.61 ; 1.13 ; 1.14 ; 1.16 ; 0.87 ; sugar signals: \u03b4 6.32 ; 4.20 ; 4.25-4.44* ; 4.02  *overlapping signals; 13C-NMR: \u03b4 47.8 (C-1); 69.0 (C-2); 79.7 (C-3); 47.8 (C-4); 48.2 (C-5); 19.2 (C-6); 32.5 (C-7); 40.0 (C-8); 48.3 (C-9); 38.1 (C-10); 23.3 (C-11); 122.7 (C-12); 144.1 (C-13); 42.1 (C-14); 28.1 (C-15); 24.1 (C-16); 46.9 (C-17); 41.7 (C-18); 46.1 (C-19); 30.7 (C-20); 33.7 (C-21); 33.1 (C-22); 64.3 (C-23); 62.8 (C-24); 17.3 (C-25); 17.4 (C-26); 26.0 (C-27); 176.5 (C-28); 33.0 (C-29); 23.6 (C-30).terature . 1H-NMR:Chebuloside II (5): colorless amorphous powder; 1H- and 13C-NMR data in accordance with the literature [1H-NMR: 4.43 ; 4.25 ; 5.10 , 3.17 ; 4.04 ; 4.40 ; 1.74 ; 1.78, 3H, s, H-25; 1.71 ; 1.16 ; 0.86 ; 0.85 ; sugar signals: \u03b4 6.27 ; 4.20 ; 4.28 ; 4.30 ; 4.00 ; 4.40 and 4.45 ; 13C-NMR: \u03b4 50.1 (C-1); 69.0 (C-2); 78.2 (C-3); 44.5 (C-4); 48.7 (C-5); 67.5 (C-6); 41.0 (C-7); 39.4 (C-8); 48.8 (C-9); 38.1 (C-10); 24.0 (C-11); 124.2 (C-12); 143.5 (C-13); 42.7 (C-14); 28.2 (C-15); 23.4 (C-16); 47.0 (C-17); 41.8 (C-18); 46.2 (C-19); 30.7 (C-20); 34.0 (C-21); 32.4 (C-22); 66.1 (C-23); 15.9 (C-24); 18.8 (C-25); 19.0 (C-26); 26.0 (C-27); 176.4 (C-28); 33.0 (C-29); 23.6 (C-30) .terature .1H-NMR: d-glucopyranosyl 2\u03b1,3\u03b2,6\u03b2-trihydroxyolean-12-en-28-oate\u03b2- (6): colorless amorphous powder; 1H- and 13C-NMR data in accordance with the literature [1H-NMR: \u03b4 4.30 ; 3.40 ; 4.82 , 2.50 ; 5.50 ; 3.19 ; 5.54 ; 1.44 ; 1.76 ; 1.71 ; 1.68 ; 1.23 ; 0.92 ; 0.88 ; sugar signals: 6.27 ; 4.18-4.45 ; 4.00 ; 13C-NMR: \u03b4 50.0 (C-1); 68.7 (C-2); 84.0 (C-3); 39.5 (C-4); 57.0 (C-5); 67.5 (C-6); 41.2 (C-7); 40.6 (C-8); 48.6 (C-9); 38.3 (C-10); 23.9 (C-11); 123.2 (C-12); 143.5 (C-13); 42.7 (C-14); 28.1 (C-15); 23.4 (C-16); 46.9 (C-17); 41.7 (C-18); 46.2 (C-19); 30.6 (C-20); 34.0 (C-21); 32.4 (C-22); 29.1 (C-23); 18.5 (C-24); 18.7 (C-25); 19.1 (C-26); 26.0 (C-27); 176.3 (C-28); 33.0 (C-29); 23.5 (C-30); sugar signals: \u03b4 95.7 (C-1\u2019); 74.0 (C-2\u2019); 78.6 (C-3\u2019); 71.1 (C-4\u2019); 79.1 (C-5\u2019); 62.0 (C-6\u2019).terature . 1H-NMR:Bellericoside (4) and d-glucopyranosyl 2\u03b1,3\u03b2,23,24-tetrahydroxyurs-12-en-28-oate\u03b2- (9) were obtained as a mixture: colorless amorphous powder. Compound (9): 1H-NMR (pyridine-5d): \u03b4 5.45 ; 3.18 ; 4.05 ; 4.30* ; 4.24 ; 4.90 ; 3.95 ; 4.62 ; 1.15 ; 1.19 ; 1.10 ; 0.91 ; 0.87 ; sugar signals: \u03b4 6.30 ; 4.20 ; 4.25-4.44* ; 4.02  *overlapping signals;. 13C-NMR (pyridine-5d): \u03b4 47.4 (C-1), 69.0 (C-2); 79.7 (C-3); 47.8 (C-4); 48.0 (C-5); 19.2 (C-6); 33.5 (C-7); 40.2 (C-8); 48.2 (C-9); 38.0 (C-10); 23.9 (C-11); 125.9 (C-12); 138.4 (C-13); 42.4 (C-14); 28.6 (C-15); 24.6 (C-16); 47.8 (C-17); 53.2 (C-18); 39.3 (C-19); 39.1 (C-20); 30.7 (C-21); 36.7 (C-22); 64.3 (C-23); 62.9 (C-24); 17.3 (C-25); 17.4 (C-26); 23.7 (C-27); 176.2 (C-28); 17.5 (C-29); 21.2 (C-30); sugar signals: \u03b4 95.7 (C-1\u2019); 74.0 (C-2\u2019); 78.8 (C-3\u2019); 71.2 (C-4\u2019); 79.1 (C-5\u2019); 62.3 (C-6\u2019).Betulinic acid (10): colorless amorphous powder; 1H- and 13C-NMR data in accordance with those of an authentic sample and with literature [1H-NMR (pyridine-5d): \u03b4 3.47 ; 3.55 ; 4.78 ; 4.96 ; 1.24, ; 1.02 ; 0.83 ; 1.07*; 1.08* ; 1.80  *interchangeable signals; 13C-NMR (pyridine-5d): \u03b4 39.3 (C-1), 28.3 (C-2); 78.2 (C-3); 39.6 (C-4); 56.0 (C-5); 18.8 (C-6); 34.9 (C-7); 41.2 (C-8); 51.0 (C-9); 37.6 (C-10); 21.3 (C-11); 26.2 (C-12); 38.7 (C-13); 42.9 (C-14); 31.3 (C-15); 32.9 (C-16); 56.7 (C-17); 47.8 (C-18); 49.8 (C-19); 152.5 (C-20); 30.3 (C-21); 37.6 (C-22); 28.7 (C-23); 16.4 (C-24); 16.5 (C-25); 16.5 (C-26); 15.0 (C-27); 178.9 (C-28); 110.0 (C-29); 19.5 (C-30).terature ,17. 1H-NQuadranoside I (11): colorless amorphous powder; 1H- and 13C-NMR data in accordance with the literature [1H-NMR: \u03b4 4.25 ; 3.43 ; 4.81 ; 5.50 ; 4.76 ; 4.90 ; 1.76 ; 1.45 ; 1.58 ; 1.78 ; 1.04 ; sugar signals: 6.42 ; 4.22 ; 4.27 ; 4.35 ; 4.06 ; 4.38-4.45 ; 13C-NMR: \u03b4 50.4 (C-1), 69.0 (C-2); 84.1 (C-3); 40.7 (C-4); 56.6 (C-5); 67.9 (C-6); 42.4 (C-7); 40.7 (C-8); 51.7 (C-9); 38.6 (C-10); 21.4 (C-11); 26.2 (C-12); 37.5 (C-13); 43.0 (C-14); 30.2 (C-15); 32.2 (C-16); 57.0 (C-17); 49.9 (C-18); 47.4 (C-19); 150.9 (C-20); 30.8 (C-21); 36.8 (C-22); 28.8 (C-23); 19.0 (C-24); 19.3 (C-25); 17.0 (C-26); 15.2 (C-27); 174.9 (C-28); 110.0 (C-29); 19.4 (C-30); sugar signals: \u03b4 95.4 (C-1\u2019); 74.3 (C-2\u2019); 78.8 (C-3\u2019); 70.9 (C-4\u2019); 79.3 (C-5\u2019); 62.2 (C-6\u2019).Candida albicans (ATCC 90028), C. krusei (ATCC 6258) and Cryptococcus neoformans (ATCC 32045). The antifungal activities were determined using microbroth dilution assays in 96-well microplates, in duplicate, following the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [The following strains from the American Type Culture Collection (ATCC), Rockville, MD, USA were used for the antifungal evaluations: e (CLSI) . The low"}
+{"text": "We contrasted the event-related brain potentials (ERPs) to visual narratives depicting an explicit event, an action star, or a \u201cnoise\u201d panel of scrambled lines. Both action stars and noise panels evoked large N400s compared to explicit-events (300\u2013500\u00a0ms), but action stars and noise panels then differed in their later effects (500\u2013900\u00a0ms). Action stars elicited sustained negativities and P600s, which could indicate further interpretive processes and integration of meaning into a mental model, while noise panels evoked late frontal positivities possibly indexing that they were improbable narrative units. Nevertheless, panels following action stars and noise panels both evoked late sustained negativities, implying further inferential processing. Inference in visual narratives thus uses cascading mechanisms resembling those in language processing that differ based on the inferential techniques.Research in verbal and visual narratives has often emphasized backward-looking inferences, where absent information is subsequently inferred. However, comics use conventions like star-shaped \u201caction stars\u201d where a reader  Inference has been a primary focus of studies of discourse  posits that a narrative structure operates in parallel to semantics, where it organizes meaning at a discourse level  implicate similar mechanisms in visual narratives as in language processing Cohn . When enInformation extracted from the visual surface then activates semantic memory to process the meaning of the immediate representation in relation to what came before in the sequence. This process is indexed in ERPs by the N400, a negative deflection that peaks around 400\u00a0ms after the onset of a word or image  narratives  and descriptive words (Punch!) in action stars, which were both attenuated compared to anomalous words, suggesting that they were both sufficient as event-cues given the context of the prior visual sequence , third panel (5), fourth panel (13), and fifth panel (40). Action star sequences substituted an action star panel for the explicit Peak panels. Because action star panels can have less visual information than explicit panels, we therefore designed action star panels with a similar degree of visual complexity as our explicit Peak panels. We calculated the \u201cgray value\u201d of all pixels across all explicit Peak panels (M\u2009=\u2009213.2) and designed three action star panels with similar values (M\u2009=\u2009213.0). These three action star panels were distributed randomly across sequences within the action star sequence types. Our final sequence type then created \u201cnoise\u201d panels by selecting subdivisions of action star panels (via a grid and iterated circular pixel selections), and then rotating these subdivisions randomly such that no discernable representational information was conveyed, though the quantity and character of the visual lines remained the same. Action stars were used as the base for noise panels rather than explicit panels in order to ensure they remained asemantic, and also involved similar line segments. Noise panels had a grey value comparable to other critical panels (M\u2009=\u2009213.0).From these strips, we created three sequence types, as in Fig.\u00a0These 60 sequences were counterbalanced into three lists using a Latin Square Design such that participants viewed each sequence only once, but across lists all participants viewed all sequences in all conditions. An additional 96 fillers, half of which contained sequencing discontinuities, were used to introduce additional heterogeneity into the experimental stimuli.F-values above 3.2 for our within-subjects design. All participants gave their informed written consent prior to the experiment and the study was approved by the Tilburg University School of Humanities and Digital Sciences Research Ethics and Data Management Committee. Participants were right-handed, taking no psychiatric medications, with no history of head trauma, and with normal or corrected-to-normal vision. Prior to experimentation, participants filled out the Visual Language Fluency Index (VLFI) questionnaire which assessed their frequency of reading and drawing visual narratives like comics, along with experiencing other media like movies and written books. The VLFI score generated by this assessment has been a consistent predictor of individual differences in visual narrative comprehension . Although statistical power was not computed a priori, a power analysis in G*Power indicated that with 3 conditions in a sample of 24 participants, to achieve a medium effect size of 0.25, it would require The experiment was conducted in a soundproof chamber, where a participant sat in a chair across from a computer screen. Lights in the chamber were turned off, except for backlighting behind the screen used to prevent a flashing effect of stimuli on the screen that could induce blinks. We used PsychoPy2 ?\u201d.EEG was recorded using a Brain Products ActiChamp system at a sampling rate of 250\u00a0Hz and high cutoff filter of 70\u00a0Hz. EEG recordings were made with 32 channel Standard actiCAPs, referenced online to electrode Fz. Eye movements and blinks were monitored using electrodes placed beneath the right eye and beside the left eye. Electrode impedances for all electrodes were kept below 10\u00a0k\u03a9. We analyzed the data using the ERPLAB plugin for EEGLAB in MATLAB , critical panel position (p\u2009=\u20090.095), or their interaction (p\u2009=\u20090.210).Trials with excessive blink or muscle artifact were isolated and removed. Across conditions at the critical panels, 6% (range 3\u20138%) of trials were rejected per participant, an average of 1.25 trials . A 3 \u2009\u00d7\u20092  ANOVA showed no significant differences in the rejection rates across sequence types . Additional analyses in epochs of 0\u2013100\u00a0ms and 100\u2013200\u00a0ms investigated the potential for early, stimulus-driven physical differences at the critical panels. A broad coverage of the scalp was examined using five regions of interest which each averaged the amplitudes across four electrodes. As in Figs. Behavioral results were compared using a repeated-measures ANOVA with three levels of Sequence Type , with post hoc tests using a Bonferroni correction. Finally, to investigate any possible influence of comic reading experience, we used Pearson's correlations with an alpha level set to 0.05 between VLFI scores and ratings and the mean amplitude differences averaged across all electrode sites at the critical and subsequent panels.F\u2009=\u200912.54, p\u2009<\u20090.001. Explicit sequences  were rated as more comprehensible than sequences with action stars  or noise panels , all ts\u2009>\u20094, all ps\u2009<\u20090.005, but these latter types did not differ from each other (p\u2009=\u20091). Nevertheless, all sequences were rated as comprehensible on the whole, rated above the midway point of 4 on a 1\u20137 scale . Finally, in open response post-test questionnaires, 35% of participants commented on observing action stars, while 33% noticed noise panels and that they differed from action stars , including one who drew a noise panel.Analysis of participants\u2019 ratings suggested differences in the sequences\u2019 comprehensibility, Fs\u2009<\u20092.7, all ps\u2009>\u20090.079, all \u03b72p\u2009<\u20090.105). No significant differences arose in the 100\u2013200\u00a0ms epoch.We first analyzed ERPs at the critical panels, depicted in Fig. Fs\u2009>\u20097.6, all ps\u2009<\u20090.001, all \u03b72p\u2009>\u20090.25) and in the right posterior region, F\u2009=\u20093.3, p\u2009<\u20090.05, \u03b72p\u2009=\u20090.13. Pairwise comparisons suggested that noise panels evoked greater negativities than both explicit panels and action stars in the central and anterior regions .In the 200\u2013300\u00a0ms epoch, a main effect of Sequence Type arose in the central region and interactions in the peripheral regions were found between Sequence Type and Hemisphere. Follow up ANOVAs in each region revealed main effects of Sequence Type in both anterior regions . Across central and anterior regions, pairwise comparisons showed a greater negativity indicative of an N400 appeared to action stars and noise panels than explicit panels , but no difference between action stars and noise panels.In the 300-500\u00a0ms epoch, main effects of Sequence Type appeared in both central and peripheral regions. An additional Sequence Type\u2009\u00d7\u2009Hemisphere interaction was followed by analyses revealing main effects in both anterior regions . Pairwise contrasts suggested that action stars evoked a greater negativity than both explicit and noise panels in the left anterior region , and a greater negativity than the noise panels alone in the right anterior region (p\u2009<\u20090.05).Later effects were first suggested in the 500-800\u00a0ms epoch by Sequence Type\u2009\u00d7\u2009Hemisphere and Sequence Type\u2009\u00d7\u2009Hemisphere\u2009\u00d7\u2009AP Distribution interactions in the peripheral regions. Main effects of Sequence Type were subsequently found in both anterior regions . Pairwise comparisons in the left anterior region suggested that noise panels evoked a frontal positivity that was close but did not exceed the threshold of significance compared to action stars (p\u2009=\u20090.050) and explicit panels (p\u2009=\u20090.058). In the right posterior region, post-hoc comparisons implicated a greater posterior positivity to action stars than noise panels (p\u2009<\u20090.05), which is more visible in the topographic maps of Fig. Additional differences were suggested in the 800\u20131100\u00a0ms epoch with an interaction between Sequence Type and Hemisphere. Follow up ANOVAs suggested main effects of Sequence Type in the left anterior and right posterior regions  and in the left posterior region of the 800\u20131100\u00a0ms epoch, F\u2009=\u20093.6, p\u2009<\u20090.05, \u03b72p\u2009=\u20090.138. These differences arose because action stars and noise panels evoked a larger rightward posterior negativity than explicit panels , which extended into the left posterior for only noise panels compared to explicit panels (p\u2009<\u20090.05).At the panel following the manipulated, critical panel, no differences occurred between sequence types in the 200\u2013300\u00a0ms epoch, but interactions between Sequence Type and Hemisphere appeared in the 300\u2013500, 500\u2013800, and 800\u20131100\u00a0ms epochs (Table r(22)\u2009=\u20090.47, p\u2009<\u20090.05, but when correcting for multiple comparisons this correlation was no longer significant. No significant correlations were found for sequences with action stars or noise panels, nor the differences between them. No significant correlations were observed between VLFI scores and ERP results.The analysis of expertise correlated only marginally with behavioral results. A positive correlation between ratings of explicit sequences and VLFI scores suggested that greater comic reading frequency lead to higher ratings, Inference has been a hallmark of the study of visual narrative processing. Here, we asked about such processing both for representationally impoverished action stars and noise panels and at the subsequent image. Our first apparent finding was that panels omitting information in a sequence elicit a large N400 effect compared to explicit depictions of an event. These N400s were evoked both by the highly conventionalized action stars and the not-conventionalized noise panels. That these inexplicit panels did not differ in amplitude of the N400 implies that they both trigger a greater demand for accessing semantic memory given the absence of overt representations. Under one interpretation, this N400 could indicate inferential processing, accessing the semantic features of the unseen information  when compared to onomatopoetic or event-descriptive words in lieu of explicit events in visual narratives  appear different than those observed at later epochs. In addition, despite differences prior to 300\u00a0ms, action stars and noise panels had similar amplitudes and latencies of the N400 on visual inspection, including topologically in its phasic ascent and descent. If such stimulus differences sponsored downstream effects, then these relative differences do not seem consistent across time windows in a sustained way  elicited larger LFPs than onomatopoetic sound effects (Pow!), regardless of congruity  that was greater than both action stars and explicit panels. In research on language, LFPs have been associated with violated lexical or semantic predictions, such as when a particular word is expected in the sentence, but participants are presented with a different, yet congruent word  or less conventional (noise panels) properties of these panels. However, the absence of depicted events in both seemed to sponsor similar attempts to reconcile this missing information. Altogether, these findings provide an initial look at the neurocognition of inference generation across sequential images, further demonstrating that inference must balance the implicit of what is omitted and the explicit of what is provided, even when what is shown remains inexplicit."}
+{"text": "Plagiomnium ellipticum and Polytrichum formosum were also documented; proximal parts had higher levels of endopolyploidy than distal parts. Endopolyploidy is spatially and temporally differentiated in the gametophytes of endopolyploid mosses and follows a pattern similar to that seen in angiosperms.Somatic polyploidy or endopolyploidy is common in the plant kingdom; it ensures growth and allows adaptation to the environment. It is present in the majority of plant groups, including mosses. Endopolyploidy had only been previously studied in about 65 moss species, which represents less than 1% of known mosses. We analyzed 11 selected moss species to determine the spatial and temporal distribution of endopolyploidy using flow cytometry to identify patterns in ploidy levels among gametophytes and sporophytes. All of the studied mosses possessed cells with various ploidy levels in gametophytes, and four of six species investigated in sporophytic stage had endopolyploid sporophytes. The proportion of endopolyploid cells varied among organs, parts of gametophytes and sporophytes, and ontogenetic stages. Higher ploidy levels were seen in basal parts of gametophytes and sporophytes than in apical parts. Slight changes in ploidy levels were observed during ontogenesis in cultivated mosses; the youngest  parts of thalli tend to have lower levels of endopolyploidy. Differences between parts of cauloid and phylloids of  Arabidopsis thaliana (L.) Heynh.  Triton X-100, pH 7.0) [\u22121), propidium iodide (30 \u03bcg\u00b7mL\u22121), and with \u03b2-mercaptoethanol (2 \u03bcL\u00b7mL\u22121) as described in Goga et al. [P. undulatum, E. striatum), there is a small risk of misinterpreting the 2C peak as the 1C peak on FCM records.Samples for flow cytometry (FCM) were prepared according to a standard method by chopping plant material in 1 mL of general purpose buffer (0.5 mmol\u00b7L pH 7.0)  in a Pet pH 7.0) ,57. Aftea et al. . Samplesa et al. . The peaa et al.  and reprBased on the number of nuclei of the individual peaks of the FCM histograms, the endoreduplication index  of each gametophyte (reduced haploid phase) was calculated according to the following formula ,60: EI =1C, n2C, n4C, n8C, n16C\u2026 represents the number of nuclei of the respective ploidy levels. Tissues with an EI higher than 0.1 are considered endopolyploid [Since the sporophyte represents an unreduced diploid phase, the EI was calculated according to the formula:P. patens and P. drummondii). A two-way ANOVA test was used to analyze the spatial distribution of endopolyploidy in organs (study of P. ellipticum and P. formosum). Plots were visualized in ggplot2 ver. 2.2.1 [Results were evaluated in the statistical software R, version 3.6.1 . To testr. 2.2.1 .Atrichum undulatum, Brachythecium rivulare, Bryum moravicum, Dicranum scoparium, Eurhynchium striatum, Physcomitrella patens (laboratory culture), Plagiomnium ellipticum, Plagiomnium undulatum, Pohlia drummondii (laboratory culture), Polytrichastrum alpinum, and Polytrichum formosum. All the studied mosses had 1C, 2C, and 4C nuclei present in the gametophyte, except P. patens  values for gametophytes were recorded in B. rivulare , while the highest values were recorded in P. ellipticum   . It is w compare .A. undulatum, B. rivulare, B. moravicum, D. scoparium, P. alpinum and P. formosum) had developed sporophytes. In all cases, sporophytes had lower levels of endopolyploidy compared to gametophytes. Mostly 2C, 4C and 8C nuclei were represented in the analyzed sporophytes, with 8C nuclei absent only in the sporophytes of A. undulatum  to the highest values in D. scoparium   .P. formosum, A. undulatum and B. moravicum, and EI values were lower in sporophytes compared to gametophytes in these cases  were statistically significant between gametophytic and sporophytic tissues (whichever parts are considered) in se cases B.P. ellipticum and P. formosum, the later collected from locality near Kamenica  in all the species investigated except Kamenica B. In conB. rivulare, E. striatum, A. undulatum or P. drummondii, had cells of one dominant ploidy level; this could be 1C or 2C in gametophyte or 2C in sporophyte. Other endopolyploid cells were found often only at quite low levels. Other species, including P. formosum, P. unudulatum, D. scoparium or P. patens, showed a pattern of almost continuous increase or decrease of nuclei counts in subsequent ploidy classes. This resulted into almost equal proportions of two or three nuclei classes, as in case of P. formosum.To generalize our results, two within-tissue ploidy level distribution patterns were observed. Some species, such as P. formosum represented here by two spatially and timely different samples. While individuals lacking sporophytes collected in Milpo\u0161 (in 2018) were variable in three gametophytic parts, those with sporophytes and collected in Kamenica (in 2020) were not. This result points to the possibility that site or seasonal differentiation, genotype or population differentiation, or possibly sexual differentiation, may shape overall plant endopolyploidy level.Interestingly, we have found variable EI pattern in P. patens and P. drummondii were grown under laboratory conditions and were compared at three stages of development. In P. patens, 8C nuclei were observed in all parts of the moss (and stages) except for the apical portion developed in the third stage. In P. drummondii, 8C nuclei were recorded only in first stage of plants and the basal part of plants in both the second and third stages. In the other samples, the highest ploidy level was 4C. Generally, P. patens showed slightly higher EI values.To analyze changes in endopolyploidy in relation to organ or tissue age, P. patens in the first stage of analysis, and 2C nuclei were present in smaller proportions. Only a few dozen of 4C nuclei were detected (p < 0.05) but not for middle part of gametophyte  (The proportion of 1C nuclei was high in detected . Similardetected A. Statis > 0.05) .p < 0.001), but not for middle part of gametophyte   .P. formosum and P. ellipticum, were used to analyze the distribution of endopolyploidy within the cauloids and phylloids originating from different parts of the gametophyte. Both species had relatively high levels of endopolyploidization, with 1C to 8C nuclei present in their gametophytes. In the gametophytes of P. ellipticum, 16C cells were also represented in the basal and middle parts of the cauloids. In general, the degree of endopolyploidization in the cauloid and phylloids of both species decreased from base to apex. Plagiomnium ellipticum had a higher endopolyploidy level in both the cauloids and phylloids compared to P. formosum.Two mosses, In all three parts of gametophytes of P. formosum, 1C to 8C cells were represented irrespective of organ, cauloid or phylloids. These four cell categories were also present in the analyzed parts of all phylloids, except in the apical portion of the middle and apical phylloids of the gametophytes. Only three peaks were observed in these sections; the fourth peak, representing 8C nuclei, was absent. Most of the basal parts of the cauloids were composed of 4C nuclei; 2C and 8C nuclei were present in smaller amounts, and 1C cells were least abundant. 4C cells again dominated in the middle part of the cauloid, but the number of 1C nuclei dominated in the cauloid base .p < 0.001) and among organ parts   = 23.3; p < 0.001) were statisticaly significant, but interaction between them was not  = 0.8; p > 0.05). A Tukey post hoc test revealed significant pairwise differences between cauloid and all three types of phylloids, showing 1.17 up to 1.20 higher EI values, and between the basal and both the middle (- 0.23 EI) and apical parts of organs (- 0.35 EI).In phylloids, the 1C nuclei were most abundant, regardless of which part of the moss from which the phylloid was sampled. Within individual phylloids, a trend of decreasing ploidy level from base to apex was observed. While the number of 1C nuclei increased, there was a decrease in the number of 8C nuclei A, which P. ellipticum, five peaks in cauloids and three to four peaks in phylloids were detected by FCM. In addition to 1C, 2C, 4C, and 8C nuclei, 16C nuclei were observed throughout the cauloid except in the apical part  were not statisticaly significant, while differences among organ parts   = 38.1; p < 0.001) were. Interaction between them was not statisticaly significant  = 1.0; p > 0.05). A Tukey post hoc test revealed significant pairwise differences between the apical part of organs and both basal and middle portions .This species had relatively high EI values and similar pattern of variation for both cauloid and phylloids. Within these organs, endopolyploidy decreased from the basal and middle parts to the apical parts . DifferePhyscomitrella patens [Endopolyploidy, or increased ploidy in somatic cells beyond inherited organism ploidy level, is widespread in some plant groups, e.g., mosses and angiosperms, and has been reported to have various effects from the cellular to whole organism level. Recent studies have primarily focused on the study of endopolyploidy in higher plants or the model moss a patens ,63. To da patens ). TherefLevels of endopolyploidy vary depending on different factors, including the type of tissue or organ ,6. In anGametophytes are established in the life cycle of mosses before sporophytes ; therefoPlagiomnium ellipticum and Polytrichum formosum. While the part of the moss thallus from which organs were removed was always significant, the differentiation in EI between organs  was documented only in P. formosum. The variability of endopolyploidization is specific to each species. For instance, in P. formosum we have observed that the cauloid has much higher EI values compared to the phylloids from any location on the gametophyte. In contrast, P. ellipticum did not show this pattern of differentiation. It is necessary to analyze more moss species to generalize trends in endopolyploidy patterns, particularly in organs and their positions on moss thalli. Cytohistological techniques coupled with confocal microscopy may be beneficial.Divergences in eventual endopolyploidy levels were observed in individual organs and parts of sporophytes and gametophytes in the present study. Endopolyploidization in apical parts of gametophytes and sporophytes was lower than in basal parts and tended to decrease along these organs from base to apex. Although the effect of interaction between factors on endopolyploidy in different parts of moss organs (organ vs. part of an organ) was investigated in the present study, the results cannot be generalized yet. This topic was investigated in two different species, Physcomitrella patens, P. drummondii, B. moravicum, and P. ellipticum were characterized by replication of the whole genome in most cells in the gametophytes in the present study. In several samples, the first 1C peak was represented by very few or no nuclei, which may results into misinterpretation of FCM records [B. moravicum, which had developed sporophytes at the time of analysis, the dominant and often the first peak in gametophytes was identified as a 2C peak. This suggests that 1C cells may often be absent in the gametophytes of some moss species. This is very interesting because it suggests that almost all gametophyte cells may harbor a completely reduplicated genome. However, here we consider an alternative explanation of reduplicated genome. Endopolyploidy in mosses is poorly explored so far. Most data come from studies on the model moss P. patens. This species is characterized by a specific, tissue-dependent cell cycle. The protonema of this moss consists of two types of tissues, caulonema and chloronema, but only the latter form in young individuals. Most chloronema cells undergo DNA synthesis during the S phase of the cell cycle early after mitosis and are usually found \u201ctimely arrested\u201d just before transition from G2 phase to M phase. Therefore, screening for nuclei ploidy levels in this moss revealed mostly 2C nuclei [1 and the S phase, the proportion of 1C nuclei is shown to increase in tissue FCM cytotyping. Later, as the cells of the caulonema age, DNA endoreduplication can be detected [2/M phase). Indeed, Bainard et al. [2/M arrest of the cell cycle may lead to misinterpretation of FCM data in genome size estimation. Although Bainard et al. [2/M phase. More detailed, future studies are needed to investigate this phenomenon in relation to endopolyploidy of mosses and further if the G2/M phase arrest of cells is ploidy-dependent. records ,46. In tC nuclei . Gradualdetected , confirmChenopodium quinoa Willd. [Trifolium pratense L. [Beta vulgaris L. [As in various organs and tissues, endopolyploidy changes during ontogenesis in an individual. Endoreduplication in cells can continue until maturation  and thera Willd. , Trifolitense L. , and Betgaris L. . P. drummondii and P. patens, a decrease in endopolyploidy from the base to the apex of gametophytes was demonstrated. In both species, higher ploidy levels were recorded in ontogenetically older parts of the thalli. The same patterns have been observed in higher plants [P. patens) of gametophytes may be alternatively the result of faster thalli growth via increasing cell division leading to the detection of a higher proportion of cells entering the M phase compared to other stages. The observed inconsistent patterns of endopolyploidy variation in the part of a gametophyte during ontogenesis found in two selected species invites further in-depth analysis of ontogenetic patterns in several mosses, preferably those with a large thallus.This phenomenon occurs not only in higher plants, but also in mosses, as documented in the present study. As exemplified by r plants ,57. A hiB. moravicum, P. patens, P. ellipticum, and P. drummondii). Nuclei that were in the baseline 1C state were of minimal proportions compared to those in the endopolyploid states.There were statistically significant differences in the levels of endopolyploidy between different parts of gametophytes and sporophytes in the studied mosses. In general, we observed higher degrees of endopolyploidization in gametophytes than in sporophytes. Based on the present study of 11 moss species, the level of endopolyploidy tends to be higher in basal parts of mosses than in apical parts. Also, endopolyploidy tends to decrease from basal to apical parts of the cauloids and phylloids. In addition, during analysis of endopolyploidy in selected moss species, it was found that some species replicated nearly all  of the nuclear DNA content in gametophyte cells (Research on endopolyploidy has so far been directed toward higher plants, although endopolyploidy is present in several moss species. As such, this group of plants is promising in terms of research on endopolyploidy as a part of adaptation to stress in plants or on the evolutionary patterns of endopolyploidy. Recent reports that endopolyploidy is common in mosses and in moss thalli and organs (, presentThe present study shows that certain cytological features associated with thalli development in mosses have just started to be explored, and that further elucidation of these features may help to better understand moss biology and evolution. Mosses are resilient and ubiquitous organisms and play an important role in several ecosystem processes, including net primary productivity, decomposition rates, above-ground microclimate modifications, and carbon and nutrient cycling . Bryophy"}
+{"text": "This prognostic study compares the diagnostic equivalence of patient-collected lower nasal specimens with that of health care worker\u2013collected oropharyngeal specimens for sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing. However, specimen collection must be done by health care workers and requires extensive use of personal protective equipment. To minimize the risk of exposure during testing, reduce personal protective equipment use, and increase access to testing, we compared the diagnostic equivalence of a modified specimen collection method, patient-collected lower nasal swabs, with that of the current clinical standard, health care worker\u2013collected oropharyngeal swabs. If the 2 methods proved to be diagnostically equivalent, patients would be able to collect specimens themselves without exposing health care workers to respiratory secretions.Since the emergence of the sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) outbreak in Wuhan, China, in December 2019, the virus has spread to 173 countries, resulting in 3\u2009855\u2009788 confirmed cases and 265\u2009862 deaths as of May 9, 2020.STARD) reporting guideline.This prognostic study was approved by the Stanford University institutional review board. Participants provided oral informed consent to clinical research coordinators and then signed a consent form with the physician who collected the oropharyngeal swab. This study complies with the Standards for Reporting of Diagnostic Accuracy , a physician-collected lower nasal swab , and a physician-collected oropharyngeal swab . During the visit, participants received a $20 incentive. The 3 specimens were placed in separate 3-mL tubes of viral transport medium , stored in a cooler, and delivered to the Stanford Clinical Virology Laboratory. Specimens were tested for SARS-CoV-2 using reverse transcriptase\u2013polymerase chain reaction targeting the envelope or open reading frame 1 ab genes.We report participant demographic characteristics, sensitivity and specificity, and discordant probabilities with 95% binomial CIs of patient-collected lower nasal compared with physician-collected oropharyngeal specimens. All analyses were performed with SAS statistical software version 9.4 (SAS Institute). Data analysis was performed in May 2020.Of 129 eligible SARS-CoV-2\u2013infected patients, 30 participated in the study . We do not have reasons for refusal for nonparticipants. Participants were predominantly white (20 participants [66%]), with no sex or age group predominance. Twelve participants (40%) self-reported possible exposure to SARS-CoV-2. Cough (20 participants [67%]), fever (13 participants [43%]), and sore throat (8 participants [27%]) were commonly reported symptoms during the first visit. Approximately one-half of the participants had documentation of chronic medical conditions (16 participants [53%]), and 5 participants (17%) tested positive for coinfections with other respiratory viruses .We observed diagnostic equivalence across the 3 methods of specimen collection . Eleven 4 that lists patient-collected lower nasal swab as an acceptable specimen collection method for SARS-CoV-2 testing. Self-collected lower nasal swabs could also be used for home- or office-based testing of asymptomatic patients. However, these preliminary findings are limited by small sample size, have limited generalizability, and need to be validated further in diverse clinical settings. These validation efforts are currently under way at our institution.These findings contribute to the recently released US Food and Drug Administration guidance"}
+{"text": "The dentinogenic ghost cell tumor (DGCT), a locally invasive benign neoplasm, is one of the rarest odontogenic tumors, usually developing in the maxilla or mandible. It can be classified into 2 types: intraosseous  and extraosseous . Here, we describe the first case of a peripheral DGCT located in the ethmoid sinus.An 8-year-old boy presented to our department with a longer than 7-month history of nasal obstruction, purulent secretion, and reduction in sense of smell in the right nasal cavity.The patient was diagnosed with peripheral DGCT of the ethmoid sinus based on computed tomography scan and pathology.Functional endoscopic sinus surgery was performed.With 2 years of follow-up, there was no evidence of recurrence.Peripheral DGCT can occur in the paranasal sinus and the need to consider this entity as a possible diagnosis by the clinicians. The dentinogenic ghost cell tumor (DGCT) is the solid variant of CCOT and is characterized by ameloblastoma-like epithelial cells associated with ghost cells and dysplastic dentin.The calcifying cystic odontogenic tumor (CCOT), a term formally used since it was named by the World Health Organization in 2005, The intraosseous DGCT is reported to be locally invasive, and patient age ranges from 12 to 75 years, whereas the extraosseous type is extremely rare and less aggressive, with an age range of 7 to 92 years. No matter what type, the DGCT usually occurs in the maxilla and mandible.,5The DGCT is one of the rarest odontogenic tumors, regarded as a locally invasive benign neoplasm. It may present as an intraosseous  or extraosseous  process.Here, we present a case of peripheral DGCT located in the right ethmoid sinus in an 8-year-old child. To our knowledge, this is the first report of a primary DGCT in the paranasal sinus in the available English literature. The patient's parents have provided informed consent for publication and this report was also approved by the Ethics committee of the West China Hospital, Sichuan University.2An 8-year-old boy presented to the pediatric department with a longer than 7-month history of nasal obstruction, purulent secretion, and reduction in sense of smell in the right nasal cavity. After drug treatment was ineffective, he was advised to visit our Ear, Nose, and Throat department. The patient had no history of nasal foreign bodies, fever, headache, facial pain, or bloody secretion. No lymphadenopathy was noted.Considering that the patient might not be able to cooperate with nasal endoscopy, a computed tomography (CT) examination was performed and revealed soft tissue density mixed with multiple discrete hyperdense calcification in the right posterior ethmoid sinus, without bony destruction Fig. . Based oWe performed endoscopic sinus surgery for the patient under general anesthesia, and found that the lesion was a solid mass of about 1.9\u200acm\u200a\u00d7\u200a1.2\u200acm\u200a\u00d7\u200a1.1\u200acm with a smooth surface involving the olfactory area. After complete resection, the specimens were sent for histopathological analysis. The hematoxylin and eosin-stained section showed proliferative surface epithelium without any dysplasia and 1 or 2 islands of odontogenic epithelium with eosinophilic material resembling dentin and numerous ghost cells  and extraosseous .DGCT is a locally aggressive neoplasm that usually develops in the maxilla or mandible, accounting for less than 1% of all odontogenic tumors.,9 Most peripheral DGCTs originate from the gingiva in dentate patients and alveolar mucosa in edentulous patients, usually appear as a sessile, sometimes pedunculated, exophytic mass.,10 In our case, the lesion might be derived from the mucosa of the nasal or ethmoid sinus, and involved the nasal cavity and olfactory area. Therefore, nasal symptoms similar to the symptoms of chronic sinusitis became the chief complaints. Nasal congestion and reduction in sense of smell caused by mechanical obstruction, and the purulent secretion might be complicated with the maxillary sinusitis. In addition, DGCTs usually appear radiographically as a cystic lesion or soft mass mixed radiolucent\u2013radiopaque lesion depending upon the degree of calcification, which are easily confused with the CT features of fungal ball in nasal sinus. Therefore, it might be misdiagnosed as fungal sinusitis. However, fungal sinusitis in children is very rare, so differential diagnosis should be considered, such as ameloblastoma, ossifying fibroma, and osteodysplasia fibrosa.The central DGCTs are usually located centrally in the bone (the maxilla and mandible), mainly occur in the canine to first molar region and exhibit as an asymptomatic swelling, although some may present with slight numbness and pain.,11 The ghost cells appear as eosinophilic epithelial cells that have lost their nuclei. The islands of ameloblastoma-like epithelium with ghost cells can distinguish DGCT from ossifying fibroma and osteodysplasia fibrosa but they may be difficult to separate from ameloblastoma. If it happens, dysplastic dentin can provide more information because the dentin and ghost cells are generally not present in ameloblastoma simultaneously. Lack of cystic structure and atypical mitosis can be used to distinguish from CCOT and ghost cell odontogenic carcinoma, respectively.Accurate diagnosis depends on pathological findings. Actually, both central and peripheral variants of DGCTs exhibit similar histological features in that the tumors are composed of ameloblastoma-like islands of odontogenic epithelium with clusters of ghost cells and variable amounts of dentinoid material in the surrounding tissue or near the epithelium. Peripheral DGCTs are less aggressive and can be controlled by local complete excision. No recurrences have been reported.,12 Regardless of the type, radiotherapy and chemotherapy are not recommended. On rare occasions, the DGCT transforms into ghost cell odontogenic carcinoma. Therefore, a long-term follow-up of no less than 3 years is suggested. There are no significant differences in clinical manifestations, pathology, and treatment between adults and children, according to current reports.Therapeutically, central DGCTs have an aggressive behavior and a high recurrence so that extensive surgical resection with an adequate safety margin is recommended.In summary, peripheral DGCT can occur in both adults and children without gender predilection. The sinus DGCT may be confused with fungal sinusitis based on the clinical manifestations, which reminds clinicians to consider this entity as a possible diagnosis.Conceptualization: Guo Liu.Data curation: Guo Liu, Jin-nan Li.Methodology: Feng Liu.Resources: Feng Liu.Supervision: Feng Liu.Writing \u2013 original draft: Guo Liu.Writing \u2013 review and editing: Feng Liu."}
+{"text": "Background and Purpose: Radiation therapy has long been contemplated as an important mode in the treatment of rectal cancer. However, there are few ideal tools available for clinicians to make a radiotherapy decision at the time of diagnosis for rectal cancer. The purpose of this study was to assess whether biomarkers expressed in the biopsy could help to choose the suitable therapy and provide predictive and/or prognostic information. Experimental Design: In total, 30 biomarkers were analyzed in 219 biopsy samples before treatment to discover the possibility of using them as an indicator for radiotherapy selection, diagnosis, survival and recurrence. Results: Twenty-two biomarkers  had diagnostic value. For survival, four biomarkers  were significant in regulating gene promoter activity and overall survival, while four had a trend . Three biomarkers  correlated with disease-free survival, while eight had a trend . Four biomarkers  were independent prognostic factors for recurrence. NFKBP65 and SIRT6 were significantly correlated with lymph node metastasis regardless of radiation. Patients with high AEG1, LOX, NFKBP65, PPAR and SATB1 had or showed a positive trend for better survival after radiotherapy, while those with positive PINCH and WRAP53 expression would not benefit from radiotherapy. Conclusions: AEG1, LOX, NFKBP65cyto, PPAR and SATB1 could be used as indicators for choosing radiotherapy. COX2-RT, COX2-NonRT and some other biomarkers may provide additional help for diagnosis. Multidisciplinary management has improved the prognosis of rectal cancer in recent years. The role of radiotherapy in the treatment of rectal cancer has also been initiated in the last few decades. Advances in preoperative radiotherapy have reduced the disease relapse and markedly increased the survival. However, there are few ideal tools available for the clinicians to make a treatment decision at the time of diagnosis. In this study, we analyzed the Astrocyte elevated gene-1 (AEG-1), CD163, COX2, Forkhead box M1 (FOXM1), ki-67, LIVIN, lysyl oxidase (LOX), Musashi-1 (MSI-1), nuclear factor-kappa Bp65 (NFKBP65), particularly interesting new cysteine-histidine-rich protein (PINCH), peroxisome proliferator-activated receptor (PPAR), PRb2/P130, p53, p73, phosphatase of regenerating liver (PRL), RNA-binding motif protein 3 (RBM3), special AT-rich sequence binding protein 1 (SATB1), sirtuin 6 (SIRT6), Tafazzin (TAZ) and WRAP53 biomarkers in 219 biopsy samples before treatment to discover the possibility of using them as an indicator for radiotherapy selection, diagnosis and prognosis. The results indicate that five biomarkers could serve as potent indicators for radiotherapy. Some biomarkers may provide additional aid for diagnosis, especially for those cases that are difficult to draw conclusions on with the traditional Hematoxylin\u2013Eosin staining. Only a few biomarkers could provide prognostic information for survival or recurrence, suggesting that it is unsuitable to predict prognosis excessively depending on the biomarkers in the biopsy. This study provides valuable information for the predictive or prognostic significance of biomarkers in biopsy and may give useful information to clinical practice.Colorectal cancer is among the three most common cancer diagnoses and causes of cancer death in women and men in Sweden and China ,2. MultiAccurate diagnosis and preoperative staging are essential to determine the optimal therapeutic plan for a patient. Computer tomography (CT) or magnetic resonance imaging (MRI) for polyp characterization is standard for preoperative staging but sub-optimal sensitivity and specificity values for detection of nodal metastasis are reported ,8,9. To Although alterations in numerous genes and pathways such as the APC/\u03b2-catenin/Tcf pathway have been indicated as possible risk factors for RC progression, the studies were mainly focused on tumor specimens obtained after operation and might be of little value to the initial management of the patients ,12. BiopAssessing the status of biomarkers in biopsy samples may provide additional diagnostic and predictive information, compared to traditional diagnosis with histopathology ,17. Howen = 127) or surgery alone . RT was given at 25 Gy in 5 fractions during a median of 6 days . Surgery was performed within a median of 8 days after RT . The expression of different biomarkers in biopsy samples  and in surgical samples including distant normal mucosa , primary tumor (PT) and lymph node metastasis (LNM) were determined by immunohistochemistry. The number of the samples varied for different biomarkers depending on available pathological material. The required informed consent was given by all participants. None of the patients received preoperative or adjuvant chemotherapy. There were no significant differences between the RT group and Non-RT group concerning the clinicopathological characteristics  was performed at our laboratory for the following proteins: astrocyte elevated gene-1 , CD163 , COX2 , Forkhead box M1 (FOXM1), Ki-67 , LIVIN, lysyl oxidase , Musashi-1 , nuclear factor-kappaBp65 , particularly interesting new cysteine-histidine-rich protein , peroxisome proliferator-activated receptor , PRb2/P130 , P53 , P73 , phosphatase of regenerating liver , RNA-binding motif protein 3 , special AT-rich sequence binding protein 1 , sirtuin 6 (SIRT6), Tafazzin/wwtr1  and WRAP53 . The IHC with the different antibodies was performed on the same patient samples at our laboratory. The intensity of staining was classified as 0 (negative staining: \u22645% positive cells), 1 (weak staining: weak yellow), 2 (moderate staining: yellow-brown) and 3 (strong staining: brown) and/or the proportion of staining was scored into five grades as 0%, 1\u201325%, 26\u201350%, 51\u201375% and 56\u2013100%. A case was evaluated as positive if it contained any positive cancer cells and negative otherwise. The staining patterns were graded as cytoplasmic, nuclear or stromal staining. The immunostaining was independently evaluated by two researchers including one pathologist without clinical and pathological knowledge about the patients. In the case of discrepant scoring results, a consensus score was reached after re-examination. The re-analysis was performed as described previously ,19,20,21n = 63) and RT group (n = 56), respectively, to exactly evaluate their predictive and diagnostic significance and, more importantly, find out whether the biomarkers in BS could be used as RT indicators. For the protein expressed in the cytoplasm, nucleus and/or stroma, we distinguishingly named them as \u201c-cyto\u201d, \u201c-nucl\u201d and \u201c-strom\u201d in \u201c-RT\u201d or \u201c-NonRT\u201d group, respectively, e.g., \u201cFOXM1cyto-NonRT\u201d and \u201cFOXM1nucl-RT\u201d. Thus, with 30 biomarkers in each group, in total 60 biomarkers were analyzed.We mainly implemented the study on three different levels. Firstly, we evaluated whether the radiation had an impact on the expression of the biomarkers. Secondly, we compared the staining of the different biomarkers BS, DN, PT and LNM between the Non-RT group and RT group, respectively, to see whether there was a correlation among those samples. Thirdly, we compared the expression of biomarkers in matched samples (the samples from the same patients) in Non-RT group (2) test or Fisher\u2019s exact test. For ranked data, a nonparametric test is a suitable statistical option [p-value < 0.05 was considered to be statistically significant. All statistical analyses were performed using IBM SPSS Statistics 23.0 software.The clinicopathological characteristics between RT and Non-RT groups were analyzed by Chi-square (\u03c7l option  The diffp = 0.007), MSI1 , RBM3cyto , RBM3nucl , TAZ  and WRAP53cyto   . The exp= 0.010) .We analyzed the difference of 60 biomarkers  between BS and PT in the unmatched or matched cases to find out whether BS can represent PT, especially focused on those in the matched samples, as the results from the matched samples would be more dependable.p < 0.05; 10/60, 16.67%), while the expression of the other biomarkers in BS were equal to or more than those in PT tissue . This additionally confirmed that, in most cases, biomarkers in BS could be those of PT.Then, we used correlation analysis to further discover the relationship between BS and PT in the RT group or Non-RT group, respectively. The results show that most biomarkers in BS were related to PT, of which 24 biomarkers had close relationships . In other words, they were not suitable for diagnostic analysis since there was no noticeable difference between BS and DN. The other 47 biomarkers were different when compared  and would be further analyzed for their diagnostic value. Comparing the expression of biomarkers between BS and DN highlighted that 22 biomarkers  in BS had diagnostic value for RC patients preoperatively , while the other 48 biomarkers in BS were equal to or higher than those in LNM (80.00%). Further correlation analysis highlighted that, among the 48 biomarkers, six were closely related to those in LNM , P130cyto-NonRT (p = 0.048), PINCH-NonRT (p < 0.001) and PPARcyto-RT (p = 0.049). AEG1-RT (p = 0.074), LOXnucl-RT (p = 0.096), SATB1-NonRT (p = 0.062) and SIRT6nucl-RT (p = 0.052) tended to be related to OS. The other biomarkers in BS had no prognostic value for OS , PINCH-NonRT (p = 0.005) and WRAP53cyto-NonRT (p = 0.007). AEG1-RT (p = 0.091), COX2-NonRT (p = 0.084), Ki67-NonRT (p = 0.089), LOXnucl-RT (p = 0.088), NFKBP65cyto-RT (p = 0.054), PPARcyto-RT (p = 0.067), SATB1-NonRT (p = 0.094) and WRAP53nucl-NonRT (p = 0.077) had the tendency to be related to DFS. Patients with positive expression of these biomarkers had reduced DFS time compared to those with negative tumors , NFKBP65cyto-RT , P130cyto-NonRT  and PPARcyto-RT  in BS were independent prognostic factors for local/distal recurrence; patients with high expression of these four biomarkers in BS were more prone to suffer from tumor recurrence  were not considerably changed by RT, while six biomarkers were significantly increased or reduced in PT after preoperative RT: FOXM1nucl, MSI1, RBM3cyto, RBM3nucl, TAZ and WRAP53cyto. Among them, MSI1 had diagnostic significance in both the Non-RT and RT groups; RBM3nucl trended to have diagnostic significance in patients who received RT; TAZ was helpful for the diagnosis in the Non-RT group; and WRAP53cyto was related to DFS and had a trend with recurrence in Non-RT group patients. We then analyzed the varied expression of biomarkers between BS and PT in different levels  to observe whether BS could represent PT. We observed that the expression of 10 biomarkers in BS was less than those in PT, while the 50 other biomarkers (83.33%) expressed in BS were equal to one or more of those in PT. Correlation analysis showed that most biomarkers in BS were related to PT, of which 24 biomarkers had a close relationship. The results further confirm that, in most cases, BS markers could be of PT, although in 16.67% of cases BS was less than PT.In the last few years, several studies have demonstrated high concordance between biopsy and resection samples in CRC and other cancers using IHC and molecular testing such as cytogenetic testing and microarray analysis . Shia etp < 0.001) in RC patients. Routine forceps biopsy, causing tissue fragmentation in a few cases, was of limited value especially in identifying residual cancer cells after nCRT. (3) The third caveat relates to the possibility that some of the driver mutations are occasionally selected late, and may thus be presented in a sub-clone that has not yet gained dominance when the biopsy is carried out, although that might happen at a very low frequency. (4) IHC performed in tissue microarrays rather than whole-tissue sections may also, to a small extent account, for the different percentages of positive results [The difference might be caused by multiple factors: (1) It could be a result of the less tissue sampled for the biopsy, i.e., the tissue obtained may be insufficient to represent the overall tumor, in addition to CRCs being known for their intra-tumor heterogeneity . The uti results . (5) It Rossum et al.  found thOn the other hand, if the expression of an oncoprotein in BS is lower than that in DN, it does not possess any diagnostic value. We analyzed the diagnostic value of biomarkers in BS and confirmed that 22 factors (36.67%) could be used as diagnostic biomarkers. Four biomarkers (6.67%) had the tendency to be meaningful for diagnosis and might need more studies for further validation.Several research groups have indicated the predictive value of biomarkers in the biopsy of various cancers. Sato et al.  demonstrIn the present study, we revealed that the expression of NFKBP65nucl, SIRT6cyto and SIRT6nucl in BS significantly correlated with those in LNM regardless of RT. Four biomarkers in BS correlated with OS: NFKBP65cyto-RT, P130cyto-NonRT, PINCH-NonRT and PPARcyto-RT. AEG1-RT, LOXnucl-RT, SATB1-NonRT and SIRT6nucl-RT tended to be related to OS. The other 86.67% (52/60) of biomarkers had no predictive value for OS. Among the 60 biomarkers, three were related to DFS: COX2-RT, PINCH-NonRT and WRAP53cyto-NonRT. AEG1-RT, COX2-NonRT, Ki67-NonRT, LOXnucl-RT, NFKBP65cyto-RT, PPARcyto-RT, SATB1-NonRT and WRAP53nucl-NonRT tended to be related to DFS. The patients with positive expression of these biomarkers were observed to have reduced DFS time compared to those with negative tumors. The other 49 biomarkers (81.67%) had no predictive significance for DFS. Further analysis indicated that only four biomarkers (6.67%), COX2-RT, NFKBP65cyto-RT, P130cyto-NonRT and PPARcyto-RT, in BS were independent prognostic factors for local/distal recurrence; the patients with high expression of these biomarkers were more prone to suffer from tumor recurrence. Eight biomarkers had the trend to be related to local/distal recurrence; however, there was no relationship between the other 48 biomarkers (80.00%) and recurrence. The patients with high COX2 or WRAP53cyto expression tended to have worse OS and DFS regardless of RT, while high P130cyto in BS tended to predict better OS and DFS in both Non-RT and RT groups. Importantly, patients with positive AEG1, LOXnucl, NFKBP65cyto, PPARcyto and SATB1 expression had or tended to have better OS and DFS after RT, positive Ki67 expression tend to have a close correlation with better DFS after RT and those with positive SIRT6nucl expression tend to have a better OS after received RT. In contrast, patients with high PINCH and WRAP53nucl did not benefit from RT; thus, these biomarkers might be used as RT indicators.The present analysis confirmed that: (1) Well-known oncogenes including AEG1, LOXnucl, NFKBP65cyto, PPARcyto and SATB1 can also be used as indicators for RT selection. (2) Only a few biomarkers in BS could provide predictive information for survival or recurrence, so we should be vigilant of their limited value. It might be unsuitable to predict prognosis egregiously depending on the expression of biomarkers in BS. (3) Some biomarkers could provide additional information and be helpful for diagnosis, especially for those cases on which it is difficult to draw diagnostic conclusions with routine Hematoxylin\u2013Eosin staining. As many biomarkers in BS were not meant for diagnosis, it is inappropriate for the clinicians to excessively rely on biomarkers in BS to make their diagnosis. (4) Biopsy has limitations in clinical practice, which might be unavoidable but could be improved. Clinicians should be aware of this and reasonably use it.There are several limitations to the present study. The sample size was not large enough due to the fact that biopsy samples were not available for all the subjects we selected from the local cancer registry, which might be not reprehensive of the entire corresponding populations. In addition, due to the paucity of biopsy samples, it was not possible to analyze all the selected biomarkers in all samples. Thirdly, owing to the lack of complete information on the family history, we could hardly evaluate whether there were any special characteristics in hereditary RC such as Lynch syndrome-associated RC.Our results provide valuable information for refining the previously debatable description of the value of biomarkers in biopsy in RC and would give useful information to clinical practitioners. Further studies, focusing on those screened biomarkers in a larger population, are needed to precisely verify the efficiency of pretreatment biopsy specimens in helping the clinician to make a therapeutic selection and predicting the outcome of RC patients."}
+{"text": "Combustible tobacco users appear to be at greater risk for serious complications from COVID-19. This study examined cigar smokers\u2019 perceived risk of COVID-19, quit intentions, and behaviors during the current pandemic. We conducted an online study between 23 April 2020 to 7 May 2020, as part of an ongoing study examining perceptions of different health effects of cigars. All participants used cigars in the past 30 days (n = 777). Three-quarters of the sample (76.0%) perceived they had a higher risk of complications from COVID-19 compared to non-smokers. The majority of participants (70.8%) intended to quit in the next six months due to COVID-19, and almost half of the sample (46.5%) reported making a quit attempt since the start of the COVID-19 pandemic. Far more participants reported increasing their tobacco use since COVID-19 started (40.9%) vs. decreasing their tobacco use (17.8%). Black or African American participants, participants who reported using a quitline, and participants with higher COVID-19 risk perceptions had higher intentions to quit using tobacco due to COVID-19, and higher odds of making a quit attempt since COVID-19 started. More research is needed to understand how tobacco users are perceiving COVID-19 risks and changing their tobacco use behaviors. Cigarette, cigar, and other combustible tobacco users appear to be at greater risk for developing serious complications from COVID-19 ,3,4. At Due to the potential negative health outcomes associated with COVID-19, it is possible that many tobacco users feel more vulnerable to its health effects and are trying to quit using tobacco\u2014which aligns with both previous research and theory on risk perception ,13. HoweThe goals of this study were to examine cigar smokers\u2019 perceived risk of COVID-19, quit intentions, and behaviors during the current pandemic. In the United States (US), cigars are one of the most commonly used tobacco products with 7.2% of adults reporting smoking cigars in the past 30 days , and oveWe also examined how other correlates were associated with COVID-19 quit intentions and behaviors, including demographics, such as age and race, and tobacco use variables, such as other tobacco use and nicotine dependence. We did so because research has found several factors in the US to be associated with risk of acquiring COVID-19 and experiencing complications, including race/ethnicity, age, and other comorbidities. Specifically, African Americans in the US are dying of COVID-19 at greater rates than white people, and are experiencing more complications . IncreasWe recruited participants as part of an ongoing study examining perceptions of different health effects of cigars. Specifically, this study presented different text statements about the health effects of cigars  to participants, and asked them to answer if they were aware of this health effect and how effective it was at discouraging their cigar use. This study will be used to develop effective warnings for little cigar and cigarillo packages. We expanded eligibility to include all cigar users to be more representative of the cigar smoking population. Participants were recruited and compensated through Qualtrics Services\u2014a leader in the field of survey software, deployment, methodology, and consumer research. Qualtrics has existing panels that can be used for social science research ,29,30, aWe had two primary outcomes: (1) quit intentions in the next 6 months due to COVID-19 , and , perceived risk of complications due to COVID-19 compared to non-smokers (newly developed item), COVID-19 risk perceptions , and frWe conducted analyses in SAS V.9.3 . We first examined bivariate associations with our two outcomes (quit intentions and quit attempts) and all correlates of interest\u2014participant characteristics, tobacco use variables, and other COVID-19 variables. We then examined bivariate associations among our two primary outcomes and changes in tobacco use since COVID-19 started . Finally, we examined associations among all correlates and our two primary outcomes in separate multivariable models. As our first outcome\u2014quit intentions\u2014was continuous, we entered all participant characteristic, tobacco use, and COVID-19 correlates in a multivariable linear regression model. As our second outcome\u2014quit attempts\u2014was dichotomous, we entered all correlates in a multivariable logistic regression model. We included all correlates in the adjusted multivariate regression models, because we did not have any a-priori hypotheses for which correlates would be associated with our outcomes. While we planned to examine changes in tobacco use as a third outcome in a multivariable, multinomial regression model, we did not run this model due to small cell counts in some of the tobacco use response options. Results from the first model include Beta coefficients (B) and standard errors (SE). If the B is positive, the interpretation is that for every 1-unit increase in the correlate, the outcome variable will increase by the B value. Results from the second model include adjusted odds ratios (aOR) and 95% confidence intervals (CI). An aOR of more than 1 means that there is a higher odds of the outcome happening with exposure to the correlate. An aOR of less than 1 means that there is a lower odds of the outcome happening. For all analyses, we set \u03b1 = 0.05 and used 2-tailed statistical tests.Participant characteristics and tobacco use variables are presented in COVID-19 variables are presented in p < 0.001 for both comparisons). Similarly, participants who reported decreasing their tobacco use were more likely to report making a quit attempt (63.0%), and participants who reported increasing their tobacco use were also more likely to report making a quit attempt (57.6%), compared to participants who reported keeping their tobacco use the same (28.4%) (p < 0.001). Finally, participants who reported making a quit attempt had higher quit intentions  than participants who did not report making a quit attempt  (p < 0.001). All bivariate associations among correlates of interest and our two outcomes (quit intentions because of COVID-19 and quit attempts since COVID-19 started) are presented in Bivariate associations among our two outcomes with changes in tobacco use are presented in p = 0.002) and had higher odds of making a quit attempt since COVID-19 started , compared to White participants (p = 0.005).Black or African American participants had higher quit intentions due to COVID-19 , compared to participants who smoked cigars and did not smoke cigarettes. In addition, participants who smoked cigars and used smokeless tobacco had higher odds of making a quit attempt since COVID-19 started , compared to participants who smoked cigars and did not use smokeless tobacco.p < 0.001), and had higher odds of making a quit attempt since COVID-19 started , compared to participants who did not report calling a quitline. Participants who believed they had a higher risk of complications due to COVID-19, compared to non-smokers had higher quit intentions due to COVID-19  than participants who believed they had the same risk of complications. In addition, having higher risk perceptions of COVID-19 was associated with higher quit intentions due to COVID-19  and higher odds of making a quit attempt since COVID-19 started . Finally, greater social distancing efforts were associated with higher quit intentions due to COVID-19 .Participants who reported calling a quitline had higher quit intentions due to COVID-19  than decreasing their tobacco use 18%). Only one previous study, to our knowledge, has examined how tobacco users have changed their behaviors in response to COVID-19 and reported similar results. In this study of 345 dual cigarette and e-cigarette users, 30.3% of participants reported increasing their cigarette use and 29.1% reported increasing their e-cigarette use since learning of COVID-19 %. Only o,13. However, we also found that people who reported increasing their tobacco use also reported higher quit intentions and had higher odds of making a quit attempt\u2014in line with research on the \u201cstress hypothesis\u201d. It is possible that these people want to quit, but are stressed or anxious, and are increasing their tobacco use despite their \u2018good\u2019 intentions. To this point, we did find that better mental health status was associated with higher intentions to quit. In addition, people may be bored or have stockpiled tobacco products before sheltering in place orders, which could have increased their tobacco use. Finally, it is also possible that people may want to quit but are not able to easily access evidence-based cessation resources like pharmacotherapy or behavioral support . We also found that over a fifth of participants in our sample reported calling a quitline because of COVID-19, and that those who reported calling a quitline had higher quit intentions and attempts. Although the majority of US smokers are aware of quitlines ,42, theyFinally, we identified several characteristics that were associated with COVID-19 quit intentions and attempts. For instance, Black or African American participants had higher quit intentions and attempts due to COVID-19. African Americans in the US are dying of COVID-19 at greater rates than white people and experiencing higher rates of complications . This maThere are several limitations to this study. First, all data were self-reported, which introduces threats of social desirability bias. Second, this was a one-time cross-sectional study, which means that we were unable to assess temporality of associations or trends over time. Future longitudinal research is needed to understand how COVID-19 is changing participants\u2019 smoking and quitting behaviors. Third, all participants were recruited online and are not representative of the US population or of tobacco users, which means that study findings may not apply to other countries or groups of tobacco users. Fourth, since this was a cross-sectional study and there was no possibility of randomizing participants to different exposures, we cannot make any claims of causality. We are careful throughout this article to use language like \u201ccorrelates\u201d rather than \u201cpredictors\u201d and \u201cassociated with\u201d rather than \u201ccauses.\u201d Fifth, we created several measures in this study that have not been previously used  given the novelty of COVID-19 and the rapid research needed to understand it. Many tobacco users included in our study perceived that they were at risk higher risk of COVID-19 complications, reported intending to quit due to COVID-19, and reported making a quit attempt since COVID-19 started, however, more tobacco users reported increasing their tobacco use due to COVID-19 than decreasing it. More research is needed to understand how tobacco users are perceiving risks of COVID-19 and changing their tobacco use behaviors."}
+{"text": "The perceived health risks of tobacco products may change during the ongoing COVID-19 pandemic. This study aimed to examine the perceived risks of tobacco use on COVID-19 infection and severity, and possible COVID-related changes in perceptions of tobacco use and overall health.We conducted an online survey of adults in the United States in June 2020 (n=2097). The survey covered cigarettes, cigars, e-cigarettes, and hookah. We also assessed changes in the use of any of the four tobacco products. Multivariate logistic regression models were used to estimate the odds of agreeing with the perceived risks for each risk and each product, with the adjustment of demographic and COVID-19 related variables.For all four tobacco products, the perceived risks to general health were slightly higher during the pandemic than before the pandemic (77% vs 79.5% for cigarettes) and the perceived risk of COVID-19 severity was larger than the perceived risk of COVID-19 infection (73.3% vs 56.2% for cigarettes). All risk measures varied with tobacco products consistently, with the risks highest for cigarettes, then cigars, followed by e-cigarettes and hookah. Females and people with higher income or education were more likely to endorse the risks of tobacco use than their counterparts. People who perceived higher risks of using cigarettes  and cigars  to COVID-19 severity were more likely to have decreased or quit their use.Tobacco/nicotine use was perceived to increase the risk of COVID-19 severity and the perceived risk of tobacco/nicotine use to general health was high during the pandemic, particularly for cigarettes. The change of perceived risks appeared to be prompting harm-reducing changes in tobacco product use. In tobacco regulatory science, findings of health risk misperceptions that can promote harmful tobacco use behaviors are used to prompt and inform corrective action through new public education efforts or new requirements relating to product characteristics (e.g. flavors or appearance), product packaging, and labeling design, advertising and other marketing, warning labels, and educational package inserts5.Perceptions of health risks and harms are associated with tobacco product experimentation, initiation, frequency, and intensity of use, as well as quit attempts7, it is not clear if current smoking is associated with a reduced risk of COVID-19 infection10. This initial research seems to contradict the hypothesis that smokers are at higher risk of disease severity due to mechanisms such as pre-existing lung disease or reduced lung capacity or higher rates of various other diseases among smokers compared to non-smokers, and have an increased transmission risk due to greater contact between fingers and lips and inhaling deeply near one\u2019s fingers. These seemingly controversial findings, together with misleading information on social media, may confuse the public, particularly smokers. Similarly, the influence of other tobacco product use such as cigars, e-cigarettes, and hookah on COVID-19 is largely unknown. The uncertainty and controversy may impact the perceived risk of tobacco use among both tobacco users and non-users.Perceived health risks of tobacco products may change during the ongoing COVID-19 pandemic, given the uncertainty surrounding the association between smoking, vaping, and COVID-19 incidence and outcomes. Preliminary evidence indicates that although smoking may be associated with a higher mortality rate and suffering more severe consequences of COVID-1912. Another study found that the majority of people who used tobacco/nicotine believed that their tobacco/nicotine use did not increase the risk of COVID-19 infection but increased the severity of COVID-19 if infected13. Further, among tobacco/nicotine users, a high perception of the risk of tobacco/nicotine use to COVID-19 was associated with a reduction in tobacco/nicotine use or an increase in the desire to quit tobacco/nicotine use14. However, these studies were conducted either in relatively small regions or having a small sample size thus limiting the generality of their findings.Research on the perceived health risks of tobacco/ nicotine use during COVID-19 pandemic is currently limited. Initial evidence showed the majority of people who used cigarettes and e-cigarettes believed that their tobacco/nicotine use increased their risk of COVID-19 in generalThis study aimed to examine the perceived risks of tobacco/nicotine use on COVID-19 infection and COVID-19 severity, as well as any possible COVID-related changes in perceptions of tobacco-nicotine use and overall health harms and risks. Specifically, we examined how perceived risks were distributed within the population and varied by tobacco products, related factors, and their influence on tobacco use behaviors.https://www.mturk.com/), an online crowdsourcing platform to conduct surveys. Inclusion criteria were: 1) ever use (as of 31 December 2019) of at least one tobacco product among the four categories of cigarettes, cigars , e-cigarettes, and waterpipe (i.e. hookah); 2) residence in the US; and 3) 90% or above approval rating from previous MTurk tasks. Eligible participants were given access to the survey, hosted by Qualtrics . The survey was active in MTurk between 8 and 18 June 2020. Each eligible participant was compensated $0.25. The Institutional Review Board at the University of Memphis approved this study.We conducted a survey of adults in the United States using Amazon Mechanical Turk , participants reported the COVID-19 status of themselves and their family, whether they had experienced a COVID-19 death among family members, friends, or colleagues, and the zip-code where they lived. Using the home zip-code, the urbanization level of the neighborhood was classified as urban, suburban, or rural, using the rural-urban commuting area (RUCA) codesThe survey focused on four types of tobacco-nicotine use: e-cigarettes or smoking either cigarettes, cigars, or hookah \u2013 and asked about the perceived risks of each on: 1) likelihood of COVID-19 infection; 2) severity of COVID-19 if infected; and 3) general health. For COVID-19 infection, participants were asked: \u2018Do you agree that the use of each of the following tobacco products will increase the risk of getting COVID-19?\u2019, and the response options were based on a 5-point Likert scale . For COVID-19 severity, the question was: \u2018If a person gets COVID-19, do you agree that the use of each of the following tobacco products will make him/her sicker, or sicker for a longer period of time?\u2019. For general health, we assessed perceived risk both before and during the COVID-19 pandemic. The questions were: \u2018Before the COVID-19 pandemic, for example, during the year of 2019 in general, to what extent did you agree that the use of each of the following tobacco products causes serious illness is very convincing?\u2019 and \u2018Right now, to what extent do you agree that the use of one tobacco product causes serious illness is very convincing?\u2019.We also assessed changes in the use of any of the four tobacco products during the COVID-19 pandemic, including new initiation, cessation, switching, or changes in consumption levels.To analyze the data, we first summarized the percent of participants who agreed or strongly agreed with the perceived risks for each tobacco/ nicotine product. Second, multivariate logistic regression models were used to estimate the odds of agreeing with the perceived risks for each risk and each product. Finally, multivariate logistic regression models were used to estimate how the perceived risks influenced the odds of decreasing or quitting the use of each tobacco product compared with increasing or initializing it. All multivariate logistic regression models were adjusted by demographic variables including gender, age, race/ethnicity, educational level, and household income, urbanization level of the neighborhood, COVID-19 related variables such as if any COVID-19 infection for self and family and if any COVID-19 death among social network, and the use status of the specific tobacco/nicotine product in 2019. Analyses were performed using SAS 9.4 .Among the 2097 participants who completed the survey, 53% were male, 57% were aged 18\u201334 years, 63% were White, 10% were Black, 16% were Hispanic, 8% were Asian, 60% had at least a Bachelor\u2019s degree, and 81% lived in urban areas. Most (91%) participants and their family members did not have any COVID-19 symptoms, and 22% reported a COVID-19 death among family members, friends, or colleagues. Among the participants, the prevalence of ever using (considering both exclusive use and co-use or poly-use with other tobacco products) cigarettes, cigars, e-cigarettes, and hookah in 2019 was 67%, 23%, 39%, and 16%, respectively (Supplementary file Table S1).As As As 18 and less traditional combustible products such as hookah20. For all four products, the perceived risks to general health during the pandemic were higher than the perceived risk to general health before the pandemic. Because the two risks were measured at the same time, the difference may not reflect an increase of the perceived risk of tobacco use to general health. However, such \u2018perceived\u2019 increase in perceived risks, although moderate, were consistent across the four major tobacco products and is consistent with the findings that adults generally perceive that tobacco-nicotine use is linked with increased COVID-19 incidence and severity . Unlike smoking-related cancer, heart disease, and lung disease, which generally take decades to develop, thereby obscuring their causal links, the effect of COVID-19 is much more immediate and tangible. The perceived risks of tobacco use, even from exposure to secondhand smoke or e-cigarette aerosols may increase markedly due to COVID-19.For all three risk measures, perceived risks were higher for cigarette and cigar smoking compared with e-cigarette and hookah use, consistent with the general relative perceived risk that traditional combustible tobacco products were more harmful than non-combustible tobacco products22. Likewise, higher income and education are associated with higher awareness of the harms of smoking and nicotine24.We found that females and people with higher income or education level were more likely to endorse the risks of tobacco use. This is consistent with existing literature where perceived risk of tobacco use and many other outcomes rated higher in females than in malesIt was noteworthy that reporting a COVID-19 death within a respondent\u2019s social network was consistently associated with lower perceived risk from tobacco-nicotine use. This may be because those who had a higher perceived risk towards certain tobacco products tend to use less or do not use this product, as persons in their social network. Thus their lower tobacco use may decrease their risk of COVID-19 death. It is also possible that the salience of identifiable risk factors for COVID-19 other than tobacco use among members of one\u2019s social networks reduces the attributions of tobacco as a causal agent for the disease.We found that decreasing or quitting the use of certain tobacco products was associated with a greater perceived risk of severity of COVID-19 infection but not with the perceived risk of COVID-19 infection. This indicated that the concern of the severity of COVID-19 if infected was more effective as a deterrent from using the product than the concern of becoming infected. It could also be that people generally think that avoiding COVID-19 infection is largely a matter of chance or, alternatively, engaging in social distancing, mask-wearing, and handwashing, with tobacco use simply not part of the equation.One limitation of this study is that the study sample was recruited from a crowdsourcing platform. Rather than a national representative sample, our participants tended to be younger and more highly educated compared to the average US adult. Secondly, as a cross-sectional study, we may not be able to accurately capture the changes in the perceived risk of tobacco use to general health. For example, as aforementioned, the perceived risks to general health before the pandemic and during the pandemic were measured at the same time, thus the difference may not reflect a real increase of the perceived risk of tobacco use to general health. Lastly, we did not capture detailed information on the history of tobacco use and current use status, which is potentially correlated with perceived risks. For example, the perceived risk is likely to be different between new and long-term users, between heavy and light users, between people who use one product, and those who are dual and poly-users.13, the perceived risk of tobacco use on COVID-19 severity was much higher than the perceived risk of COVID-19 infection, and the majority of participants endorsed that tobacco use increases COVID-19 severity. Females and people with higher income or education level were more likely to perceive the risks of tobacco use than their counterparts. The finding that the perceived risk of tobacco use to COVID-19 infection fluctuated around 50% for various tobacco products reflects confusion among the adult public. The positive aspect is that these perceptions \u2013 whether accurate or not \u2013 appear to be prompting harm-reducing changes in tobacco product use14.Overall, COVID-19 is increasing perceived risks among the public from using the four types of tobacco products. Consistent with the initial evidenceClick here for additional data file."}
+{"text": "With the continuous deepening of medical reforms and the continuous attempts and explorations of various management models, the traditional health care model is undergoing tremendous changes, and patients' needs for medical institutions are becoming more and more comprehensive. Medical institutions are meeting the needs of providing medical services to patients at the same time. It is even more necessary to change our thinking and enhance the service concept. This article is based on case-based deep learning hospital nursing business process reengineering and the application and feasibility study of integrated nursing information construction in nephrology nursing. This article uses the literature analysis method, the social survey method, and other methods to discuss the construction of integrated nursing information. On the one hand, the content of this article uses the concept of process reengineering to analyze the current development status and existing problems of the hospital care industry and find countermeasures to solve problems. On the other hand, the main research content of this article is the construction of integrated nursing information and its analysis of the application and feasibility of nursing in the nephrology department. At the same time, under the background of the rapid development of the mobile Internet, we will carry out extended thinking on the continuous transformation of the construction of nursing information. According to the survey results, 87.5% of patients in the nephrology department are dissatisfied with the current hospital's work efficiency, and 85.7% of the nursing staff in the nephrology department are generally satisfied with the information management of the current department. After the implementation of the hospital information integration system, patient satisfaction is as high as 98.2%, and the satisfaction of medical staff reached 94.2%. The construction of integrated nursing information has played a great role in the application of nephrology nursing. Under the background of information integration, with the rapid development of computer technology, network technology, and communication technology, information construction has become a hot spot of concern to the whole society. The popularization of the Internet has provided unprecedented opportunities for the development of information technology. While the field brings opportunities, it also intensifies competition. The essence of the integrated construction of nursing information is that many medical systems are integrated horizontally, and data and information are seamlessly linked, so that hospitals, departments, and positions can be interconnected, breaking the traditional medical mode of vertical and gradual information transmission mode . InformaWith the continuous development and progress of society and the reform of the medical system, many experts and scholars began to discuss \u201chow to continuously improve the level of medical care\u201d . This inn\u2009=\u200940 before and after n\u2009=\u200980) was used to measure changes in nurse satisfaction. Whalen et al. believes that when nursing and nursing services are integrated, real-time pickup and agency tasks also increase, which may lead to an increase in database queries, thereby increasing the amount of information transmitted [In recent years, as the informatization has gradually penetrated our daily lives, it has brought us convenience in various fields. All regions are exploring how to use informatization and processes to better provide patients with convenient and effective medical services. The Zhu and Gu study examined the impact of the comprehensive nursing handover system  on nurses' satisfaction with handover and practice changes . Backgronsmitted . It can The integrated construction of nursing information relies on the development of informatization, intelligence, and platformization. The information platform is used to reengineer the conventional traditional business processes effectively and thoroughly, accompanied by the third characteristic of informatization and intelligent technology . The conWith the continuous promotion of the concept of \u201chigh-quality nursing service\u201d throughout the country and related research and development, the concept of \u201cconsolidating basic nursing and providing satisfactory services\u201d has been continuously deepened and developed in the nursing work of nurses. Nurses continue to improve themselves and perform their duties as nurses. Patients provide effective, high-quality, and satisfactory nursing services. Patient satisfaction has been included in the evaluation process of hospital standardization construction and has become an important index for evaluating hospitals . As an iWith the reform of the medical system and the state's repositioning of the nature of hospitals, competition in the medical industry has become increasingly fierce. Affected by the external environment, the competition among major hospitals has changed from a single factor to a comprehensive factor. Therefore, hospitals must use medical resources scientifically and rationally, and think and solve problems from the perspective of patients, to effectively enhance their competitiveness. The nursing work flow is based on an in-depth understanding and scientific analysis of the main business processes of the organization and is guided by customer needs. Effective planning, reorganization, or modification of the main business processes is carried out to improve the links in the business process to improve the quality of the entire operation, with the purpose of improving labor efficiency and reducing the overall cost of operations . In shorx to x\u2009+\u20091. This process requires analysis [x to x\u2009\u2212\u20091. The first process is the end of the needs analysis. The admission procedure to the discharge procedure is a system, and the general state of the system can be expressed as follows:Tx represents the time distribution of the arrival of customers during the input process, Tp represents the time distribution of the patient's stay in the hospital or the patient's information in the hospital system, and R represents the information system capacity [Rn\u22121 represents the overall information capacity after excluding the nth patient's remaining capacity [Entering the hospital's information system through the patient's personal information makes the system state from analysis ; when thcapacity . When a capacity .r represents the total nursing business demand after the system is updated [\u03bb, T0) represents the demand status when different patients end business services and start new nursing services. The data is sorted by the genetic algorithm, which can be efficiently arranged for process reengineering after monitoring the data. Process reengineering is just the beginning. Continuous improvement is the core of maintaining process reengineering.Because there is the possibility of another patient's information output at the same time as the patient's information is entered, in order to ensure the effective reintegration or redesign of the overall hospital care business in this situation, we choose to use genetic algorithms to select, cross, and cross over. Mutation and other operations are adjusted to ensure accurate data update. The method of data integration of the genetic algorithm in nursing business process is  as follo updated , and , the solution of the recursive formula is as follows:Equation  is a typThus, the following is obtained:Si represents the reliability index of the parallel system; Fi represents the reliability index of the hybrid system; and Se is the reliability of the individual nursing information or the basic information system of a single patient that makes up the parallel system [It is very convenient to build an integrated nursing information system program with a high-efficiency development program, operation efficiency, and operational functionality . Analyzil system . The nur\u2009 Step 1: nephrology patients who meet the diagnostic criteria during the recovery period\u2009 Step 2: stable vital signs\u2009 Step 3: patients with cognitive dysfunction who cannot cooperate with the investigator are excluded\u2009 Step 4: nephrology nursing work for more than half a year\u2009 Step 5: pass the annual assessmentInclusion criteria of experimental subjects are as follows:This article investigates the patients' satisfaction of the nursing business process in the nephrology department of a third-class hospital in Guangzhou. Through literature research, expert consultation, and interviews, the preparation of the questionnaire was repeatedly revised, and the questionnaire was designed and carried out for the satisfaction of patients in nephrology and related nursing staff, and the reliability and validity of the questionnaire were tested to ensure the validity of the questionnaire. Through the descriptive statistics of the questionnaire and the satisfaction results of medical and nursing integration, a comprehensive evaluation of the construction of information integration is carried out.Using the nursing business process reengineering theory to build a nursing business process model and using genetic algorithms to analyze the model will have a certain effect on the analysis of the role of integrated nursing information construction, medical staff, and nephrology patients' satisfaction.wo experiments were conducted, which are as follows:\u2009 Step 1: expert consultation and interview on information literacy indicators of nursing staff\u2009 Step 2: analysis of the missing items of nursing information in the Department of NephrologyThe survey results show that 87.5% of patients in the nephrology department are dissatisfied with the current hospital's work efficiency, and 85.7% of the nursing staff in the nephrology department are generally satisfied with the information management of the current department. After the implementation of the hospital's information integration system, patient satisfaction is as high as 98.2%, and the satisfaction of medical staff reached 94.2%. Through the analysis and design of each function in the mobile nursing system based on the Internet of things, the following functions are mainly realized . The nurThe introduction of modern computer and communication technology into the medical field has extremely practical value. The integrated nursing information system adopts the operation mode of sending and receiving at the same time. It can ensure that the nursing work is carried out in an orderly manner and that the nursing needs of patients are met. Build a network-based medical care information service platform in a short time and gradually integrate relevant information resources of the hospital. The improvement in business management work is a feature of current research for the research of intelligent nursing path information management systems, which has important economic and social value , 32. In The nursing business process model diagram shows that the nursing work is based on the basic information of nephrology patients entered into the hospital database. The patient's medical care information terminal fills in the patient's personal medical record information through the patient's family members and submits medical care online, which shortens the complicated procedures of traditional care and saves manpower and material resources to a large extent. However, the staff is required to classify the patient care information filled out by the patient personnel and then input the data into the computer for data processing \u201335.Nurses can read or enter patient information through the system at any time, and the computer will generate the dynamic changes of the patient's important physical signs and doctor's orders. While saving manpower, it also avoids the introduction of errors in the transmission and entry process. At the same time, nursing staff can analyze specific problems and flexibly change nursing methods and nursing items so that patient care is more objective and fairer. Nurses can more accurately grasp the doctor's diagnosis and treatment intentions based on computer prompts, and all payment, medicine withdrawal, and dispensing information are entered into the system, reducing communication barriers between departments. In this regard, we compared the work efficiency of nephrology nurses before and after the implementation of integrated information management and recorded the data in According to the comparative analysis of the data compiled in The main function of the system test is to analyze, summarize, and correct the errors of the system in the test so that the functional requirements in the mobile nursing information management can be correctly realized in the system, and the nonfunctional requirements can also be improved. The mature system can better meet people's working habits and make the operation easier. According to the actual operation users of the integrated nursing information system in the overall medical unit, combined with the analysis of the needs of doctors and patients, and combined with various business operations, the specific user roles involved in this research have been determined. They are medical patients and medical staff (this experiment is mainly for nursing staff) and information system managers. This article selects the nephrology department of a third-class hospital in Guangzhou to conduct questionnaire surveys and interviews with patients to study the application of integrated nursing information. The personnel participating in this experiment include the specific user roles in the system, medical patients, medical staff, and system administrators. In the first round of experiment, a questionnaire survey was conducted for medical staff, and the basic statistics of the people who received the questionnaire survey are shown in In the first step of this round of experiment, the 15 questionnaires prepared were distributed to 15 nurses from the clinical nursing department, and they were invited to fill them in truthfully to ensure the authenticity of the experiment. After the subject to be investigated was completed, the content of the questionnaire was evaluated to confirm whether it meets the feasibility standard of this experiment. The evaluation is recorded in In order to follow the principles of goal, science, objectivity, measurability, and simplicity, this questionnaire has a recovery rate of 100% and an effective rate of 100%. The experimental process and experimental results carried out have reference and certain research value.Information literacy is the foundation of evidence-based nursing and the key standard of nursing information ability. In addition, information literacy can improve the lifelong learning skills of nurses. The information ability of nurses requires nurses to have basic information technology skills and use these information technologies to carry out clinical practice and scientific research to achieve the best nursing practice. According to the search data, 73% of nurses in my country currently choose colleagues when they encounter clinical problems, 89% of nurses have never used the hospital library, 73% of nurses do not use survey reports, and 92% of nurses consider problems when they encounter problems. Some of them use the Internet and the World Wide Web, but more than 80% of this group have never used a database. Due to the knowledge gap in information literacy, the limitation of access to high-quality information resources, and the attitude of not paying attention to research, nursing staff are not fully prepared to provide qualified nursing work. To this end, we conducted statistics on the information literacy of these 15 personnel, and the data is recorded in The data shows that 15 nursing staff's ability to acquire nursing information is insufficient. Even though they are aware of the importance of nursing information, under the influence of existing conditions, the use of nursing information and the ability to evaluate it after use cannot be exercised. The level of nursing informatization in our country is still at a low level, and the level of information literacy of nursing staff needs to be improved. The integration of nursing information provides powerful conditions, a broad platform for improvement, and unlimited development space to promote nurses to understand the value of information literacy and improve their own information literacy.In the second round of experiments, in response to the common problems in current nephrology care, such as lack of timely drug supply, insufficient nurse-patient communication, patients not in the ward or uncooperative, or family members interrupted, interviews were conducted with these 15 interviewees. And to sort out and analyze the missing nursing information items in the department of nephrology, the data is sorted in During the interview, it was learned that nurses often ignore patients' physiological symptoms and psychological symptoms such as anxiety and depression. Nursing assessments such as pain assessment, nutritional assessment, psychosocial assessment, adverse reaction assessment, condition observation, and regular assessment need to be carried out regularly due to work. The amount of work is too large, and the work is too complicated to take care of; when arranging the work flow, life care will be placed at the end, and life care is prone to omissions after the treatment operation is completed; the psychological care and the nursing intervention involved in the nursing intervention during the nursing continuation work the end-of-life care is still not enough. With the integrated nursing information system, various detailed nursing work can be carried out in an orderly and stable manner. This conclusion can be demonstrated in Nursing staff can quickly and accurately record the patient's personal information data, which greatly improves the satisfaction of patients and nursing staff. Integrated information can refine and decompose nursing work, optimize each step from the smallest unit of quality problems, and establish a medical model that can be controlled in real time. This can greatly reduce the working time of nursing staff, rationally arrange the work process, and play a role of supervision. The implementation of the integrated information system platform can seamlessly connect medical treatment, dispensing, basic nursing, and other processes. Nurses and patients can share all the patient's medical treatment information so that the communication between doctors and nurses can be more accurate and timelier. The work process was optimized, the cooperation between medical staff was promoted, the health guidance was further implemented, the quality of nursing service was deepened, and the nurses' enthusiasm for work was aroused.As the recipient of the nursing work flow and the director of the nursing staff, the patient occupies an important position in the integrated nursing information system. Five inpatients with renal disease in the department of nephrology were selected using statistical methods to participate in this study on the integration of nursing information in the department of nephrology . We talked with these 5 patients. After the information integration system was adopted, they analyzed the nursing work satisfaction evaluation of the nurse staff and solved the problems of the patients' concerns in nursing work after information integration. According to data analysis, these five interviewees believe that the construction of an information integration system has had a great effect on the nursing work of nurse staff, and that the staff's work is in place; before the information integration management, there is, for example, the caller fails to receive a response from the nursing staff within 5 minutes, fails to monitor on time according to the doctor's advice, lacks health education, and their physical and psychological care needs are not met. These problems have been properly resolved through the integration of nursing information, which has improved patients' satisfaction with treatment.With the development of the social economy and the advancement of medical technology, the medical model centered on diseases in the past is far from being able to meet the medical needs of current patients. The medical model of \u201cbio-psychosocial\u201d centered on patients is more suitable for current medical care. Serving patients and effectively solving the problems encountered by patients in the process of hospital visits is the purpose of the development of an integrated nursing information system, and it is also its primary task. While the system can conveniently display relevant information about patients' visits and hospitalizations, it can also collect detailed patient clinical data information and provide medical staff with the right to retrieve and check patient-related information without being restricted by time and place, so that it can be used by the medical industry. Scientific research, health care, and medical levels provide effective diagnosis and treatment information. The current situation we are facing is a very complex situation with various heterogeneous data and different application systems. The lack of unified planning, unified interface standards, and a unified data storage platform will cause overlap of various functions and duplication of nursing business processes. The integrated information platform is based on the existing information system and established in accordance with the requirements of joint integrated operations. The knowledge and management methods of the medical system can be enhanced through the clinical information management system in the ward. Improving in business management is a feature of current research for the study of integrated nursing information management systems, which has important economic and social value."}
+{"text": "In order to improve the nursing effect of emergency general surgery, this paper combines computer algorithms to carry out the intelligent management of general surgery nursing, and realizes the standardization of nursing information, the electronic nursing file, the precision of nursing workload, and the intelligentization of nursing quality control by means of informatization. This truly and objectively reflects the nursing operation and treatment situation, prevents the occurrence of some adverse events, and effectively reduces the workload of nursing care. Moreover, this paper uses a standardized software design method to define the software concept, and then conducts a detailed demand analysis of the nursing display function through detailed investigation, class work, discussion and analysis, and comparison decision-making methods. In addition, this paper compiles the software through strict coding standards, and finally designs test cases to test and improve the software. Through actual case studies, it can be seen that the computer-assisted emergency general surgery nursing method proposed in this paper has a certain progress compared with the traditional nursing method. The pre-examination and triage of emergency general surgery is relatively mature abroad. A large number of studies have proved the effectiveness and practicability of their respective triage standards, and the corresponding emergency triage process and path have been established for clinical use. The establishment of modern triage standards began in the 1990s [These triage standards are all five-level triage, and patients are divided into five levels according to the degree of criticality, of which level I is the most critical and urgent patient, and level V is the mildest nonemergency patient. Regarding the evaluation of the American ESI standard, there are still some studies that indicate that the triage standard is not perfect. Although it is based on the allocation of emergency resources, it still has shortcomings . First oComputer information technology gradually began to be applied to hospital management in the form of a single computer. In the early 1990s, hospitals began to introduce computer health information systems (HIS). The creation and adoption of clinical decision support (CDS) system tools have changed the way medical professionals traditionally manage patient health, disease data, healthcare records, and diagnosis and treatment plans . PrelimiIn order to improve the nursing effect of emergency general surgery, this paper combines computer algorithms to carry out the intelligent management of general surgery nursing so as to improve the nursing effect of general surgery and provide a reference for the further improvement of the quality of follow-up general surgery nursing service.In the early 1970s, hospitals in the United States, Japan, and other hospitals began to try to apply large-scale commercial computers to hospital management, began to research and develop hospital information systems, and established HIS system prototypes. By the mid-1980s, nursing information systems began to be used in some large hospitals in developed countries. With the development and maturity of various new network information technologies, foreign nursing-related information systems have been relatively complete. With the strengthening of the degree of integration of hospital nursing information and hospital management systems, all aspects of hospital nursing can be effectively managed, and the safety of nursing behavior is protected to a certain extent . The earIn general, the nursing system is developing rapidly toward specialization, mobility, and diversification. The nursing system has evolved from a single, submodule subordinate to HIS to an integrated mobile nursing system, that includes traditional nurse workstations, nursing medical records, nursing management, and nursing care. Specialized clinical nursing information systems are being developed for planning and nursing tasks . ForeignWith the continuous improvement of the level of nursing specialization, it has also promoted the development of nursing specialties. Since the introduction of the modified early warning score (MEWS) system in the United Kingdom, the observation and early warning of critically ill patients in nursing work have played a very important role . The MEWThe hospital's intelligent nursing display system is a business system that applies and serves the internal use of the Arab League Central Hospital. At the same time, the hospital also uses the intelligent nursing display system as an important means and tool to promote the improvement of nursing management. From the perspective of the development of medical informatization, the construction of nursing informatization is gradually developing toward mobile, intelligent, and wearable models. The mobile nursing information system uses mobile devices such as tablet computers, PDAs, smart phones, and other portable mobile devices to collect and distribute data. It also includes the use of large-screen display devices for data display and push. This can effectively help medical staff to grasp patient diagnosis and treatment information in a dynamic, comprehensive, and timely manner, scientifically arrange and execute diagnosis and treatment and nursing plans so as to solve the query, and enter vital signs data and medical care data by medical staff anytime and anywhere. Moreover, it helps hospitals realize a patient-centered service concept and a quality-centered management model. The development of the hospital's intelligent nursing display system can enable nurses to perform medical behaviors in clinical nursing work, such as fluid infusion, physical sign collection, intake and output, treatment execution, daily care, etc., to follow the patient to complete the collection work at the bedside. Moreover, the application of mobile nursing information technology rationally optimizes the original work flow, thereby improving the efficiency and the quality of nursing work, meeting the management requirements of high-quality nursing, and handling various emergencies  in a timely and accurate manner.(1) Comprehensive nursing information integration: It connects various business application systems in the hospital through the hospital data integration platform. Moreover, it integrates multiple systems such as clinical nursing, nursing management, mobile nursing, intensive care, medical order system, inspection, cost accounting, and material management. In addition, it collects relevant data and incorporates it into the clinical data center to form a data warehouse for the main body of nursing information, which solves the problems of repeated data entry and data inconsistency in multiple systems, and displays it on demand after processing. (2) Automatic generation of nursing plan: It is a predefined nursing plan knowledge base, and intelligently formulates nursing plans based on doctors' orders, nursing evaluation results, special physical signs, and patient conditions, and displays the patient's nursing plan dynamically in real time with a graphical interface. (3) Intelligent reminder of emergencies: The system can remind various problems in nursing work in real time, and intelligently, such as drug allergy and drug contraindications, and promptly remind emergency events such as crisis value to avoid medical errors. (4) Multi-level information display: The system can use electronic whiteboards, handheld terminals, tablet computers, electronic large screens, and other intelligent terminal devices to display corresponding information to doctors, responsible nurses, head nurses, and nursing department managers as needed to meet the information needs of people at different levels.The hospital's intelligent nursing display system should combine the development needs of the hospital's clinical business, adhere to the demand-led approach, refer to the best practices of domestic and foreign industry informatization, and explore the informatization development model with low cost, good results, and outstanding highlights. It is necessary to gradually promote the hospital's information planning and construction in clinical diagnosis and treatment, medical quality control, operation management, decision analysis and other related aspects, and introduce scientific, professional, and refined management methods and means to drive the hospital's management innovation and service innovation. At the same time, it is necessary to realize the standardization of the management process, the networking of work communication, the electronicization of documents and materials, the sharing of knowledge and experience, and the openness of public welfare information so as to realize the new work pattern of \u201corderly management, free communication, and perfect service\u201d in the hospital. At present, nursing information management systems and mobile clinical information systems based on mobile nursing work, electronic nursing documents, and labeling of nursing items have been put into use in many large- and medium-sized hospitals in China. The hospital intelligent nursing display system is a part of the nursing system, and the overall goal of the system is to promote the improvement of the hospital's nursing quality capabilities. With the least participation of the nursing staff, it can provide the nursing staff with the greatest help, or assist the nursing staff to achieve the following functions: (1) It can realize the automatic check of drugs and patients to avoid medical errors; (2) it can greatly reduce the workload of nursing and reduce the work intensity of nurses; (3) it can improve the efficiency and quality of nursing, and improve the level of nursing service; (4) it standardizes the clinical medical care process and improves the hospital's clinical medical care management level; (5) it provides medical and nursing staff message reminders to provide faster nursing assistance for nursing work; (6) it realizes patient review and provides comparison of average admission data for nursing management; (7) it implements a high-quality nursing information technology support platform to ensure that medical staff can communicate with each other anytime and anywhere; (8) it establishes a fair assessment mechanism to increase the enthusiasm of nursing staff. In general, the construction of the hospital's intelligent nursing display system will bring brand-new changes to nursing work, optimize the nursing workflow, and provide patients with better nursing services.The conceptual model design is actually the realization process of the conceptual model. The conceptual model is an abstraction of the real world, i.e., it artificially processes actual people, objects, things, and concepts, extracts the necessary characteristics needed to build the system, ignores some nonessential details, and accurately describes these characteristics with various concepts.The above is a detailed description and research of each functional module in the community intelligent remote monitoring system. Based on the analysis and abstraction of system developers, end users, and customers, it is concluded that the community intelligent remote monitoring system should include six use cases including physiological parameter monitoring, alarm information processing, nursing, information query, information management, and data interface. With reference to the above-detailed analysis of each use case of the system, we gradually refine the data objects of this part of the system, design a partial conceptual model, and determine the relationship between them. The partial conceptual model of the system is shown in The partial conceptual model of the system is mainly composed of the client's basic information object, the client's relative information object, the doctor's information object, and the client's multi-physiological parameter object. The numbers on the relationship line indicate the corresponding relationship between the data objects. For example, there is a one-to-many relationship between basic customer information and multiple physiological parameter data, i.e., a customer object contains multiple physiological parameter information. Through the detailed design of the partial conceptual structure, the relationship between the data objects is truly reflected, and the foundation for the subsequent logical model design is laid.The system hardware framework is shown in The driver framework of 88W8686 is shown in \u2009 Risk management for patients: Medical staff need to strengthen the management of neurosurgery emergency, surgery, elderly and critical patients, and use bed guards for patients with inconvenient mobility to prevent patients from falling from beds and other injuries. Therefore, the floor should be kept dry, handrails should be added to the bathroom, related warning signs should be present in wet areas, and the wheels, brakes, and guardrails of the patient's bed should be checked regularly. The nurse's shift needs to be 15\u2009minutes in advance, and the content of the shift at the bedside must be effectively verified. For patients with more serious illnesses, the nursing staff need to strengthen communication with their families and provide relevant psychological counseling.\u2009 Risk management for nurses: It is necessary to strengthen professional training and risk management awareness of nurses so as to effectively improve the quality of nursing work. At the same time, it is also necessary to strengthen the communication skills of nurses to promote a more harmonious relationship between the doctors and the patients. Hospital units can regularly organize nurses to conduct neurosurgery technical training and theoretical learning so as to improve the proficiency of nurses in nursing operations. At the same time, it is also necessary to strengthen the nursing level, communication level, and drug use methods of the nursing staff to improve the quality of nursing work. In addition, it is necessary to organize nurses to conduct academic exchange meetings on a regular basis to realize the sharing of information resources. For nursing staff with low qualifications, guidance and supervision need to be strengthened.\u2009 Environmental risk management: The environment of the nephrology ward needs to be kept clean and comfortable so as to create a comfortable and quiet atmosphere for patients to recuperate. It is necessary to effectively implement the ward visiting system to prevent patients from being disturbed in their recuperation. At the same time, the environment needs to be disinfected to prevent infections in patients.\u2009 Risk management for the pipeline: Patients in the Department of Nephrology generally have more catheters indwelled, and the main purpose is to observe the patient's condition. Therefore, the catheter needs to be kept unobstructed, and a fixing device should be added to prevent the catheter from twisting and pulling. At the same time, it is also necessary to sterilize the pipelines to strengthen the operational hygiene awareness of the medical staff and prevent patients from becoming infected. In addition, the indwelling time of the catheter needs to be communicated with the physician to prevent infection caused by excessive indwelling time.\u2009 Risk management for emergency treatment: It is necessary to carry out effective and reasonable treatment of emergency plans for intravenous extravasation of special drugs, emergency plans for blockages, emergency plans for pressure sore prevention, emergency plans for cross-infection, and emergency plans for power outages and water cuts. At the same time, it is necessary to carry out relevant emergency drills to improve the emergency awareness of nurses, strengthen the awareness and understanding of emergency handling, improve proficiency, and effectively guarantee the quality of emergency work. On this basis, the effects of computer-aided care in emergency general surgery are calculated, and the results are shown in Tables 1We select the emergency general surgery inpatients admitted to the emergency department of this hospital from June 2019 to December 2020 as the research sample. According to the method of random allocation, they are divided into the experimental group and the control group. Among them, the control group used traditional general surgical nursing methods, and the experimental group used the intelligent computer-aided nursing system constructed in this paper. In addition, other intervention methods are the same. On this basis, we collect relevant data for comparative analysis.It can be seen from the above research that the computer-assisted emergency general surgery nursing method proposed in this paper has a certain progress compared with the traditional nursing method. Therefore, the system of this paper can be used as an auxiliary nursing system in the follow-up hospital emergency general surgery nursing to improve the nursing effect of emergency general surgery.Emergency Department of General Surgery is a very important department, which is highly specialized and involves many types of diseases. In addition, the condition of some patients with craniocerebral trauma changes rapidly and the onset is more rapid; thus, medical personnel are more required to have skilled emergency handling capabilities and operational skills. The professional nature and the importance of the departments also determine the trend of nurses' high work intensity and workload. Under this highly stressful working state, the occurrence of medical care risks is also increasing. Therefore, nurses need to strengthen the analysis and management of risk factors in nursing work so as to prevent risk events. At the same time, hospital units also need to combine the items complained by patients and medical disputes, strengthen in-depth analysis and investigation of these events, understand and recognize the information content related to risk events, and evaluate the consequences of them so as to summarize the factors associated with risk events. In order to improve the nursing effect of emergency general surgery, this paper combines computer algorithms to carry out the intelligent management of general surgery nursing so as to improve the nursing effect of general surgery. Through actual case studies, it can be seen that the computer-assisted emergency general surgery nursing method proposed in this paper has a certain progress compared with the traditional nursing method."}
+{"text": "This study aims to design a set of the visual artificial intelligence system based on medical information mining for hospital emergency care management. A visual artificial intelligence emergency first aid nursing management system is designed by analyzing the needs of the emergency first aid nursing management system. The results show that system personnel allocation, comparative management, record management, query management analysis, basic setup analysis, nursing management basis, and nonfunctional requirements all need to be optimized for the emergency first aid management system. In this study, the comparative management module, log management module, and the query management module are designed, and the emergency first aid management system of different APP terminal functions in different modules is described in detail. The nursing document query business is tested, and the corresponding time of query of nursing assessment sheet, nurse shift record, nurse record, and physical sign observation sheet is 375.50\u2009ms, 351.48\u2009ms, 336.36\u2009ms, and 245.57\u2009ms, respectively. It shows that the visual artificial intelligence emergency nursing management system based on medical information mining can provide convenience for clinical work to a large extent and has potential application value in hospital emergency nursing work. Medical information (MI) is a combination of medicine and computer science that aimed to facilitate the acquisition, storage, retrieval, and utilization of health and biomedical information . As an eThe medical treatment of the emergency department is the key of the key in the hospital. The emergency department mainly serves to patients with acute and severe diseases, and the diagnosis and treatment level of this department is directly related to the quality of life of patients in the later period and the degree of satisfaction of patients with the hospital. However, the large flow of emergency patients and the high management cost and the difficulty of emergency care require the rational allocation of resources of the emergency department. Buscema M used automatic compressed mapping (Auto-CM) to find out the subtle associations between various variables . This meThen, a visual artificial intelligence nursing system is developed. It has functions of comparative management, query management, record management, nursing document management, basic settings, and basic nursing management, which are mainly realized through an APP end , 13. TheAbove all, this study focuses on the design of the medical information visualization emergency first aid system. With first aid measures in hospital emergency departments as the breakthrough point, the work procedures of the nurse, head nurse, and nurse directors are analyzed. According to specific requirements, the APP end of the emergency first aid management system is designed, and the submodules of the visual artificial intelligence emergency first aid management system are described in detail. Finally, the system is tested. The study provides a more practical management system for hospital emergency nursing and convenience for clinical work and improves the work efficiency of medical workers.P(X) represents the probability that transaction X appears in transaction set D.The probability-based interest model algorithm is as follows:P(Y) represents the probability that transaction Y appears in food set D.P(Y/X) represents the probability of simultaneous occurrence of transactions X and Y in transaction set D.X\u27f6Y)), Supp(Y) is an evaluation standard, which is used to ensure that Int(X\u27f6Y)<1 is established.The interest model based on differences is as follows.The interest model based on correlation is as follows.X\u27f6Y) illustrates the relationship between the preceding and subsequent items X and Y in the association rule (X\u27f6Y).The degree of interest based on relevance Int(The information-based interest model is as follows.X\u27f6Y), the amount of information, and the similarity of the probability distributions of the preceding item X and the subsequent item Y, and the probability of the preceding item is used as a measure of the conciseness of the preceding item of the association rule.The interest degree model based on the amount of information comprehensively considers the conciseness of the association rule (The influence-based interest model is as follows.Through the comparison and analysis of various interest degree models, as well as the mining of medical information, the improved algorithm is as follows.There are many problems in the hospital emergency nursing system. Nurses are not efficient in the processes of infusion and dispensing, and it is difficult for nurses to quickly query the patient's basic information, doctor's orders, and other medical information in their daily work. There lacks an effective way to manage nursing document information, and basic nursing management also has various shortcomings. Therefore, a visual nursing system for the emergency department based on artificial intelligence is developed in the form of an APP. The system requirements are shown in In the APP end of the medical information-based visual artificial intelligence emergency nursing system, many new services have been added to meet the various needs of nurses, such as management of prescribing, infusions, and doctor orders; in record management module, nurses can view and deal with physical signs and documents; the query management module includes patient information, medical order information, and treatment of patients; and statistical performance, monitoring of nursing links, and application settings are included in the basic settings module. The business flowchart of the visual artificial intelligence emergency nursing management system based on medical information is shown in The visual artificial intelligence emergency nursing management system includes nurses, head nurses, and nursing department. The system APP enables the operator to carry out a series of operations, such as comparison, query, and record management. The roles involved in the of emergency nursing management system are shown in In the functional section of the APP, nurses in the emergency department can enter the comparison management interface through the APP to process fluid dispensing and infusion-related services to achieve rapid emergency nursing. The comparison management module is shown in The record management module reflects a series of nursing-related requirements, and it can collect nursing signs, record nursing sign information, generate nursing documents, process document information, and manage nursing measures. The record management module is shown in The query business concerns patient information, doctor's order information, and diagnosis and treatment expenses. The basic information above can be obtained through query module. The query management module is shown in The basic setting module reflects the relevant needs of statistical nursing work, monitors nursing links, and obtains notifications. In addition, users can also set according to their own habits, such as signature keyboard, automatic data reference, and writing input. The basic setting analysis module is shown in Basic nursing management involves basic management businesses, including nursing document management, human resource management, nursing quality management, adverse event management, and nursing statistical analysis. The basic nursing management module is shown in The visual artificial intelligence emergency nursing management system based on medical information mining puts forward higher requirements for adaptability and safety. Adaptability: the mobile service components of the system should have large memory storage and can adapt to the complex and changeable use environment. Safety: due to irresistible factors such as slow network communication speed, mobile users will encounter poor communication or slow speed when logging in; in order to ensure the security of user logging in, the system should identify users who are offline multiple times in per unit time. Ease of use: the hospital has a large number of object-oriented nursing tasks of complex types. Therefore, the system should be able to provide a personalized setting system, so that nurses can flexibly set up according to their own operating habits.Comparison management window and comparison management business are the basic categories of comparison management modules. The business category includes liquid dispensing management business, infusion management business, specimen collection business, medical order execution business, and liquid preparation entity, infusion management entity, specimen collection entity, and medical order execution entity. The comparison management design is given in The nurse sends an application for processing the preparation business. The system accepts the processing application, sequentially calls the comparison management window and business categories, and returns to the liquid preparation business. The processing of the liquid to be dispensed is shown in The functional category of the record management module includes record management window, record management business, nursing physical sign management business, nursing document management business, patrol management business, and nursing measure management business. The record management design is given in The query management module includes query management window, query management business, patient information management business, medical order management business, diagnosis and treatment expense management business, and special patient management business. The entity category includes patient information entity type, diagnosis and treatment expense entity type, inspection and examination entity type, special patient entity type, and doctor order entity type. The query management design is given in The basic setting module includes basic setting window, basic setting business, performance statistics business, nursing link monitoring business, information push business, application setting business, nursing link entity, information entity, and performance statistics entity. The design of basic setting is given in The monitoring business in the nursing link is shown in System test environment: Windows Server 2012 operating system, 1\u2009TB hard disk, 4G processing memory; the Web server runs Windows 7 and has 500\u2009GG hard disks and 2G processing memory; the browser kernel is IE and Safari; the APP terminal is the Android 5.0 operating system and can connect to the WiFi network. On the basis of formulating test cases, the test process is standardized, striving for the accuracy of system test results. In the study, the requirements on the visual artificial intelligence emergency nursing system are analyzed. Then, the role of the system is analyzed, each functional module is established, and the functional requirements are analyzed. As for the system design, the overall functional architecture of the system is designed.n\u2009+\u20091 item through n items. This method is mainly divided into two steps, which are called connection steps and pruning steps [The development of the APP adopts the MVC design pattern, Spring MVC framework, and other technologies. The MVC design pattern stipulates the responsibilities of the components and reduces the coupling between the components . The useng steps , 23.In the study, the steps of emergency nursing and the needs in the process of emergency nursing are analyzed. It is found that there is a lack of effective means for nurses to compare the information of infusion and liquid dispensing. It is difficult for nurses in daily work to quickly query the patient basic information, such as orders, and the head nurse and nursing managers on lack of systematic methods of management for nursing documents information. Based on these problems, the visualization APP emergency care management system is designed and tested. The results show that the visual artificial intelligence first aid nursing management system APP designed in this study has certain operability, but there are still some limitations in this study. In this study, only APP window is designed, without Web window application. In subsequent studies, Web window design should be added to meet the needs of more functions. In conclusion, this study promotes the application of the artificial intelligence-based visual nursing system for hospital emergency care in clinical work, which provides convenience for clinical work and improves the work efficiency of medical workers."}
+{"text": "Dichlorodiphenyldichloroethylene (DDE), the primary persistent metabolite of dichlorodiphenyltrichloroethane (DDT), has toxic effects on cells, but its dose-dependent impact on mitochondrial proteins involved in mitochondrial fusion and fission processes associated with cell viability impairment has not yet been analysed. Mitochondrial fusion and fission processes are critical to maintaining the mitochondrial network and allowing the cell to respond to external stressors such as environmental pollutants. Fusion processes are associated with optimizing mitochondrial function, whereas fission processes are associated with removing damaged mitochondria. We assessed the effects of different DDE doses, ranging between 0.5 and 100 \u00b5M, on cell viability and mitochondrial fusion/fission proteins in an in vitro hepatic cell model ; the DDE induced a decrease in cell viability in a dose-dependent manner, and its effect was enhanced in conditions of coincubation with dietary fatty acids. Fusion protein markers exhibited an inverted U-shape dose-response curve, showing the highest content in the 2.5\u201325 \u03bcM DDE dose range. The fission protein marker was found to increase significantly, leading to an increased fission/fusion ratio with high DDE doses. The low DDE doses elicited cell adaption by stimulating mitochondrial dynamics machinery, whereas high DDE doses induced cell viability loss associated with mitochondrial dynamics to shift toward fission. Present results are helpful to clarify the mechanisms underlying the cell fate towards survival or death in response to increasing doses of environmental pollutants. Mitochondria are vital organelles involved in ATP production and in regulating apoptotic and necrotic cell death ,2,3. SuiMitochondria can use several mechanisms to maintain their physiological homeostasis ,3,45. MiThe main aim of the present study was to analyze the dose-dependent response to DDE exposition in terms of cell viability, mitochondrial protein fusion, and fission content in the human hepatocarcinoma cell line (HepG2). Scientists consider this cellular model suitable for in vitro studies on hepatic toxicology ,60. Firsv/v) fetal bovine serum, 1% (v/v) non-essential amino acids, 0.2 mM L-glutamine, 50 U/mL penicillin and 50 \u00b5g/mL streptomycin . Cells were maintained at 37 \u00b0C in a 5% CO2, 95% air-humidified atmosphere and passaged twice a week. To perform treatments with DDE, cells were seeded at the density of 4.0 \u00d7 104/cm2 and cultured for 24 h before stimulation. A stock solution of DDE was prepared by dissolving the pesticide in dimethyl-sulfoxide (DMSO). In each treatment, the final concentration of DMSO in the culture medium was 0.1%, which represents a non-toxic concentration for hepatocytes [A HepG2 cell line was cultured in Minimum Essential Medium supplemented with 10%  was only treated with DMSO (0.1%). In the coincubation experiments with fatty acids, cells were co-treated for 24 h with the different doses of DDE and 250 \u00b5M of palmitate, oleate or eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) 1:1 mixture.v/v, respectively. The resulting 8 mM fatty acid stock solutions were filtered using a syringe equipped to filter (0.2 \u03bcm), aliquoted and stored at \u221220 \u00b0C. Concerning EPA and DHA , 40 mM stock solutions were prepared by dissolving fatty acids in methanol as indicated by the manufacturer and mixed 1:1 v/v to obtain a stock solution EPA + DHA. Then, fatty acids were co-incubated with BSA in a culture medium. The final fatty acids concentration used in the study (250 \u00b5M) was freshly prepared in a cell culture medium at the time of the treatments.Oleate and Palmitate (Merck) 40 mM stock solutions were prepared by dissolving fatty acids in NaOH 0.1 M at 70 \u00b0C for 15 min under stirring. A 10% BSA fatty acid-free  solution was dissolved at 55\u00b0 in NaCl 0.9% and mixed to fatty acid solutions 4:1 The cytotoxicity was assessed by a 3--2,5-diphenylte-trazolium (MTT) assay. This technique is based on the enzymatic conversion of MTT in mitochondria . To asseWestern blot analysis was performed as previously described . The folOil red stain quantitative (spectrophotometric) and qualitative (microscopic) analyses were performed using a classic protocol to detect lipid droplets in cells . Brieflyn = 3) or interquartile of range (n > 3). Each experiment was performed in biological triplicate (n = 3) or more (n > 3). Technical replicates (1 to 3 replicates) of the same biological samples were also performed. One-way ANOVA test followed by Bonferroni\u2019s post hoc test was used to test significant differences . Differences were considered statistically significant with a p-value < 0.05.All data were expressed as median with range , were observed with high DDE doses  A. Cell vQualitative and quantitative tests were performed using the oil-Red O method to detect total intracellular lipid accumulation in cells treated with DDE alone or associated with fatty acids. In a recent work, we analysed the time- and dose-dependent effect of palmitate or oleate on lipid accumulation and cell viability in HepG2 cells . We showTo evaluate the dose-dependent effect of DDE on mitochondrial dynamic machinery, western blot analysis was performed to measure cellular contents of proteins involved in mitochondrial fusion and fission processes, namely Mfn2 and Opa1, as a marker of outer and inner mitochondrial membrane, respectively, and DRP1 as a fission marker. As for fission marker DRP1, the relative protein content described a progressive-increase pattern in response to DDE concentrations up to the dose of 50 \u03bcM, with a significant increase (+45%) in the 5\u201350 \u03bcM range compared to control cells. On the other hand, the increase in DRP1 content was about +18% in cells treated with 100 \u03bcM of DDE . TherefoTo study the effects of DDE on the mitochondrial antioxidant system, we analyzed the levels of mitochondrial enzyme SOD2 on total cell lysates. Results showed a progressive dose-dependent reduction in SOD2 protein content with a significant decrease (\u221225%) with the highest DDE dose (100 \u00b5M), compared to DMSO treated cells . AnalyseAdverse effects of DDE, including cellular stress generation ,72, apopOur study confirmed that DDE reduced cell viability in HepG2 cells in line with literature data in other cellular models . Low DDEIt should also be noted that, in the presence of fatty acids, xenobiotics toxicity could be associated with lipo-toxicity with an additive adverse effect on cell survival . Lipo-toNoteworthy, the coincubation of cells with fatty acids and increasing doses of DDE induced a progressive decrease in lipid accumulation compared to cells treated only with fatty acids. The reduction degree was similar for all fatty acids. A 20% decrease with low DDE doses and a 50% decrease with the highest DDE dose could be observed. Further experiments are needed to evaluate whether lower doses of fatty acids could induce a similar impact. However, present finding is in line with our previous results in an animal model of hepatic steatosis, where we showed that simultaneous treatment with DDE and high-fat diet elicited a significant reduction in hepatic lipid content associated with increased beta-oxidation rate and detoxification, compared to high-fat diet treatment in the absence of DDE . It coulPresent findings in cells cotreated with increasing DDE doses and different types of dietary fatty acids suggested that it could be of interest to further investigate the interaction between DDE, as a lipophilic pollutant, and dietary fatty acids, given that they could be the main route of entry of DDE into the body. Further studies could shed light on the complex interactions among environmental pollutants and nutrients and suggest whether different dietary nutrients (such as saturated or unsaturated fatty acids) could potentiate the adverse effects of environmental pollutants or could be protective towards them.A further goal of the present work was to analyze the dose-dependent response of mitochondrial markers to increasing doses of DDE in HepG2 cells. Mitochondria are cellular targets susceptible to chemicals, such as DDE . Given tIn the present study, we also analysed SOD2 content as a marker of mitochondrial function and antioxidant system and GRP75 as a marker of mitochondria/endoplasmic reticulum interaction.SOD2 represents one of the first mitochondrial-associated antioxidant defenses used by cells to quench superoxide anion produced by mitochondria . It has 2O2 and CCl4-induced oxidative damages, ameliorating ATP production and preserving cell viability. In our study, GRP75 protein levels showed a similar inverted U-shape dose-response curve as observed for Mfn2 and Opa1 levels, with the highest significant increase (about +30%) with 5 and 10 \u00b5M DDE vs DMSO-treated cells. This result suggested that an additional adaptive phenomenon involving ER-mitochondrial contact point formation/communication could be associated with mitochondrial increases in dynamic protein in response to low DDE doses. This mechanism could be used by cells to regulate cell survival and mitochondrial function in the presence of low DDE doses. On the one hand, the increase in mitochondrial dynamic machinery and GRP75 chaperone in response to low DDE doses could play a role in the mitochondrial network and mitochondria/endoplasmic reticulum interaction maintenance to maintain cell viability. On the other hand, in cells treated with high DDE doses, decreased levels in mitochondrial fusion proteins, GRP75, and SOD2 content associated with the increase in DRP1 levels suggested mitochondrial fragmentation and dysfunction in response to high DDE doses, which could trigger the observed reduction in cell viability.We also analysed the content of intracellular chaperone GRP75, a protein involved in mitochondria/endoplasmic reticulum interaction . This chThe present study added new scientific notions regarding adaptive/toxic phenomena induced by increasing dose of DDE in hepatic cell culture. Cellular adaptation to low doses of the environmental pollutant seemed to include maintaining mitochondrial network and mitochondria-endoplasmic reticulum interaction, which could play a pro-survival role. On the other hand, high doses of DDE induced a toxic effect that reduced cell viability by impairing mitochondrial fusion/fission balance and antioxidant defense. Present results are useful to clarify the mechanisms underlying the cell fate towards survival or death in response to increasing doses of environmental pollutants. Further studies are needed to shed further light on the metabolic events associated with our findings and highlight the different roles of cellular organelles and their adaptation to environmental pollutants."}
+{"text": "Lockdown measures due to the COVID-19 pandemic have led to lifestyle changes, which in turn may have an impact on the course of headache disorders. We aimed to assess changes in primary headache characteristics and lifestyle factors during the COVID-19 lockdown in Germany using digital documentation in the mobile application (app) M-sense.We analyzed data of smartphone users, who entered daily data in the app in the 28-day period before lockdown (baseline) and in the first 28\u2009days of lockdown (observation period). This analysis included the change of monthly headache days (MHD) in the observation period compared to baseline. We also assessed changes in monthly migraine days (MMD), the use of acute medication, and pain intensity. In addition, we looked into the changes in sleep duration, sleep quality, energy level, mood, stress, and activity level. Outcomes were compared using paired t-tests.p\u00a0>\u20090.999). MMD, headache and migraine intensity neither showed any significant changes. Days with acute medication use were reduced from 4.50\u2009\u00b1\u20093.88 in the baseline to 4.27\u2009\u00b1\u20093.81 in the observation period (p\u00a0<\u20090.001). The app users reported reduced stress levels, longer sleep duration, reduced activity levels, along with a better mood, and an improved energy level during the first\u00a0lockdown\u00a0month (p\u00a0\u2264\u20090.001).The analysis included data from 2325 app users. They reported 7.01\u2009\u00b1\u2009SD 5.64 MHD during baseline and 6.89\u2009\u00b1\u20095.47 MHD during lockdown without significant changes (p\u00a0=\u20090.688 in the ANOVA). Migraine frequency, headache and migraine intensity, and acute medication use were also not different during the entire observation period.In an extension analysis of users who continued to use M-sense every day for 3 months after initiation of lockdown, we compared the baseline and the subsequent months using repeated-measures ANOVA. In these 539 users, headache frequency did not change significantly neither (6.11\u2009\u00b1\u20095.10 MHD before lockdown vs. 6.07\u2009\u00b1\u20095.17 MHD in the third lockdown month, Despite slight changes in factors that contribute to the generation of headache, COVID-19-related lockdown measures did not seem to be associated with primary headache frequency and intensity over the course of 3 months. Patients with primary headache disorders perceive external factors such as stress, sleep, and changes of everyday routine as common trigger of their headache attacks . EspeciaOn March 11, 2020, the World Health Organization (WHO) declared COVID-19 a global pandemic, and on March 22, the German government imposed strict social distancing measures on public life . The resStudies on mental health during the COVID-19 pandemic suggest a high psychosocial impact of such measures with a negative effect on quality of life, beginning in the first lockdown weeks , 7. PatiA digital headache diary is a reliable tool to track headache attacks and potential triggers. Compared to paper-and-pencil diaries, digital documentation is associated with a higher compliance and better data quality . We aimeM-sense is a commercial mobile headache application (app) available via app store for Android and iOS in Germany, Austria, and Switzerland. At the time of the data analysis, M-sense was marketed as a migraine app in a free-of-charge \u201cBasic\u201d version with approximately 85,000 registered users and an additional \u201cActive\u201d version for purchase.Functions of M-sense comprise the documentation of headache attacks in an interactive electronic diary, along with a daily entry of predefined factors, which may influence the course of headaches. Headache features are entered according to a standardized scheme, which includes start and end of the headache, pain intensity, headache localization, headache character, presence of aura, accompanying symptoms, and acute medication use. A validated algorithm classifies single headache episodes as migraine, tension-type headache (TTH) or non-migraine / non-TTH headache . A detaiThe app developers provided us with aggregated data from all M-sense users, who entered headache-related data in this app in the four-week periods before March 22 (baseline) and after March 22 (first month of lockdown) every day. Primary outcome of this retrospective analysis was the change in monthly headache days (MHD) between baseline and the first lockdown month. Our analysis also included changes in monthly migraine days (MMD), monthly days with use of acute headache medication (AMD), mean headache and migraine intensity . A headache day was defined as any calendar day with a registered headache attack, regardless of the individual headache features. A migraine day was defined as each calendar day at which the user experienced a headache attack classified as migraine by M-sense. Acute headache medication consisted of triptans and non-steroidal anti-inflammatory drug (NSAID). We also assessed changes of several predefined factors including sleep duration , self-assessed sleep quality (on a NRS 0\u201310), energy level (0\u201310), mood (0\u20134), stress (0\u201310), and level of activity (0\u201310).We then performed an extension analysis of M-sense users who continued to use the app for at least 3 months after lockdown begin. In these users, we analyzed changes in the above mentioned parameters between the baseline phase, the first, second and third lockdown month.p-value \u22640.05 was considered statistically significant. P-values were adjusted for multiple comparisons using the Bonferroni procedure. We calculated effect sizes using Cohen\u2019s d.Statistical analysis was performed using R, version 3.6.2. For data protection, we developed the statistical code on a dummy dataset. The team of Newsenselab ran the finalized code on the real dataset. By doing so, the research team did not access personal data but received only aggregated results for the predefined endpoints. Demographics and monthly headache characteristics were summarized with descriptive statistics, using frequencies and percentages or means \u00b1 standard deviation. We compared outcomes between baseline and first month of lockdown using paired, two-tailed t-tests. In the extension analysis, outcomes were compared between all months using repeated-measurement analysis of variance (ANOVA). Post hoc pairwise comparisons were performed only if the ANOVA revealed significant results. A n\u00a0=\u20092325 users (mean age 38.69\u2009\u00b1\u2009SD 11.09\u2009years) entered data in M-sense every day. The sample consisted of n\u00a0=\u20091699 females (73.1%), n\u00a0=\u2009271 males (11.6%), and n\u00a0=\u20092 diverse sex (0.1%), while n\u00a0=\u2009353 (15.2%) did not provide information about sex.During the primary observation period, p\u00a0=\u20090.999). MMD, headache and migraine intensity did also not change significantly over time .Headache frequency did not show any statistical difference, with 7.01\u2009\u00b1\u20095.64 MHD during baseline and 6.89\u2009\u00b1\u20095.47 MHD in the first lockdown month . MMD, AMD, migraine or pain intensity did also not show any significant change. Stress levels remained numerically below baseline in every month, but without statistical significance. Table\u00a0They reported 6.11\u2009\u00b1\u20095.10 MHD during baseline and 6.07\u2009\u00b1\u20095.17 MHD in the third lockdown month .Previous research on sleep patterns during the COVID-19 pandemic is scarce: In an Indian cohort, 325 students reported an increased sleep duration during lockdown, while 203 office workers indicated reduced sleep hours . Our finA major concern for patients with primary headache disorders in the COVID-19 pandemic may be the worsening of their condition due to reduced access to medical care . HoweverThe reduction of physical activity due to home confinement in the pandemic has already been reported in large survey-based cohort studies , 19. We This study is an example on how headache data collected by an app can be used for research purposes in specific situations like the COVID-19 pandemic. Digital data collection offers the possibility of a large sample size. Nevertheless, several methodological limitations should be considered when interpreting our results. The M-sense algorithm classifies the single headache attacks, but cannot provide a final headache diagnosis. Due to the analysis of aggregated anonymous data patients could not be assessed for individually diagnosis. Previous assessments have shown that more than 70% of users have headaches compatible with the diagnosis of migraine . TherefoIn conclusion, we did not observe a change in headache frequency and intensity in patients suffering from migraine and/or TTH during the first 3 months of the lockdown in Germany. A small stress reduction in the first lockdown month was not associated with an improvement in these primary headache disorders."}
+{"text": "FH) cells. Postpartum memory B cells of MS patients expressed higher levels of CCR6 and preferentially developed into plasma cells under\u00a0TFH-like\u00a0in vitro\u00a0conditions. These findings imply that memory B- cell differentiation contributes to postpartum relapse risk in MS.In MS, pathogenic memory B cells infiltrate the brain and develop into antibody-secreting cells. Chemokine receptors not only define their brain-infiltrating capacity, but also assist in their maturation in germinal centers. How this corresponds to pregnancy, as a naturally occurring modifier of MS, is underexplored. Here, we aimed to study the impact of pregnancy on both ex vivo and in vitro B-cell differentiation in MS. The composition and outgrowth of peripheral B cells were compared between 19 MS pregnant patients and 12 healthy controls during the third trimester of pregnancy (low relapse risk) and postpartum (high relapse risk). Transitional, and not naive mature, B-cell frequencies\u00a0were found to drop in the third trimester, which was most prominent in patients\u00a0who experienced a pre-pregnancy relapse.\u00a0Early after delivery, these frequencies raised again, while memory B -cell frequencies modestly declined. CXCR4 was downregulated and CXCR5, CXCR3 and CCR6 were upregulated on postpartum memory B cells, implying enhanced recruitment into germinal center light zones for interaction with T follicular helper (T In the relapsing phase of MS, naive B cells escape peripheral tolerance checkpoints2 and develop into memory populations that activate CNS-infiltrating, IFN-\u03b3-producing CD4+ T cells3. This is further supported by the reduction in T-cell activation in MS patients after receiving anti-CD20 therapy4. Normally, memory B cells interact with T cells within germinal centers (GCs) of peripheral secondary lymphoid organs. Our group recently demonstrated that B cells utilize IFN-\u03b3 to drive their CXCR3-expression, boosting their ability to recruit to MS brain tissue as well5. Hence, in MS, CNS-infiltrating memory B cells are probably reactivated by T cells in perivascular spaces6 and ectopic lymphoid structures in the cerebral meninges7 to further mature into plasmablasts/plasma cells and contribute to focal inflammation6.MS is a chronic inflammatory and demyelinating disease of the CNS, for which a key role for B cells in the pathogenesis has been shown in recent years9. This is further controlled by the expression of other chemokine receptors, such as CXCR4, CXCR5 and CCR6. CXCR4 and CXCR5 orchestrate the GC response by guiding B cells into dark and light zones respectively. Centroblasts undergo several rounds of proliferation and somatic hypermutation in the dark zone, while centrocytes interact with follicular dendritic cells and T follicular helper (TFH) cells to undergo antigen-specific selection in the light zone10. CCR6 has been reported to promote their development and antigen responsiveness in the light zone12. Therefore, specific aberrancies in chemokine receptor profiles may affect the outgrowth of B cells into memory and plasmablasts/plasma cells13. Currently, it remains to be determined whether this is related to relapse risk in MS.IFN-\u03b3-induced CXCR3 expression on B cells is not only associated with increased homing to inflamed tissue and the formation of ectopic lymphoid structures, but also with aberrant plasma cell differentiation within GCs14. Although current diagnostic and treatment strategies probably contribute to an attenuation of this fluctuation15, these specific phases of pregnancy can still be considered periods of relatively low and high relapse risk in MS. Pregnancy is known to cause a shift from TH1 to TH2 responses16 and promote the expansion of circulating TFH cells17, therefore likely affecting the maturation of B cells in GCs.Notably, in pregnant MS patients, relapse risk is reduced by approximately 70% in the third trimester. This increases in the first three months after delivery, with almost 30% of patients having a postpartum relapseFH-like cell culture system to investigate how in vitro memory B-cell differentiation into plasmablasts or plasma cells differs between periods of relatively low (third trimester) and high (early postpartum) relapse risk.In this experimental study, our primary aim was to determine the effects of pregnancy as naturally occurring disease modifier on peripheral B-cell differentiation in MS. We assessed the frequencies and GC-related chemokine receptor profiles on ex vivo B-cell subsets in paired first trimester, third trimester and early postpartum blood samples of MS patients and healthy controls. In addition to this, we used a T18 and retrospectively validated to match the most recent McDonald 2017 criteria19. Patients did not use any immune modulatory medication before, during and early after pregnancy. Two patients gave birth via a cesarean section and all others had vaginal deliveries. No relapses were observed during pregnancy. During the early postpartum period, 6 patients developed a clinically defined relapse20. These patients did not differ from non-relapsing patients in their third trimester expanded disability status scale (EDSS) scores. No MRI evaluations for disease activity were performed. Additionally, 12 age-matched healthy pregnant women were included, who did not have central nervous system or inflammatory disease and were seen at the outpatient obstetric clinic at Erasmus MC. None of the healthy pregnant women used immunomodulatory medication before or during the study, were hypertensive, experienced recurrent abortions or were diagnosed with diabetes mellitus. Clinical characteristics of patients and controls are depicted in Table Nineteen pregnant women with RRMS were included at the MS Center ErasMS as part of our previous study\u00ae tubes containing sodium heparin according to manufacturer\u2019s instructions . After isolation, cells were taken up in RPMI 1640  containing 20% fetal calf serum (Lonza) and 10% dimethyl sulfoxide  and stored in liquid nitrogen until further use. Cells were pre-incubated with Fixable Viability Stain 700 (BD Biosciences) for 15\u00a0min at 4\u00a0\u00b0C. The following monoclonal antibodies were used stained for 30\u00a0min at 4\u00a0\u00b0C: CD27 , CD138 , CXCR4 , CXCR5 , IgD , IgG , CCR6 , CD19 , CD38 , CXCR3 , CXCR4  and IgM . Stained cells were measured using an LSRII\u2010Fortessa flow cytometer. For in vitro culture experiments, memory (CD19+CD3-CD27+) B cells were purified using a FACSAria III sorter. Data were analyzed using FACS Diva software, version 8.0.1 . Since ex vivo B-cell phenotyping of the relapsing MS group was performed in a separate study, it was not possible to compare the MFI of functional markers with the non-relapsing MS or control groups.PBMCs from patients and controls were collected in the first and third trimester of pregnancy as well as 4\u20138\u00a0weeks after delivery (postpartum). For sample collection, we used Vacutainer CPT21. In short, irradiated murine 3T3 fibroblasts expressing human CD40L were co-cultured with sorted memory B cells in the presence of IL-21 . After 6\u00a0days of culturing, viable CD19+ cells were analyzed using flow cytometry and supernatants were collected and stored at -80\u00a0\u00b0C until further use.In vitro B-cell differentiation assays were performed as recently describedIgM and IgG levels were determined in supernatants of memory B cells cultured for 6\u00a0days using ELISA. After overnight coating with goat anti-human Ig  at 4\u00a0\u00b0C, flat-bottom 96-well plates  were washed with PBS/0.05% Tween-20 and subsequently blocked with PBS/5%FCS for 2\u00a0h at RT. Samples were added for 1.5\u00a0h at room temperature. After washing, peroxidase-conjugated goat anti-human IgG (Thermo Fisher Scientific) or rabbit anti-human IgM  were used to detect bound antibody. 3,3\u2032,5,5\u2032-Tetramethylbenzidine substrate (Thermo Fisher Scientific) was used to reveal peroxidase activity. Reactions were stopped with sulfuric acid and optical densities were measured at 450\u00a0nm using a BioTek Synergy 2 reader . Concentrations were calculated using standard curves for IgM and IgG.P values\u2009<\u20090.05 were considered statistically significant.Graphpad Prism software (version 8) was used for statistical analyses. Both percentages and mean fluorescent intensity (MFI) are shown as individual data points together with the corresponding mean. We compared paired data using Wilcoxon signed-rank tests and data between clinical groups using 2-way analysis of variance (ANOVA) with Bonferroni\u2019s multiple comparison tests, unless stated otherwise. 18 to determine their relation to MS. Finally, we verified whether relapses are associated with alterations in B-cell profile and compared B-cell development into antibody-secreting cells in vitro for samples from low and high relapse risk periods .Our overall aim was to explore how pregnancy as naturally occurring disease modifier influences the peripheral B-cell lineage in MS. To properly address the impact of pregnancy without relapses as a potential confounder, we first analyzed ex vivo B-cell differentiation profiles in paired first trimester T1), third trimester (T3) and early postpartum (PP) blood samples from 13 MS patients without a postpartum relapse. These profiles were compared with blood samples from the same periods of 12 healthy controls , naive mature (CD38-/dimCD27-IgM+) as well as IgM+ and IgG+ memory (CD38-/dimCD27+) B cells using flow cytometry  implies an increased potential of these cells to recruit and further develop in the GC light zone.Together with the increased Ig surface levels Fig.\u00a0E, the diFH cells within the GC light zone. After reaching the CNS, memory B cells are likely reactivated to develop into potent antibody-secreting cells in MS patients. To assess whether B cells are prone for such recall responses in the high-risk postpartum period in MS, memory (CD27+) B cells were purified from paired third trimester and postpartum samples of 11 MS patients and compared for their outgrowth into plasmablasts/plasma cells in vitro. Under IL-21- and CD40L-inducing conditions, which mimic a TFH cell response, more antibody-secreting cells (CD38highCD27high) were formed in cultures with postpartum versus third trimester memory B cells  frequencies, these were mainly increased for postpartum memory B cells from MS patients, which were significantly higher than those from healthy controls  were more induced in both third trimester and postpartum memory B-cell cultures  found for ex vivo postpartum B cells implied a marked propensity to recruit to the TFH cell-containing GC light zone11. The elevated expression of CCR6 on ex vivo postpartum memory B cells of MS compared to healthy females pointed toward increased, T cell-dependent recall responsiveness23. This was supported by their ability to differentiate into plasma cells under TFH-like conditions in vitro.Pregnancy causes a transient period of immune suppression, which is lost early after delivery in MS. Here, we studied the functional impact of pregnancy as a disease modifier on peripheral B-cell subsets (primary aim), whether this is differentially regulated between MS patients and healthy controls (secondary aim) and if this is affected by a pre- or post-pregnancy relapse (tertiary aim). The chemokine receptor expression pattern 25, a glycoprotein which pumps steroids out of cells. We recently found a similar impact of steroid treatment in patients with MS, AQP4-IgG+ neuromyelitis optica spectrum disorder and MOG-IgG-associated disease26. Because of the rise in IgMhigh transitional B cells early after delivery, one could speculate that this results in increased entrance of potentially autoreactive naive mature B cells to GCs, resulting in the development of pathogenic memory subsets that are destined to enter the CNS and contribute to an MS relapse. Although we did not touch upon their CNS-infiltrating ability in this study, we can at least assume that the postpartum increase in CXCR3 expression mediates local B-cell enrichment in MS5. This may be further induced by the observed abundance of CCR6 on postpartum B cells of MS patients.Regarding naive B cells, the proportion of circulating transitional B cells was found to be decreased in third trimester compared to postpartum samples. A similar observation was made in a previous study27. Our group previously reported an upregulation of CXCR4 during MS onset in non-pregnant patients28. The observed reduction in CXCR4 expression during the high relapse risk postpartum period is therefore counterintuitive. A possible explanation for this discrepancy may be that during a primary response, CXCR4high naive mature B cells escape from TFH-mediated selection in the GC light zones of peripheral secondary lymphoid organs13, while CXCR4low memory B cells are more prone to interact with TFH cells and develop into long-lived plasma cells during a recall response in the CNS. CCR6 could facilitate such recall responses23 and, together with CXCR3, has also been associated with the production of high affinity antibodies29.The postpartum upregulation of CXCR5 especially seen in MS patients is likely involved in B-cell organization rather than recruitment in the CNS30. The clinical relevance of local Ig production has become apparent from the increased risk of CIS to MS conversion in patients with CSF oligoclonal bands31, which are present in more than 95% of MS patients and indicates ongoing IgG production in the CNS. Consistently, the absence of B cells in brain lesions of MS patients is associated with a lack of CSF oligoclonal bands, a lower intrathecal IgG production, and a more favorable outcome32. The observation that anti-CD20 treatment reduces CSF B cell numbers while oligoclonal bands persist33 suggests that intrathecal IgG are mainly produced by (CD20-) long-lived plasma cells in the CNS. Recently, it has been shown that MS myelin is bound by IgG and that IgG immune complexes trigger human microglia, resulting in enhanced production of pro-inflammatory cytokines34.Thus far, the potential of functionally distinct memory B cells to develop into plasma cells has been relatively understudied in MS. Despite the current focus on antibody-independent B-cell functions, this is of high relevance as long-lived plasma cells reside within the chronically inflamed CNS of MS patients35. Furthermore, we did not perform in vitro B-cell cultures with pregnancy-related sera or hormones to verify whether the observed GC light zone phenotype is controlled by extrinsic factors. Finally, although beyond the scope of this study, intrinsic factors such as memory B-cell EBV load could contribute to the increased CXCR3 expression and plasma cell formation21 as seen early after delivery.Our study has some limitations. The relatively low numbers of included subjects hampered the analysis of MS risk groups based on disease activity before and after pregnancy. Sequential data collection of such patients is difficult, but the accumulating evidence for the safe continuation during pregnancy of various disease modifying therapies may increase optionsTogether, this work provides new insights into how B-cell development is affected during high and low risk periods associated with pregnancy in MS. We demonstrate a first link between chemokine receptor expression profiles and the capacity of  memory B cells to differentiate into plasma cells, which should be further studied in the near future. This may not only help to decipher underlying mechanisms of local B-cell accumulation and antibody production, but also offer new tools to better predict disease activity in patients with MS.Supplementary Figures."}
+{"text": "Background: Multiple sclerosis (MS) patients are protected from relapses during pregnancy and have an increased relapse risk after delivery. It is unknown how pregnancy controls disease-contributing CD4+ T helper (Th) cells and whether this differs in MS patients who experience a postpartum relapse. Here, we studied the effector phenotype of Th cells in relation to pregnancy and postpartum relapse occurrence in MS.Methods: Memory skewing and activation of effector Th subsets were analyzed in paired third trimester and postpartum blood of 19 MS patients with and without a postpartum relapse and 12 healthy controls. Ex vivo results were associated with circulating levels of pregnancy-induced hormones and mirrored in vitro by exposing proliferating Th cells to corresponding serum samples.Results: Based on HSNE-guided analyses, we found that effector memory proportions of Th cells were increased in postpartum vs. third trimester samples from MS patients without a postpartum relapse. This was not seen for relapsing patients or healthy controls. CXCR3 was upregulated on postpartum memory Th cells, except for relapsing patients. These changes were verified by adding sera from the same individuals to proliferating Th cells, but did not associate with third trimester cortisol, estradiol or progesterone levels. For relapsing patients, activated memory Th cells of both third trimester and postpartum samples produced higher levels of pro-inflammatory cytokines.Conclusion: Effector Th cells are differentially regulated during pregnancy in MS patients, likely via serum-related factors beyond the studied hormones. The pro-inflammatory state of memory Th cells during pregnancy may predict a postpartum relapse. Women with multiple sclerosis (MS) have a reduced relapse rate of ~70% in the third trimester of pregnancy , which c+ T helper (Th) and not CD8+ T cell clonotypes are reduced in the blood  containing sodium heparin for cell-based analysis. Serum and peripheral blood mononuclear cells (PBMCs) were isolated according to manufacturer's instructions. PBMCs were taken up in RPMI 1640  containing 20% fetal calf serum  and 10% dimethyl sulfoxide  and stored in liquid nitrogen until further use. Serum samples were stored at \u221280\u00b0C.highCD127low) and defined naive, central memory and effector memory populations based on CCR7 and CD45RA expression. Th1, Th2, Th17, Th17.1 and Th17 \u201cdouble-positive\u201d (DP) subsets were discriminated based on differential expression of CCR6, CCR4, and CXCR3  using CD4 microbeads and the autoMACS Pro Separator , frozen as described above and stored in liquid nitrogen until further use. Thawed cells were labeled with CFSE  for 10 min at 37\u00b0C. After washing twice with RPMI 1640 containing 100 U/ml penicillin and 100 \u03bcg/ml Streptomycin  and 5% FCS (Thermo Fisher Scientific), cells were plated at a concentration of 1 \u00d7 10E6/ml in RPMI1640 containing Pen/Strep. Anti-CD3/-CD28 dynabeads (Thermo Fisher Scientific) were added (1:5) together with paired third trimester or postpartum serum samples from MS patients or healthy controls till a concentration of 5%. After 72 h of culture, dynabeads were removed and cells were assessed by flow cytometry.CD4+CD4+CD8\u2212CD25dim\u2212/CD45RA\u2212) were isolated using a FACSAria-III machine (BD Biosciences) and plated at a concentration of 0.5 \u00d7 106/ml in RPMI 1640 containing 5% inactivated human AB serum (Sanquin) and Pen/Strep. Cells were rested overnight (37\u00b0C) and were stimulated with phorbol 12-myristate 13-acetate  and ionomycin  for 5 h. Subsequently, supernatants were harvested and stored at \u221280\u00b0C until further use. Supernatants were diluted 2-fold and analyzed for GM-CSF, IFN-\u03b3, IL-2, IL-6, IL-17A and TNF-\u03b1 using a custom Luminex multiplex bead immunoassay . Measurements were performed on a Bio-Plex MAGPIX machine and data were analyzed using Bio-Plex Manager MP software .Memory Th cells . Both steroids were calibrated using commercially available calibrators ; article number P-069 (progesterone) and C-106 (cortisol). Trueness was verified for progesterone using reference materials ERM-DA347 and ERM-DA348. For cortisol, trueness was verified using the ERM-DA451 IFCC cortisol reference serum panel (34 levels). Both analytes were measured without bias when compared to the reference materials. Measurement uncertainty was assessed for both analytes by measurement of tri-level matrix matched controls . For progesterone, the coefficients of variation (CVs) over a period of 1 year were 5.2, 6.6, and 10.4% at 1.4, 17.9, and 65.3 nM, respectively. For cortisol, CVs were 3.6, 3.7, and 4.0% at 40.0, 98.6, and 573.0 nM, respectively. Estradiol levels were measured by automated immunoassay on . CVs over 19 runs were 4.4 and 3.9% at 345 and 1,099 pM, respectively.p-value of < 0.05 (*) after multiple testing correction was considered significant. Categorical data were analyzed using Chi-squared, Kruskal-Wallis or Wilcoxon rank-sum tests.Generalized linear mixed models (GLMM) with a gamma distribution were used to assess longitudinal effects and the false discovery rate (FDR) method of Benjamini and Hochberg (BH) was used to correct for multiple testing . Data between clinical groups were compared using Kruskal-Wallis tests incorporating Dunn's multiple comparisons or 2-way analysis of variance (ANOVA) with Bonferroni's multiple comparison tests . Longitudinal results were displayed as connected data points for each individual. For the comparison of data between clinical groups, Box and Whisker plots were used containing individual data points and displaying the median with the interquartile range (IQR). For all tests, a In this exploratory study, pregnant relapsing-remitting MS patients visiting MS center ErasMS were included and retrospectively validated to match the most recent McDonald 2017 criteria , 17. Excex vivo flow cytometry data of CD4+ Th cells from paired third trimester and early postpartum blood of healthy controls  and MS patients without a postpartum relapse  in an unbiased manner using HSNE (lowCD45RAlow) was decreased, while cluster P1 was increased (CCR7highCD45RAhigh) in third trimester vs. postpartum blood (dimCD45RAlow) was proportionally decreased during pregnancy relative to the presence of other clusters. For the MS-NR group, this was confirmed by manual gating, showing significantly reduced effector memory (CCR7lowCD45RAlow) and increased naive (p = 0.002 and p = 0.012) Th cell frequencies. For the HC group, these frequencies did not differ (+ Th cells in vitro (ex vivo (p = 0.026 and p < 0.001). In contrast to our ex vivo results (p < 0.001). Interestingly, these frequencies appeared higher than in the MS-NR and HC group. Overall, these data suggest that effector memory Th cells are kept in check during pregnancy by serum-related factors, which is lost and may contribute to an MS relapse early after delivery.To assess shifts in phenotypic effector profiles, we first analyzed ing HSNE . For botum blood . Clustert differ  and weret differ . To detein vitro . Th cell results , reduceddim/lowCD45RAlow) Th clusters P2 and P3 (p = 0.002) and MS-NR groups (p < 0.001), but not for the MS-R group. In line with our HSNE analysis (\u2212CXCR3+CCR4\u2212), Th17 DP (CCR6+CXCR3+CCR4+) and Th17.1 (CCR6+CXCR3+CCR4dim\u2212/) cells (in vitro proliferating memory Th cells exposed to MS-NR and MS-R third trimester vs. postpartum sera (p < 0.001). This was comparable to the in vitro effects on effector memory phenotypes . As expected, mass spectrometry revealed that cortisol levels were strongly elevated in third trimester sera from each clinical group (p < 0.001). Progesterone and estradiol could not be detected in postpartum samples. Third trimester cortisol, progesterone and estradiol levels did not differ between clinical groups  cells . Interesm) cells  were decm) cells , 3. In senotypes . Althougl groups . These dhighHLA-DRhigh memory Th cells were increased in postpartum compared to third trimester samples (p < 0.001). These proportions were higher than those in the HC group (p = 0.008). To address how this is related to their ability to produce pro-inflammatory cytokines, memory Th cells were purified from paired third trimester and postpartum samples and stimulated with PMA and ionomycin. Although not significant for each cytokine, higher levels of interleukin-6 (IL-6), tumor necrosis factor \u03b1 (TNF-\u03b1), IL-2, IL-17A, interferon gamma (IFN-\u03b3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were found in supernatants from postpartum memory Th cells of the MS-R group  cells are selectively enriched in the blood of MS patients who clinically responded to nalalizumab  and Th17.1 (IL-17low) cells  cells .+ effector memory Th subsets during pregnancy corresponds to a postpartum relapse in MS. Due to the limited number of samples available for this study, our results should be validated in a larger cohort of similar well-defined treatment-naive pregnant MS patients, preferably with radiological data available to confirm the absence of immunological activity in cases not experiencing a clinical postpartum relapse. Since the composition of effector Th cells can easily be monitored in the blood, subsequent studies should clarify whether their pro-inflammatory nature could be evaluated as a prognostic cellular marker of disease activity during and after MS pregnancy.Our study provides additional insights into how the suppression of CXCR3The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Medical Ethics Committee Erasmus MC. The patients/participants provided their written informed consent to participate in this study.SK performed experiments, analyzed data, interpreted results, and wrote the manuscript. RN organized the clinical study and critically revised the manuscript. AW-W, M-JM, AR, and RW performed experiments. CM assisted with the statistical analysis. WD, BB, and SB analyzed data and critically revised the manuscript. JS and ML designed the research, obtained funding, interpreted results, and critically revised the manuscript. All authors contributed to the article and approved the submitted version.JS received lecture and/or consultancy fee from Biogen, Merck, Novartis, and Sanofi-Genzyme. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest."}
+{"text": "In addition, for the [68Ga]Ga-DOTATOC examinations, tracer uptake quantitation and liver lesion detectability were assessed.The aim of this prospective study was to evaluate a data-driven gating software\u2019s performance, in terms of identifying the respiratory signal, comparing [68Ga]Ga-DOTATOC PET/CT examinations. Prospective DDG was applied on all bed positions and respiratory motion correction was triggered automatically when the detected respiratory signal exceeded a certain threshold , at which point the scan time for that bed position was doubled. These bed positions were reconstructed with quiescent period gating (QPG), retaining 50% of the total coincidences. A respiratory signal evaluation regarding the software\u2019s efficacy in detecting respiratory motion for [68Ga]Ga-DOTATOC was conducted and compared to [18F]FDG data. Measurements of SUVmax, SUVmean, and tumour volume were performed on [68Ga]Ga-DOTATOC PET and compared between gated and non-gated images.Twenty-four patients with confirmed or suspected neuroendocrine tumours underwent whole-body [R\u2009\u2265\u200915 was exceeded and gating triggered on mean 2.1 bed positions per examination for [68Ga]Ga-DOTATOC as compared to 1.4 for [18F]FDG. In total, 34 tumours were evaluated in a quantitative analysis. An increase of 25.3% and 28.1%, respectively, for SUVmax (P\u2009<\u20090.0001) and SUVmean (P\u2009<\u20090.0001), and decrease of 21.1% in tumour volume (P\u2009<\u20090.0001) was found when DDG was applied.The threshold of 68Ga]Ga-DOTATOC PET/CT. DDG yielded significantly higher SUVmax and SUVmean values and smaller tumour volumes, as compared to non-gated images.High respiratory signal was exclusively detected in bed positions where respiratory motion was expected, indicating reliable performance of the DDG software on [ Respiratory motion has an impact on positron emission tomography (PET) imaging of the thorax and abdomen, usually in the vicinity of the diaphragm. Non-corrected respiratory motion might lead to severe lesion blurring which affects several aspects of the imaging assessment such as delineation, detectability, and quantitation , 2. ApplData-driven gating (DDG) constitutes a device-less alternative by which respiratory motion is derived directly from the PET-acquisition data. A number of different techniques for DDG have been developed during the last decade, and many have shown promising results when applied on retrospective data \u201313. DDG R. The R value is a measure of how much the signal resembles respiratory motion in each bed position and hence provides information on to what extent this motion might affect the resulting image. An R value threshold can be predefined by the user and during the scanning, when this threshold is exceeded in a bed position, motion correction is triggered automatically. This results in activation of quiescent period gating (QPG), extending the scan time and retaining images from a desired percentage, typically 50%, of the total acquisition. When the level of respiratory signal in a bed position is below the set threshold, it is assumed that there is no need for respiratory gating and the scan time is not extended FDG PET data. However, since this DDG software is based on PCA, which is a signal processing method, its sensitivity is expected to depend on noise levels and tracer distribution [18F]FDG.To the best of our knowledge, all previously published studies evaluating the MotionFree software\u2019s performance regarding identification of the respiratory signal have been conducted retrospectively on [ribution . We hypo68Ga]Ga-DOTATOC is used for PET/CT-imaging of neuroendocrine tumours (NETs) [68Ga]Ga-DOTATOC would most likely benefit from respiratory motion correction.The somatostatin analogue Ga-DOTATOC and [18F]FDG examinations conducted with the same scanning parameters. In addition, for the [68Ga]Ga-DOTATOC examinations, tracer uptake quantitation and liver lesion detectability were assessed.The aim of this prospective study was to evaluate the MotionFree software\u2019s performance, in terms of identifying the respiratory signal, comparing [68Ga]Ga-DOTATOC was produced in-house on an automated synthesis platform  using disposable cassette system (C4-Ga68-PP) and pharmaceutical grade 68Ge/68Ga generator . The product was formulated in sterile saline and sterile-filtered in-line. The quality control regarding radiochemical purity, chemical purity and quantity was conducted using high performance liquid chromatography with UV- and radio-detectors connected in series.The [68Ga]Ga-DOTATOC. The examinations were performed at Uppsala University Hospital during a one-year period, from June 2019 to June 2020, when the clinical workflow allowed for additional acquisition time. The study was approved by the regional ethics board for medical research (Dnr 2019/00092) and the need for written consent was waived.Twenty-four patients  with confirmed or suspected neuroendocrine tumours underwent whole-body PET/CT examinations with [68Ga]Ga-DOTATOC mean\u2009\u00b1\u2009SD 64\u2009\u00b1\u20098\u00a0min (range 56\u201379) prior to the examinations conducted on a Discovery MI digital 4-ring PET/CT system  with 198\u00a0mm axial field of view. Before each PET-acquisition, a CT was performed in end expiration breath hold for attenuation correction as according to our centre\u2019s standard protocol. The vendor\u2019s integrated software MotionFree  was applied prospectively for DDG on all scans and motion correction was triggered automatically when the measured respiratory signal exceeded a threshold set to R\u2009=\u200915. The threshold was chosen according to the vendor\u2019s recommendation.The patients were injected with mean\u2009\u00b1\u2009SD 1.4\u2009\u00b1\u20090.2\u00a0MBq/kg (range 1.1\u20132.0) of FDG PET data from 62 consecutive patients, scanned at Karolinska University Hospital, Huddinge, Stockholm on an identical scanner with the same acquisition time and settings as for the [68Ga]Ga-DOTATOC examinations was utilized. The [18F]FDG patients were injected with 3\u00a0MBq/kg using an autoinjector (MEDRAD\u00ae Intego), and the scans were started approximately 60\u00a0min post-injection. No further analysis was conducted for [18F]FDG PET. The [18F]FDG data was merely used to compare the DDG algorithm\u2019s performance in terms of detecting respiratory motion.For comparison of the number of triggered bed positions, [R values were extracted from the scanner's database retrospectively for each bed position. The centre of each bed position in relation to that of the liver dome was calculated for every patient. Localization of the liver dome utilized the CT performed for attenuation correction.The previously calculated 18F]FDG and [68Ga]Ga-DOTATOC were assessed using a Mann\u2013Whitney test. A P\u2009<\u20090.05 was considered statistically significant.Differences in the number of triggered bed positions for [68Ga]Ga-DOTATOC PET-images, well-defined tumour lesions located in bed positions that were triggered by DDG during live acquisition, were assessed for measurements of SUVmax, SUVmean, and volume using auto contour volume of interest (VOI) delineation with 41% of SUVmax, including up to 5 lesions per patient and a maximum of 3 lesions per organ. The VOIs were transferred to the corresponding gated images. To compare SUVmax, SUVmean and lesion volume, in gated versus non-gated images, an orthogonal regression was performed. In order to quantify the changes in background activity, a spherical VOI of 16 cm3 was placed in normal liver tissue in non-gated images and copied to the corresponding gated images. SUVmean in the spherical liver VOI and lesion-to-liver measures (lesion SUVmax/liver SUVmean) were evaluated. Statistical significance was considered for P values below 0.05 using Wilcoxon\u2019s signed rank test. Quantitative measurements were made using an Advantage Workstation Server . Statistical analyses were performed in Prism 8 (GraphPad Software Inc).In non-gated [68Ga]Ga-DOTATOC PET/CT examinations in which liver metastases were detected. Liver lesion detectability was evaluated by a senior nuclear medicine physician with 21\u00a0years of PET/CT experience. The non-gated and gated PET-image sets of each patient were assessed and compared side by side, and the number of detected metastases was registered. Patients with more than 20 liver metastases were excluded from the comparison. The physician was not blinded to gated versus non-gated image sets.In order to achieve some initial data on the clinical performance of DDG in comparison to the non-gated image sets, lesion detectability was analysed, including all [68Ga]Ga-DOTATOC PET/CT scans and 352 bed positions from 62 [18F]FDG PET/CT scans were analysed. There were significantly more triggered bed positions in the [68Ga]Ga-DOTATOC PET/CT examinations than in the [18F]FDG PET/CT scans (P\u2009=\u20090.0074). In the [68Ga]Ga-DOTATOC examinations the average number of beds per patient was 5.3. When using a threshold of R\u2009=\u200915, QPG was triggered in 22 of the 24 patients and 50/128 bed positions (39%), resulting in mean 2.1 gated bed positions per scan. Bed centre positions in which QPG was triggered were found in the range 7.1\u00a0cm superior to 29.5\u00a0cm inferior of the liver dome. In the [18F]FDG PET/CT examinations, QPG was triggered in 47 of 62 patients and in 87/352 bed positions (25%) using the same threshold (R\u2009=\u200915). The number of triggered beds for [18F]FDG scans was mean 1.4 and the centre position for the bed positions were found in the range 9.9\u00a0cm superior to 40.6\u00a0cm inferior of the liver dome . The orthogonal regression analysis, comparing gated and non-gated, of SUVmax measurements, resulted in a slope of 1.192  1.055\u20131.328). SUVmean was also significantly higher in gated than in non-gated images , with an orthogonal regression slope of 1.212 (95% CI 1.058\u20131.365). There was a mean 21.1% decrease in lesion volume in gated as compared to non-gated images (P\u2009<\u20090.0001), with an orthogonal regression slope of 0.782 (95% CI 0.628\u20130.937)  in non-gated images and 4.37 (range 2.03\u20137.65) in gated images (P\u2009=\u20090.4). The ratio between lesion SUVmax and liver SUVmean was increased by 19.6% on average in the gated as compared to non-gated images (P\u2009<\u20090.0001).The mean SUV68Ga]Ga-DOTATOC PET/CT and were included in a subsequent lesion detectability analysis. Two patients harboured more than 20 lesions and were thus excluded from the analysis. In two patients, 8 and 5 metastases were detected in the non-gated and 10 and 8 in the gated images, respectively , however, was not significantly different when comparing gated and non-gated images. The NET-patients\u00b4 eligibility for peptide receptor radionuclide therapy (PRRT) with 90Y and 177Lu labelled DOTA-peptides Ga-DOTATOC PET examinations. As illustrated by Fig.\u00a068Ga]Ga-DOTATOC PET/CT imaging routine could potentially increase lesion detection and reader confidence. This, however, has yet to be assessed in a separate trial in a larger number of patients, employing several observers and grading reader confidence scores. Also, an evaluation including different image reconstructions, such as Bayesian penalized likelihood reconstructions, could also be valuable since different reconstruction methods applied together with DDG, are likely to impact image quality and PET quantification FDG data, in terms of evaluating the respiratory signal, quantitative measurements and image quality, and where the performance of DDG was found superior Ga-DOTATOC PET/CT and gated images yielded significantly higher lesion SUV and lower lesion volumes as compared to non-gated images. This supports that DDG might be a useful tool to provide more accurate quantitation of [68Ga]Ga-DOTATOC tumour uptake.High respiratory signal was exclusively detected in bed positions where respiratory motion was expected, indicating reliable performance of the DDG software on ["}
+{"text": "Fusarium spp., is a major disease of field pea (Pisum sativum). The development of genetic resistance is the most promising approach to manage the disease, but no pea germplasm has been identified that is completely resistant to root rot. The aim of this study was to detect quantitative trait loci (QTL) conferring partial resistance to root rot and wilting, caused by five fungal isolates representing Fusarium solani, F. avenaceum, F. acuminatum, F. proliferatum, and F. graminearum. Evaluation of the root rot-tolerant cultivar \u201c00-2067\u201d and susceptible cultivar \u201cReward\u201d was carried out with the five species. There was a significant difference (p < 0.001) between the mean root rot values of the two cultivars inoculated with the F. avenaceum (F4A) and F. graminearum (FG2) isolates. Therefore, in the QTL study, the F8 recombinant inbred line (RIL) population derived from \u201cReward\u201d \u00d7 \u201c00-2067\u201d was inoculated in the greenhouse (4 \u00d7) with only F4A and FG2. The parents and F8 population were genotyped using 13.2K single nucleotide polymorphisms (SNPs) and 222 simple sequence repeat (SSR) markers. A significant genotypic effect (p < 0.05) and high heritability (79% to 92.1%) were observed for disease severity, vigor, and plant height following inoculation with F4A and FG2. Significant correlation coefficients were detected among and within all traits. This suggested that a high proportion of the genetic variance was transmitted from the parents to the progeny. However, no significant QTL (LOD > 3) were detected for the RILs inoculated with F4A. In the case of the RILs inoculated with FG2, 5 QTL for root rot severity and 3 QTL each for vigor and plant height were detected. The most stable QTL for plant height (Hgt-Ps3.1) was detected on Chrom5/LGIII. The two most stable QTL for partial resistance to FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in a 15.1-cM and 11.2-cM genomic region, respectively, on Chrom4/LGIV. The most stable QTL for vigor (Vig-Ps4.1) was found in the same region. Twenty-five major and moderate effect digenic epistatic interactions were detected. The identified region on chrom4/LGIV could be important for resistance breeding and marker development.Fusarium root rot, caused by a complex of  Fusarium spp., Aphanomyces euteiches, Pythium spp., Phytophthora spp., Rhizoctonia spp., Didymella spp. (formerly Mycosphaerella spp.), and Ascochyta spp.  in the major field pea cultivation regions in Canada and worldwide  includes the major pathogens causing Fusarium head blight (FHB) of wheat, barley, oats, and other small grain cereals , while four minor QTL were found on LGIII, IV, VI, and VII . These QTL explained 44.4\u201353.4% of the total variance for resistance  in a rough map generated with 14 SSRs. The QTL identified in most of these studies had very large confidence intervals due to the limited number of markers used. The low marker density makes it difficult to apply the identified markers in pea breeding.Genetic resistance offers the most promising way to control FRR and wilt in pea. However, there is no complete resistance to FRR in field pea, and only a few studies have reported QTL associated with partial resistance to this disease , 2019. Csistance . McPhee Fusarium spp. an important research objective.On the Canadian prairies, cereals are grown in tight rotations with canola, while the cultivation of field pea and other pulses is increasing . Boom-anFusarium spp. recovered from surveys for root rot in Alberta, Canada; (2) map the QTL associated with partial resistance to FRR using a segregating recombinant inbred line (RIL) pea population genotyped by Fusarium isolates; and (3) determine the stability of the QTL, accounting for disease severity, vigor, and plant height.Therefore, the objectives of this study were to: (1) evaluate the partially resistant pea cultivar \u201c00-2067\u201d for resistance to different 1 plants, which were then used to develop an F8 RIL population (One-hundred thirty-five RILs used by pulation  by the spulation .F. solani), F4A (F. avenaceum), F037 (F. acuminatum), F039 (F. proliferatum), and FG2 (F. graminearum), representing the Fusarium species most frequently recovered from symptomatic pea plants in root rot surveys in Alberta, were used to screen the parental cultivars \u201c00-2067\u201d and \u201cReward.\u201d Briefly, to obtain the SSIs, surface-sterilized pieces of root tissue with disease lesions were placed on potato dextrose agar (PDA) and incubated at 25\u00b0C for 2\u20133 days and then transferred to the peptone-pentachloronitrobenzene (PCNB) medium for further selection. Mycelial tips of the fungal isolates were cut from selected colonies under a stereomicroscope , and the water agar (WA) procedure was used to obtain SSI (Five single-spore isolates (SSI), S4C , with the resulting suspension decanted into 250-ml Erlenmeyer flasks, containing a 100-ml autoclaved CMC medium . The flasks were covered in aluminum foil to block light and incubated on a rotary shaker at room temperature for 2 weeks. The suspension was centrifuged to collect conidia. The concentration of conidia was estimated with a hemocytometer and adjusted to a final concentration of 2 \u00d7 106 spores ml\u20131 with sterile-deionized water.Conidial suspensions of the five isolates were generated following Plastic cups (9-cm diameter and 10.5-cm depth) were filled with a sterilized potting mixture . In the greenhouse tests with each SSI , the roots of seven 5-day-old seedlings of the partially resistant parent \u201c00-2067\u201d and the susceptible parent \u201cReward\u201d were immersed in the conidial suspension for 15 min and transplanted into the soilless mixture in a cup. An aliquot (1 ml) of conidial suspension was pipetted onto the roots before they were covered with the potting mix. The plants were kept in a greenhouse at 20\u201325\u00b0C/15\u201318\u00b0C day/night and a 16-h photoperiod with daily watering to maintain the potting mix at saturation conditions conducive for FRR development. Each experiment was repeated two times. After 3 weeks, disease severity was estimated for the parental cultivars on a scale of 0\u20134, where: 0 = completely healthy; 1 = brown or black spots on the main root; 2 = lesions covering the main root, but the rootlets still healthy; 3 = lesions spread to the entire root system; and 4 = root totally dead .Fusarium isolates was used as inoculum to screen the 135 RIL population and parents under greenhouse conditions. The inoculation and the maintenance of the plants were as described above. The pots were arranged in a randomized complete block design (RCBD) with four replicates. The greenhouse experiment was repeated four times. After 3 weeks, plant height (cm) was measured from the base of the stem to the top leaf. Plant vigor was evaluated as a measure of the wilting severity on a scale of 0\u20134  (The most virulent of the ly dead) . The plaP-value of the Shapiro\u2013Wilk test was used to determine the normality for each variable using R  searches of the Pulse Crop Database1 to determine their possible functions.Linkage analysis was carried out using the filtered SNP and SSR markers, following F. graminearum (FG2). This was then repeated for the RILs inoculated with F. avenaceum (F4A). The analysis was conducted using means for the four single greenhouse experiments, LSM, and BLUPs of the total data by Composite Interval Mapping (CIM) using WinQTL Cartographer v2.5  from the RILs inoculated with her v2.5 . The prop < 0.05 . The LODFusarium isolate was indicated, followed by \u201cPs\u201d = Pisum sativum, the first number = the pea linkage group (Fg-Ps4.1\u201d represents the QTL for disease severity caused by F. graminearum located on linkage group IV of the pea genome. The chromosomes and pseudomolecules were named in accordance with Vig-Ps2.1) and plant height .The QTL names were defined according to the QTL detection studies by ge group , and theR2) was determined for each QTL. Furthermore, QTL with R2 > 10%, 5\u201310%, and <5% were arbitrarily classified as major-, moderate-, or minor-effect QTL, respectively. The origins of favorable alleles for individual traits were assigned to different parents, following R2 > 15%, 7.5\u201315%, and <7.5%, respectively. Epistatic-effect QTL were named with the prefix \u201cE,\u201d followed by the QTL name and a serial number .Quantitative trait loci identified in at least two of the four environments were classified as stable. The percentage of variation (p < 0.001) between the mean root rot values of the tolerant parent \u201c00-2067\u201d and the susceptible parent \u201cReward\u201d, following inoculation with F. graminearum isolate FG2  (p < 0.001)  ,b. This < 0.001) ,b. The G< 0.001) ,b.t-test indicated a significant difference between the parents for disease severity in all four experiments. Frequency distribution  existed among the single experiments, pooled, and BLUPs for vigor  of \u201c00-2067\u201d were 234.5 \u00b1 54.6 cm, 157.3 \u00b1 50.6 cm, 155.5 \u00b1 59.5 cm, 159.7 \u00b1 6.7 cm, 176.7 \u00b1 56.6 cm and 158.9 cm, respectively. For \u201cReward\u201d, the plant heights were 177.5 \u00b1 36.1 cm, 120.7 \u00b1 31.5 cm, 129.5 \u00b1 26. cm, 178.5 \u00b1 34.6 cm, 151.5 \u00b1 36.9 cm, and 100.8 cm, respectively. The frequency distribution of plant height of the RIL population for all six variables was not normal and slightly skewed  was found for plant height among the single experiments, pooled, and BLUPs data  on the parental cultivar \u201c00-2067\u201d inoculated with FG2 were 1.5 \u00b1 0.7, 1.3 \u00b1 0.6, 2 \u00b1 1.2 and 1.5 \u00b1 0.7 for the four greenhouse experiments, 1.6 \u00b1 0.8 for LSM and 1.2 for the BLUPs. This was comparable with the estimated mean of 1.1 \u00b1 0.4 obtained in the preliminary screening of the parents . On the ribution  indicateribution . High coribution . The difribution . The Sharibution . A signior vigor . The heiy skewed . A high UPs data .r < \u20130.90, p < 0.001), indicating the adverse effect of FG2 on root and aboveground growth. Plant height showed low to moderate correlation with disease severity  and vigor .Collectively, the correlation analysis among traits indicated that root rot caused by FG2 was negatively correlated with vigor and plant height. High correlation coefficients were detected between disease severity and vigor in all conditions   for \u201c00-2067\u201d were 4.0 \u00b1 0.0, 3.7 \u00b1 0.5, 3.5 \u00b1 0.6, and 4 \u00b1 0 for the four individual greenhouse experiments, 3.8 \u00b1 0.5 for LSM for the pooled data and 4.0 for BULPs of the pooled greenhouse experiments (p < 0.001) (The estimated means (\u00b1SE) of disease severity for \u201c00-2067\u201d were 1.0 \u00b1 0.0, 1.0 \u00b1 0.8, 1.3 \u00b1 0.5, 1.0 \u00b1 0.0, 1.1 \u00b1 0.4, and 1.0, while, for \u201cReward\u201d, they were 3.3 \u00b1 0.5, 3.3 \u00b1 0.5, 3.5 \u00b1 0.6, 3.0 \u00b1 0.0, 3.3 \u00b1 0.4, and 3.0 for DSGH1, DSGH2, DSGH3, DSGH4, LSM of pooled data, and BLUPs, respectively . These vribution , althougribution . The cor< 0.001) . Based oeriments . The esteriments . Additioeriments . The cor< 0.001) .t-test. The estimated plant height for \u201c00-2067\u201d for the individual experiments, LSM, and BLUP was 210.8 \u00b1 128.2 cm, 174.5 \u00b1 104.8 cm, 159.5 \u00b1 13.5 cm, 210.0 \u00b1 53.1 cm, 188.7 \u00b1 82.8 cm, and 208.0 cm, respectively. The estimated plant height for \u201cReward\u201d was 118.3 \u00b1 100.2 cm, 193.5 \u00b1 104.5 cm, 125. \u00b1 98.9 cm, and 194.0 \u00b1 38.4 cm for the individual experiments, 157.7 \u00b1 88.5 cm for LSM and 133.1 cm for the BLUP. The frequency distribution for the RIL population was continuous and slightly skewed to the right. In addition, HGH2, HGH3, HGH Pooled, and HGH BLUPs followed a normal distribution   .r < \u20130.95, p < 0.001) and with plant height . Plant height was positively correlated with vigor .The correlation among the traits for plants inoculated with F4A was similar to that of plants inoculated with FG2. Disease severity was highly correlated with vigor  retained markers were as described by F. avenaceum isolate F4A. As such, no QTL likelihood profiles are shown. In the case of RILs inoculated with F. graminearum isolate FG2, a total of 11 QTL were detected for the three parameters and six variables  by the CIM using Win QTL Cartographer v2.5 (R2) values ranging from 4.05 to 36.35%  for disease severity, vigor, and plant height were detected for the RILs inoculated with her v2.5 ; Table 3F. graminearum isolate FG2, Fg-Ps4.1, and Fg-Ps4.2 were located in the middle of Chrom4/LGIV at positions 59.3\u201374.4 cM and 74.0\u201385.2 cM, respectively  of Chrom5/LGIII and with flanking markers of AA5 and PsCam036163_21311_1095) and Fg-Ps3.2  explained 9.62\u20139.88% of the total variance (Fg-Ps5.1 [detected on the top part (0.9\u20139.2 cM) of Chrom3/LGV and flanked by the SNP markers PsCam059449_39630_321 and PsCam011153_7569_125] explained 14.2% of the total variance in greenhouse Experiment 1 (Fg-Ps5.1) had a negative additive effect, indicating that genomic regions for resistance in Fg-Ps4.1, Fg-Ps4.2, Fg-Ps3.1, and Fg-Ps3.2 originated from \u201c00-2067,\u201d while Fg-ps5.1 derived its resistance from \u201cReward\u201d  > Vig-Ps3.2  > Vig-Ps3.1  both on Chrom5/LGIII  of the same chromosome or linkage group, was flanked by the SNP marker PsCam013763_9362_423 and the SSR marker AD270 . This QTL was located on the bottom segment of Chrom5/LGIII  of four greenhouse experiments (R2 = 7.04\u201320.04%). These QTL were located 142.3\u2013168.0 cM on Chrom7/LGVII and were flanked by the SNP markers PsCam002756_2184_427 and PsCam045262_28962_162  (R2 = 13.63\u201314.06). The additive effect was negative for Hgt-Ps3.1, but positive for Hgt-Ps7.1 and Hgt-Ps7.2  , was detgt-Ps7.2 . This suTwo hundred eight putative digenic epistatic pairs were identified using all variables for disease severity, vigor, and plant height. These comprised 65 (12-24) for disease severity, 57 (10\u201321) for vigor, and 86 (15\u201328) for plant height. The 208 putative digenic interactions consisted of one major epistatic effect (PVE \u2265 15%), 13 moderate epistatic effects (7.5% \u2264 PVE \u2264 15%), and 194 minor epistatic effects (PVE \u2264 7.5%). BLUPs for disease severity, vigor, and plant height detected 20, 18, and 21 putative digenic interactions, respectively. Within the 59 digenic interactions among BLUPs of all traits, epistatic analysis identified one major QTL pair, three moderate QTL pairs, and 55 minor QTL pairs . In contFg-Ps3.1, Fg-Ps3.2, and Vig-Ps3.1). The remaining 15 epistatic QTL were not related to any of the additive-effect QTL , followed by E.Hgt-Ps7 (R2 = 19.1%) and E.Hgt-Ps4 (R2 = 13.5%). The fourth was E.Hgt-Ps3 (R2 = 13.5%), which was linked to Fg-Ps3.2 and Vig-Ps3.1. Only E.Fg-Ps7 and E.Vig-Ps7 were linked to Fg-Ps3.1, showing moderate epistatic effect .Twenty-five digenic epistatic interactions with major and moderate effects were identified by 33 flanking markers, of which 10 epistatic-effect QTL with 14 flanking markers were linked to three additive-effect QTL  of all the Fusarium species isolated from 1,812 wheat, 71 durum, 596 barley, and 177 oat samples  only for the isolates representing F. avenaceum and F. graminearum. Therefore, the F8 RIL population derived from \u201cReward\u201d \u00d7 \u201c00-2067\u201d was screened with F4A (F. avenaceum) and FG2 (F. graminearum) for the detection of partial resistance to the two Fusarium species. The greenhouse experiments were repeated four times to determine the G \u00d7 E interaction for all traits. In addition, the best linear unbiased predictors (BLUPs) and LSM were applied to minimize environmental effects  was comparable. R2 = 68\u201380%) identified by F. oxysporum were also located on Chrom4/LGIV, while three minor QTL (R2 = 2.8\u20135.4%) were located on Chrom5/LGIII. Despite identifying the same chromosomes, the similarity of the location of the QTL cannot be confirmed, given the different markers used in the two studies. However, the coincidence of the QTL is not surprising, since very few partially resistant pea cultivars are used in breeding programs across the world. F. avenaceum were located on Chrom7/LGVII. F. solani were located on Chrom6/LGII, while several minor QTL were located on Chrom5/LGIII, Chrom4/LGIV, Chrom6/II, and Chrom7/LGVII.In this study, 11 QTL accounting for disease severity, vigor, and plant height were identified. The major QTL for disease resistance were located on Chrom4/LGIV, while two minor QTL were detected on Chrom5/LGIII and one QTL on Chrom3/LGV. These QTL were coincident with the QTL detected for resistance to Aphanomyces root rot . The majSignificant QTL \u00d7 QTL interactions were found between the minor QTL for disease severity and plant height but not for vigor. An interaction of the major QTL for disease severity, vigor, and height was not observed. F. graminearum. The use of high-density SNP markers and SSR anchor markers contributed to the construction of a fine linkage map and the identification of two stable QTL located on Chrom4/LGIV associated with partial resistance to F. graminearum. The identified QTL showed broad resistance to F. graminearum, F. solani, F. avenaceum, F. acuminatum, and F. proliferatum, as well as to A. euteiches. This study, together with our previous report (To the best of our knowledge, no genetic studies have been carried out to determine the genomic regions associated with the partial resistance of field pea to s report , suggestHeritability of each trait, marker information, linkage information, and QTL profiles are available in the main manuscript or as Fusarium spp., greenhouse screening of RIL population and parents for resistance to Fusarium root rot, disease rating, measurement of vigor and plant height, phenotypic data analysis, DNA extraction, PCR, genotyping with SSR markers, and writing of the manuscript. RF-A: supervision of molecular marker work, molecular data analysis, linkage map construction, QTL mapping, and writing of the manuscript. SS: principal investigator, grant application, supervision and provision of technical support to graduate student, and revision of the manuscript. K-FC: grant application, supervision, and provision of technical support for graduate students for RIL population screening in the greenhouse. S-FH: principal investigator, grant application, supervision and provision of technical support for graduate students for RIL population screening in the greenhouse, and revision of the manuscript. All authors contributed to the article and approved the submitted version.LW: inoculum preparation of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher."}
+{"text": "Most somatic mutations that arise during normal development are present at low levels in single or multiple tissues depending on the developmental stage and affected organs. However, the effect of human developmental stages or mutations of different organs on the features of somatic mutations is still unclear. Here, we performed a systemic and comprehensive analysis of low-level somatic mutations using deep whole-exome sequencing (average read depth ~500\u00d7) of 498 multiple organ tissues with matched controls from 190 individuals. Our results showed that early clone-forming mutations shared between multiple organs were lower in number but showed higher allele frequencies than late clone-forming mutations  along with less nonsynonymous mutations and lower functional impacts. Additionally, early and late clone-forming mutations had unique mutational signatures that were distinct from mutations that originated from tumors. Compared with early clone-forming mutations that showed a clock-like signature across all organs or tissues studied, late clone-forming mutations showed organ, tissue, and cell-type specificity in the mutation counts, VAFs, and mutational signatures. In particular, analysis of brain somatic mutations showed a bimodal occurrence and temporal-lobe-specific signature. These findings provide new insights into the features of somatic mosaicism that are dependent on developmental stage and brain regions. Most somatic mutations that arise during normal development are present at low levels in single or multiple tissues, and often show a degree of clonality depending on the time and origin of the mutation. Recent studies have identified the characteristics of postzygotic variants of somatic mutations at the single-cell or mono-clonal levels. However, the results may not be fully representative of the mutational processes involved. Here, we describe a comprehensive analysis of low-level somatic mutations identified after deep whole-exome sequencing in peripheral and brain tissues. We found that clone-forming mutations are uniquely defined by early and late-stage aspects in the mutational profiles. Thus, we identified reliable spatiotemporal characteristics of mosaic variants. Additionally, we found low-level clone-forming mosaic variants across multiple stages and tissues, and identified their intrinsic features. Somatic mutations persistently occur in normal cells during the entire human lifetime . AlthougTo address these issues, we performed a comprehensive analysis of low-level somatic mutations identified using deep whole-exome sequencing (WES) data (average read depth: ~500\u00d7) from 498 tissues of 190 individuals. The cohorts analyzed consisted of two or more organs, including the brain and matched-peripheral tissues, and covered a variety of neurological conditions. The pairwise analysis of age, brain locations, and diseases enabled multi-dimensional analysis and direct comparison with cancer mutations identified using the same analysis procedure.n = 301), blood (n = 100), liver (n = 60), heart (n = 13), and other peripheral tissues (n = 24). The 190 individuals from whom tissues were obtained included patients with \u2018non-tumor\u2019 neurological disorders (n = 133), brain tumors , and non-diseased controls (n = 38)  .We defined and used three different categories in the analysis: early-stage, late-stage, and tumor mutations . Early-sr = 0.84; P = 1.00\u00d710\u221242) and between the WES and TASeq data  indicated the accuracy of the calls   . Howeverr, 0.36) .De novo signature extraction of the 1494 somatic SNVs  helped identify three novel signatures   identifiWe identified functional differences between the early and late-stage mutations. In particular, early-stage mutations showed a lower ratio of non-synonymous to synonymous substitutions (quantified as the dN/dS ratio by maximum-likelihood methods) (0.79) than did late-stage mutations (0.94), tumor mutations (0.94), and common germ-line coding variants  , indicatr = 0.45; p = 2.99\u00d710\u221210; Next, we investigated the characteristics of late-stage mutations, with a particular focus on diversity among different organs and cell types. The numbers of mutations varied substantially by organ, with a smaller number in the brain  and a higher number in the blood  relative to other peripheral organs  . HoweverUnsupervised hierarchical clustering of the three signatures  in late-stage mutations showed that mutations in the brain primarily comprised signatures A (early-stage) and B2 (tumor), whereas blood mutations harbored signatures B1 (late-stage) and B2 (tumor) .+), oligogenic (Olig2+), and others (negative). TASeq of the separated cell populations showed that both early and late-stage mutations were present in multiple cell lineages, but a large asymmetry in mutation frequencies was present among cell types with late-stage mutations  contained early-stage mutations (5.47% VAFs) whereas the other (NLE-P-0225) contained five late-stage mutations (average: 8.00% VAF) , each ofutations .We further classified the late-stage brain mutations into temporal and non-temporal areas, and analyzed area-specific mutational signatures . As repoFurthermore, late-stage mutations in the blood and tumor mutations showed enrichment for T>C mutations on transcribed strands . TranscrBased on a large-scale deep whole exome sequencing data analysis using a total of 498 matched sample pairs from 190 individuals, we provided a detailed picture of low-level but clone-forming somatic mutations, the associated mutation counts, and characteristics that are distinguished in time and space. We found that early-stage mutations, which arise before gastrulation and are shared by multiple organs, are lower in number and have a reduced functional impact than late-stage mutations that are restricted within a single organ. Moreover, we showed that late-stage mutations are associated with human mutational processes in the late-embryonic and post-natal developmental stages, but vary by organ, tissue, and cell lineages. In particular, late-stage mutations in the brain showed a bimodal distribution for developmental stages and the asymmetry of mutational features across brain-cell types and regions.Using pairwise analysis of the brain and other matched organs, we identified mutational counts, variant fractions, and mutational processes with high confidence levels at each developmental or time stage. Our mutational numbers were roughly comparable to those from previous studies, which reported 0.53 and 3.15 shared and non-shared somatic mutations in the brain , respectively , 32. We In terms of mutagenic processes, we extracted three unique signatures that showed overall similarity but contained early stage, late stage, and tumor SNVs. This phenotype indicates that somatic mutations from different stages have different contexts. For mapping to aging-related COSMIC Mutational Signatures, the increased relative proportion of late-stage somatic mutations in SBS1 indicates the presence of active proliferation and clonal expansion during the late-embryonic and post-natal or aging periods , 34. Thede novo signatures of late-stage mutations showed that mutations in the brain had early-stage and tumor mutation characteristics. The former result reflects the characteristic of low stemness and little or slow progression of division/differentiation in brain cells. Further analysis of late-stage brain mutations in the temporal lobe regions revealed that these were more similar to the signature B2 (tumor signature). These results suggest that late-stage somatic mutations in the brain show a bimodal occurrence during the early embryonic and post-natal periods, with a pattern similar to that in tumor mutations. We hypothesized that tumor-like mutational signatures in the temporal lobe may originate via neurogenesis  that confers clonal proliferation properties, as reported previously . Lysis buffer (1 mL) was added to the homogenate and mixed by pipetting, after which the lysate was fixed in 1% paraformaldehyde at room temperature for 10 min. The fixed lysate was quenched with 0.125 M glycine at room temperature for 5 min. The homogenate was washed with suspension buffer (1 mM EDTA and 2% BSA), and filtered with a 40 \u03bcM cell strainer. The sample was incubated with anti-NeuN  and anti-Olig2  antibodies overnight at 4\u00b0C, followed by washing with suspension buffer and staining with the secondary antibody for 1 h at 4\u00b0C. After washing with suspension buffer, nuclei were passed through a 40 \u03bcM cell strainer and stained with 1 \u03bcg of 4\u2032,6-diamidino-2-phenylindole. Nuclei used to isolate each cell type were analyzed and sorted using a MoFlo Astrios EQ cell sorter . Nuclei pellets were centrifuged for 10 min at 1500 \u00d7S1 FigLate mutational analysis by mutagen in different organs  (A), (B) Age correlation with somatic mutation VAFs in late-stage variants and blood late-stage variants. (C) VAFs of late-stage somatic mutations in brains are classified by the diseases in individuals. (D) VAFs of late-stage somatic mutations in the heart, liver, and lipoid tissues. (E), (F) Number of mutations per individual and VAF distribution in individuals aged 50\u201375 years.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file. 1 Feb 2022Dear Dr Kim,Thank you very much for submitting your Research Article entitled 'Multi-organ analysis of low-level somatic mosaicism reveals stage- and tissue-specific mutational features in human development' to PLOS Genetics.The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.In addition to the reviewers' concerns, I would request that in a revision you also carefully evaluate terminology that refers to mutations as pre- or post-gastrulation, since on my read it was not always clear that certain mutations were present in all three germ layers  such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at data availability policy\u00a0requires that all numerical data underlying graphs or summary statistics are included with the submission, and you will need to provide this upon resubmission if not already present. In addition, we do not permit the inclusion of phrases such as \"data not shown\" or \"unpublished results\" in manuscripts. All points should be backed up by data provided with the submission.Please be aware that our\u00a0Preflight Analysis and Conversion Engine\u00a0(PACE) digital diagnostic tool.\u00a0 PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.While revising your submission, please upload your figure files to the\u00a0Similarity Check, powered by iThenticate, into its journal-wide submission system in order to screen submitted content for originality before publication. Each PLOS journal undertakes screening on a proportion of submitted articles. You will be contacted if needed following the screening process.PLOS has incorporated\u00a0To resubmit, use the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]We are sorry that we cannot be more positive about your manuscript at this stage. Please do not hesitate to contact us if you have any concerns or questions.Yours sincerely,Marshall S. Horwitz, M.D., Ph.D.Consulting Editor - PLoS GeneticsPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1:\u00a0The authors present an interesting study that leverages whole-exome sequencing (WES) data from multiple tissues from a large number of individuals (190) to gain insight into the nature of somatic mutations that occur in early and late embryonic development. The authors classify somatic mutations as early if they are shared by two different tissues and late if they are not. In addition, the authors also sequenced a subset of brain tumors for comparison. The main finding is that early mutations have higher allele frequency and lower functional impact than late mutations as revealed by multiple genetic metrics . In addition, novel mutational signatures and their contributions to different brain regions are described.While the approach is interesting and there is a high need in the field for this sort of comparative analysis, the article has some important caveats, described below.1. The study is far from being a systemic and comprehensive analysis of low-level somatic mutations, as claimed in the abstract. Only 3 tissue types  are analyzed in a large number (60+) of individuals. Authors claim multi-organ sequencing but for most individuals only two tissue types are analyzed: either brain and blood, or brain and liver. The authors should tone down the claim of multi-organ analysis since the study is essentially on brain tissue, just using different control tissues.2. The article is difficult to read because some key points are not clearly explained. For example, in the 19 individuals with brain tumors, the brain tissue analyzed was from tumor or from normal? That is unclear in figure 1 and it is not clearly explained in Material and Methods. The text only mentions: \u201cIf the source of the sample was related to a brain tumor, it was separately regarded as tumor mutations.\u201d Please clarify the origin of samples in individuals with cancer and whether brain tumor tissue was used for the case-control comparisons explained in Fig. 1A. If it was used, this could be a source of confounding because in individuals with cancer, shared mutations tumor-blood could be due to ctDNA or cancer cells disseminated in the blood stream.3. The legend for Fig. 1A needs more explanation, especially regarding what samples are considered cases vs controls and the one-sample proportion test. What are the tests for organ specificity?4. When quantifying the number of early and late-stage somatic mutations per individual, how do the authors correct for the fact that different number of samples and different tissues were analyzed in different individuals? The article might benefit from removing the \u2018other tissues\u2019 and \u2018heart\u2019 subgroups since there are not many samples in those subgroups and will make the comparisons more similar across individuals. The different sample types used for different individuals is a concern.5. It is unclear how the mutational signature analysis might be relevant when the late-stage mutations come from a mixture of different tissues including brain, heart, liver, blood and others. Different tissues harbor different mutational tissues, please see PMID: 34433965 and 34433962. In addition, the number of early-stage mutations is small (94) for robust mutational signature analysis.6. The comparison of number and VAF of late-stage somatic mutations by tissue type should take into consideration the fact that the 3 groups of tissues are not age-matched. The is no data for older individuals for \u2018other organs\u2019.7. The brain tumors included in the study are glioblastoma and ganglioglioma. Glioblastoma is an adult tumor and ganglioglioma is a child tumor. Is it possible that the unique tumor mutational signature identified is the result of the mixture of mutations derived from these two very different brain tumors? If the ganglioglioma tumors were removed, would the mutations observed resemble to the landscape of mutations (driver genes) and mutational signatures reported for glioblastoma?8. Very low resolution of figures makes it impossible to read some of the content.Reviewer #2:\u00a0Hyeonju Son and associates analyzed mosacism for SNVs and indels by means of deep whole-exome sequencing in 498 tissues from 190 individuals. Their findings were consistent with those of previous studies: somatic mutations with apparent early embryonic origin were fewer in number but had higher variant allele frequencies than variants that appear to have arisen later in life. Mutational signatures among early embryonic and later-arising variants were similar but differed somewhat.This research is well described, and the paper is generally well written, although there are occassional errors of English usage. There are a few issues that require attention:Line 233: \"Early-stage mutations were not functionally tolerated than those of late-stage mutations.\" What does this mean? Are some words left out?Discussion: The authors should discuss the limitations of this study .Line 292: \"Subjects with refractory epilepsy, including focal cortical dysplasia and non-lesional epilepsy, and who had undergone epilepsy surgery were enrolled through the Severance Children\u2019s Hospital of Yonsei University College of Medicine.\" If these samples were obtained because of regional brain tissue excision for intractable epilepsy, could the mutations found have been responsible for the seizures? If so, might the frequency of variants and/or the VAF be biased to higher values in these samples? The same question could be raised in any other group of samples obtained by surgery to remove pathological tissue. This is an issue that the authors should discuss.Reviewer #3:\u00a0Throughout its lifetime, human body accumulates mutations, which despite affecting multiple body parts rarely cause a pathology. To provide a better grasp of spatiotemporal characteristics of this genetic mosaicism, the Authors of the manuscript performed deep whole exome sequencing of human tissues comparing the pattern of clone-forming mutations in various organs. In its focus on differences between early (pre-gastrulation) and late (post-gastrulation) mutations, this well-designed study addresses an interesting, and largely unexplored problem of developmental shifts in somatic mutagenesis and makes up an important contribution to current studies about somatic mosaicism.The results include the finding that most somatic mutations are produced after gastrulation and that diversity of late-appearing mutations is higher than of the early ones. However, it is not clear if the observed numerical differences between early and late clone forming mutations reflect distinct mutation rates during these developmental stages. More details should be also provided to elucidate whether differences in mutational signatures across tissues stem from the action of distinct  mutagens in these sites. They Authors should refer to recent studies by Li et al., Nature 2021, 597 (7876): 398-403, who also considered the problem of genetic mosaicism and compared the mutational patter across tissues.I see the value of the present manuscript in its potential to contribute to ongoing studies of clonal evolution and suggest that the Authors elaborate on how their work contributes to recent studies on this subject :239-256; Swiatczak, Cell Mol Life Sci, 2021 78(21-22):6797-6806; Laconi et al. 2020, Br J Cancer 2020, 122(7):943-952). For example, the Authors hint that variant allele frequency (VAF) can be used as a measure of a degree of clonal expansion of mutated cells in various tissues but draw no conclusions pertaining to the question of somatic evolution based on the observed differences between VAF in the brain, blood and other tissues.All in all, the present manuscript provides an important contribution to cutting edge research on genetic mosaicism, which is getting a lot of attention now, but extending the present research to incorporate the above questions could help to shed light on the mechanisms underlying the observed mutational profiles.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********what does this mean?). 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You will be contacted if needed following the screening process.PLOS has incorporated\u00a0To resubmit, you will need to go to the link below and 'Revise Submission' in the 'Submissions Needing Revision' folder.[LINK]Please let us know if you have any questions while making these revisions.Yours sincerely,Marshall S. Horwitz, M.D., Ph.D.Consulting Editor - PLoS GeneticsPLOS GeneticsGregory BarshEditor-in-ChiefPLOS GeneticsReviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1:\u00a0The authors have made a substantial effort to address my questions and concerns in the responses to reviewers and have added a very extensive paragraph about the limitations of the study in the Discussion section, which I appreciate. However, I believe the manuscript still lacks some of the necessary amendments.The first one relates to the problem of different ages for the different sample types, which is very obvious from Fig. 4C. The authors provided an analysis restricted to patients aged 50 to 75 in the responses to reviewers. I believe this data should be discussed in the text and included as a supplementary figure.The second issue relates to the selection of tissues for the study, which was done based on convenience. This leads to the \u201cother organs\u201d being a random mixture of tissues, which is still not well explained in the article, and to the brain tissue including a large proportion of samples from diseased tissue. The article would greatly benefit from more clarity on the types of samples included and from additional plots (which could be included as supplementary data) in which the authors further analyze the data to demonstrate that the choice of samples is not biasing the results. For example, in response to Reviewer #2, the authors indicate that the VAF of disease tissues is not different from the normal. All those comparisons should be clearly presented with dot plots in the article. Also, the authors have included this sentence in results: \u201cVAF of late-stage mutations was inversely correlated with age, probably due to the accumulation of late-somatic mutations in blood with a low allelic fraction with aging\u201d. This plot should be shown for all late-stage mutations and also for blood mutations only.Finally, the article still requires attention to the explanation of the figures and methods. What are the numbers in the bars of Fig. 1A? what do red and blue colors indicate? In Fig. 3B, how are the relative contributions calculated? In Methods, there are some sentences that are hard to understand. For example: \u201cIn the filtering process of paired-mode somatic calling using Mutect2, \u201cnormal artifact\u201d is corresponding to it; variants are non-germline mutation, differ from reference sequence, and contained in matched pair samples. \u201c Overall, the article would benefit from careful reading to improve some language issues and to make some of the explanations in methods and results clearer.Reviewer #2:\u00a0Hyeonju Son and associates have revised and substantially improved their analysis of somatic mosaicism using deep exome sequencing. They have adequately addressed all of my previous major concerns.There are still numerous errors of English grammar and word usage; this paper would benefit greatly from copy editing for English usage.Reviewer #3:\u00a0The manuscript has been much improved in terms of clarity as the Authors have identified the origin of tumor samples and provided essential details about the mutation calling methods. They also brought the importance of their findings into light by relating their research to other somatic mutagenesis and clonal evolution studies.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1:\u00a0YesReviewer #2:\u00a0YesReviewer #3:\u00a0Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article , and click on the Fetch/Validate link next to the ORCID field.\u00a0 This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.In the meantime, please log into Editorial Manager at If you have a press-related query, or would like to know about making your underlying data available , please see the end of this email. If your institution or institutions have a press office, please notify them about your upcoming article at this point, to enable them to help maximise its impact. Inform journal staff as soon as possible if you are preparing a press release for your article and need a publication date.Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Genetics!Yours sincerely,Marshall S. Horwitz, M.D., Ph.D.Consulting Editor - PLoS GeneticsPLOS GeneticsGregory BarshEditor-in-ChiefPLOS Geneticswww.plosgenetics.orgTwitter: @PLOSGenetics----------------------------------------------------Comments from the reviewers (if applicable):Reviewer's Responses to QuestionsComments to the Authors:Please note here if the review is uploaded as an attachment.Reviewer #1:\u00a0The authors have made a substantial effort to address my concerns, including the addition of a supplementary figure that increases confidence on the results. I find a bit unusual, however, that panels C to F of Supplementary Figure 1 are not presented in Results, but in Discussion. That said, I believe that the extra figures have improved the manuscript. Clarity is still an issue and the manuscript would greatly benefit from language editing.**********Have all data underlying the figures and results presented in the manuscript been provided?PLOS Geneticsdata availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.Large-scale datasets should be made available via a public repository as described in the Reviewer #1:\u00a0Yes**********what does this mean?). If published, this will include your full peer review and any attached files.PLOS authors have the option to publish the peer review history of their article , one way to make that data available is to deposit it in the\u00a0The following link will take you to the Dryad record for your article, so you won't have to re\u2010enter its bibliographic information, and can upload your files directly:\u00a0http://datadryad.org/submit?journalID=pgenetics&manu=PGENETICS-D-21-01628R2http://www.datadryad.org/depositing. If you experience any difficulties in submitting your data, please contact help@datadryad.org for support.More information about depositing data in Dryad is available at data availability policy\u00a0requires that all numerical data underlying display items are included with the submission, and you will need to provide this before we can formally accept your manuscript, if not already present.Additionally, please be aware that our\u00a0----------------------------------------------------Press Queriesplosgenetics@plos.org.If you or your institution will be preparing press materials for this manuscript, or if you need to know your paper's publication date for media purposes, please inform the journal staff as soon as possible so that your submission can be scheduled accordingly. Your manuscript will remain under a strict press embargo until the publication date and time. This means an early version of your manuscript will not be published ahead of your final version. PLOS Genetics may also choose to issue a press release for your article. If there's anything the journal should know or you'd like more information, please get in touch via  13 Sep 2022PGENETICS-D-21-01628R2 Analysis of low-level somatic mosaicism reveals stage\u00a0and tissue-specific mutational features in human development Dear Dr Kim, We are pleased to inform you that your manuscript entitled \"Analysis of low-level somatic mosaicism reveals stage\u00a0and tissue-specific mutational features in human development\" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.The corresponding author will soon be receiving a typeset proof for review, to ensure errors have not been introduced during production. Please review the PDF proof of your manuscript carefully, as this is the last chance to correct any errors. Please note that major changes, or those which affect the scientific understanding of the work, will likely cause delays to the publication date of your manuscript. Soon after your final files are uploaded, unless you have opted out or your manuscript is a front-matter piece, the early version of your manuscript will be published online. The date of the early version will be your article's publication date. The final article will be published to the same URL, and all versions of the paper will be accessible to readers.Thank you again for supporting PLOS Genetics and open-access publishing. We are looking forward to publishing your work! With kind regards,Anita EstesPLOS GeneticsOn behalf of:The PLOS Genetics TeamCarlyle House, Carlyle Road, Cambridge CB4 3DN | United Kingdomplosgenetics@plos.org | +44 (0) 1223-442823plosgenetics.org | Twitter: @PLOSGenetics"}
+{"text": "Over the last years it has been observed that the progress in data collection in life science has created increasing demand and opportunities for advanced bioinformatics. This includes data management as well as the individual data analysis and often covers the entire data life cycle. A variety of tools have been developed to store, share, or reuse the data produced in the different domains such as genotyping. Especially imputation, as a subfield of genotyping, requires good Research Data Management (RDM) strategies to enable use and re-use of genotypic data. To aim for sustainable software, it is necessary to develop tools and surrounding ecosystems, which are reusable and maintainable. Reusability in the context of streamlined tools can e.g. be achieved by standardizing the input and output of the different tools and adapting to open and broadly used file formats. By using such established file formats, the tools can also be connected with others, improving the overall interoperability of the software. Finally, it is important to build strong communities that maintain the tools by developing and contributing new features and maintenance updates. In this article, concepts for this will be presented for an imputation service. Data isThis article highlights current challenges and possible solution strategies in plant genotyping using the example of the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben. The transformation of genebanks into bio-digital resource centers  is a proThe advantages and effectiveness of this modular approach have been demonstrated by already finished and still ongoing research projects in the field of plant genetic resources. These include the creation of a homogeneous data space of plant genetic resources stored in genebanks and their web-based interactive exploration , the digEspecially genotyping and imputation as a subfield in particular, highly depends on the quality of the underlying RDM. So, based on the experience from these projects, an eye is cast on a current workflow for imputation of genotypic data. In particular, data for imputation is currently stored locally and processed sequentially on virtual machines. With a transition to a cloud-based, distributed solution, the efficiency as well as sustainability is expected to be increased significantly. In the following, the individual services involved are described, before explaining the current workflow in detail. Afterwards, suggestions are made to modernize the current imputation services as well as discussing the benefits and newly arisen challenges. This is enabled and accompanied by the modular approach and the specification of well-defined interfaces as well as the usage of standardized formats. Finally, it is shown how further synergies can be generated through integration into international as well as national programs such as the National Research Data Infrastructure (NFDI). In particular, the IPK is involved in the consortium for biodiversity research NFDI4Biodiversity, where one goal is to establish a cloud computing infrastructure with the Research Data Commons (RDC) to provide analysis services.2IPK\u2019s RDM strategy aims to support a federated scenario of research groups under the umbrella of a central service portfolio to support all steps of the RDM data life cycle , 20, 21.the Research Data Management Organiser (RDMO)  as a quethe LIMS to support data collection in the labs, greenhouses and in the field,the data pre-processing according to common guidelines andthe provisioning of staging areas, a Galaxy instance to stage commonly used data analysis pipelines and a Slurm cluster for individual software pipelines,sharing through an ORACLE Application Express  for decldata sharing through data steward-based data publishing pipelines in central repositories, such as EMBL-ENA, EMBL-EVA or e!DAL-PGP , and lasthe collaboration and dissemination of international standards for data and metadata, such as MIAPPE , BioScheThe seven steps of the research data lifecycle  are fulfIn this section, the focus will be on the three steps Store, Share and (Re)Use of the data lifecycle. The selection refers to outcomes from various projects with IPK participation that are connected to the genotyping domain and are related to the use case of imputation. However, the scope could be extended further, but will be limited for the purposes of this article to the aspect of released or ongoing developments from IPK or community developed outcomes with IPK contribution.2.1As a generic data management backend, a stack of a highly available commercial relational database management system (RDBMS) and a file storage appliance, a hierarchical storage management system (HSM), is operated by core funded IT to host all IPK research data.The actual implementation of project specific data structures is in majority brokered by an institute-wide available laboratory information management system (LIMS) for structured data and an electronic laboratory book (ELN) for the documentation of experimental work . The comThe European Variation Archive (EVA) , which iA submission to EBI-EVA requires a valid Variant Call Format (VCF)  file thaThe recommended workflow for a FAIR compliant genotyping dataset submission: [if not yet available: (0) Submit reference assembly to INSDC public repository], (1) Registering sample metadata at EBI-BioSamples, (2) Submission of raw read files at EMBL-ENA by using previously registered Biosamples records, (3) Prepare, validate and submit VCF file; previous ENA run IDs obtained from step 2 should be linked to the EVA study; BioSample IDs from step 1 are re-used as genotype names.2.2One option to share data is by using files in established file formats. For genotypic diversity data, the Variant Call Format (VCF)  allows tIn addition to the above-mentioned sharing of data by using files, the same can also be fulfilled via domain-specific APIs. The Breeding API (BrAPI)  is a com2.3To analyze the diversity of various genotypes the method imputation is the attempt of completing sparse SNP data. The concept is based on the idea that unrelated samples in small regions are identical by descent (IBD). The differences between samples can be attributed to recombination of the originally underlying chromosomes. The goal of an imputation is now to identify those original haplotypes of the target haplotypes from a reference panel and to fill the gaps of the samples with the markers of the reference. In the following, reference markers are understood to be those markers that occur in the reference panel. Similarly, target markers are those from the target set and imputed markers are those that are not set in the target set and are taken from the reference panel . Target Imputation enables and enhances several variant data analysis methods. For example, it enables the prediction of missing data, which has a positive impact on data analysis of only variant data. However, imputation also strengthens the expressiveness of inter-domain analysis methods, such as Genome Wide Association Studies (GWAS). It has been shown that the use of imputed data gives a 10% better result than an GWAS analysis on the raw data . ImputatVarious algorithms are available to perform imputation. Most of them are based on Hidden Markov Models (HMM), but also on positional Burrows-Wheeler transformation or SNP-tagging approaches. HMM approaches have better accuracy, since the whole chromosome, as well as all mosaic configurations of the haplotype are considered. Among the most used methods are IMPUTE , FastPHATo visualize and analyze the genotypic diversity data before and after the imputation, an inhouse developed web tool called DivBrowse  is used.33.1The workflow for the imputation service, as depicted in 3.2Two different options are offered for the imputation service. On the one hand, it is possible to impute only on the target set, on the other hand via a reference panel. The reference panel are dense variant calls that are used to fill the gaps in the target set. Here it is important that there is an intersection between the markers in the reference panel and the targets. For the reference panel, a sequence of steps is provided as a script for a minimal quality control, which is based on steps suggested in . This enThe preferred process is the imputation on a reference panel. To ensure that the chromosome, position and reference as well as allele nucleotides are identical, an additional program is provided. If there is no reference panel available the Beagle software can be run without one, imputing the missing data from the other samples of the target set.3.3The described workflow was tested on a dataset to obtain a runtime comparable to the proposed improvements. For this test, a dataset consisting of 8070 wheat samples with a total of over 1.6\u00a0M unphased variants was used. The data set is submitted to the EMBL-EVA repository with the project identifier PRJEB52759. The imputation was performed on this data on a virtual machine in the de.NBI Cloud  that has3.4Following imputation, the resulting VCF can be sent back to the local machine, so that it is available for further processing. Possible uses have already been mentioned in the previous section and, as far as they exceed the domain of genotypic data, will not be discussed further. A direct use case solely on the imputed data would be to load the resulting VCF file into a DivBrowse instance for visualization and analysis.4To enable the best user experience for a cloud based imputation service, we implemented a handy data import and optimised the performance using a distributed, cloud based execution pipeline. This improved on one hand, the user-friendliness, on the other hand, the runtimes of the individual imputations. The following sections outline additional possible future steps to be taken into those directions.4.1A potential hurdle in usability is that the imputation service runs on a virtual machine that is controlled via the command line. The user then executes a number of scripts in a predefined order with individualized parameters, thereby semi-automatically managing the jobs. This whole process can be simplified by providing a web interface. Such an interface should allow the user to (1) submit the target data, (2) change the reference, (3) start and manage jobs, (4) monitor currently running jobs, and (5) request the imputed data. This spares the user the need to use the command line and familiarize themselves with the parameters of the scripts. In addition, this automation of job submission and management would minimize the risk of user-caused errors in the workflow.Furthermore, the service can be connected to API-based data sources, which allow the data set to be pre-filtered by requesting only a small slice rather than the entire data set. By making several requests, either a merged VCF file can be generated, or the individual regions can be stored as independent VCF files. This would depend on the further processing. With DivBrowse, a BrAPI endpoint is offered for genotypic data, as described before. In order to be able to use this source, a consumer  is devel4.2Bottleneck for the imputation on a single node is the construction of the HMM for the regions on the dataset. By running the imputation as described in the previous section, those regions get imputed sequentially without any parallelization due to hardware limitations. However, by simply extending the environment to a multi-node setup and running independent imputation jobs for the different chromosomes in parallel, the run-time will decrease significantly. Furthermore, it is possible to run independent jobs in parallel for genomic windows on the chromosomes based on the identical by descent (IBD) segments that can be identified beforehand, decreasing the run-time even more. In DivImpute, the parallelization by chromosome-based job batches is already available, the imputation on genomic windows is yet to be implemented.To reduce the work the user has to put in to start the imputation, one can decide to use workflow languages like Common Workflow Language (CWL)  or nextfWith SparkBeagle  there ex4.3To test the procedure, the imputation of the test data set from the previous section was repeated. This time, however, the environment was extended by 2 additional virtual machines. Firstly, a head node was created, which is smaller in specification and is only used to access the computational nodes and manage the jobs. This node has 4 VCPUs, 4\u00a0GB RAM and 20\u00a0GB disk as well as 2\u00a0TB additional volume storage. This head node has access to 2 computational nodes. Both of these nodes are identical and have the same specifications as before in the single node test run.In order to start the imputation, the data set was first divided according to chromosomes in the head node and then distributed batch wise to the computational nodes. The batches are based on the three sub genomes of wheat, so that the A, B and D genomes were imputed separately. The A and B genomes were imputed in parallel, and the D genome as soon as one node finished imputing either the A or B genome. The A genome was imputed in almost 128\u00a0h and the B genome in almost 182\u00a0h. When the imputation of the A genome was finished, the D genome jobs were started, taking roughly 69\u00a0h. The significant differences in imputation time for the three sub genomes can easily be explained by taking a look at the number of markers for the different chromosomes. Comparing the number of markers for chromosome 7 of all sub genomes reveals that with 58k markers the D genome has almost half the number of markers of A (ca. 101k) and B (ca. 110k) genomes cf. . The tes4.4In summary, some advantages and disadvantages can be derived for the described concept. In addition to the improvements in usability, the acceleration or the reduction of the runtime of the imputation is a main advantage. Moreover, this is expected to happen without a loss of accuracy of the results. In addition, the introduction of modern infrastructure solutions such as Spark and Kubernetes allow jobs to be automated and the workload to be distributed among the computational nodes. This can reduce the runtime even further. By running ready-to-use solutions such as SparkBeagle, the management of merging the individual partial results, as well as cleaning up the executor is implemented in a fault tolerant way. In general fault tolerance is ensured by the usage of for example Kubernetes, as pods will be rescheduled, when a node is lost. In the current solution a node fault will result in the processes to be lost and need to be started again manually.However, by using ready-to-use solutions, one is less flexible and limited in the decisions and customization of the system. SparkBeagle requires Hadoop as well as Hadoop Distributed File System. Therefore, one would need to deploy as well as maintain those additional systems. Besides technical requirements, such solutions also limit the usage of the imputation software, as SparkBeagle seems, based on the documentation, to not support imputation solely based on the target set, but only based on a reference panel. Therefore, reducing the flexibility of the service. Furthermore, it seems the active development of SparkBeagle has been stopped, showing \u2013 with stopped maintenance \u2013 another disadvantage of ready-to-use solutions. Apart from using ready-to-use solutions, the introduction of Kubernetes and Spark require one to coordinate the complex interplay of the additional components, maintain their functionality as well as introduce security policies on those systems. One way of dealing with this, is to rely on specialized services providing such environments in a platform-as-a-service (PAAS) style, which allow for an easier and faster deployment of such an imputation service.5In this article, a use case for the analysis of genotyping data in the field of crop plant research has been described. Here, the transfer of analyses pipelines from local, institutional IT systems to external distributed infrastructures has been presented. As an exemplary application, the research field of genotyping with the subfield of imputation was considered. For an research institute like the IPK Gatersleben, which holds over 150,000 different samples of cultivated plants in its genebank, their development and activation is an important task and challenge in order to further develop the IPK Genebank in particular as well as the whole IPK in general into a bio-digital resource center. Currently, these genebank samples are systematically genotyped in many projects including following publication of the results in scientific articles and the provision of the data sets regarding the FAIR criteria in international repositories, in information systems hosted at IPK and accessible via the web as well as comprehensive data publications. Further analysis and integration of this published information with datasets from interesting users in the scientific community is currently one of the greatest challenges. Thus, the easy reuse of the research data is currently in the focus.Hence, the availability of research data that meets the FAIR criteria is thus increasingly fulfilled. This is achieved by consistently applying metadata standards for description, by offering the data in standard formats, and by providing access via standardized APIs. But how can reuse of the research data be further advanced? The current task is to build up infrastructures, offer them sustainably and operate them permanently. A single research institute cannot fulfill this task alone, because the necessary resources are not available locally or cannot be acquired.In the area of life sciences, a decentralized, federated infrastructure was established with the mentioned de.NBI network, which is currently being sustained. One component of de.NBI is the de.NBI cloud, which allows the scientific community to perform its analyses in data centers distributed all over Germany. Thereby, the offered compute resources can be used with publicly available as well as own data. The de.NBI cloud was used to compute the imputation described in the use case in this present article.It was recognized that the development to build sustainable infrastructures needs to go further. In Germany, for example, the National Research Data Infrastructure (NFDI) has been under establishment since 2020. Here, nearly all scientific domains far beyond the life sciences are working together to offer sustainable solutions so that research data can be published in accordance with the FAIR criteria, remain accessible and also be reused. Through this collaboration across all scientific disciplines, it is expected to exploit appropriate synergies in building and operating the infrastructures to achieve sustainability more effectively. This concept is called the Research Data Commons (RDC).The IPK Gatersleben is contributing actively to the NFDI in the consortia NFDI4Biodiversity and FAIRagro and is working together with NFDI collaborators to deploy and sustain the imputation use case described in this article into the NFDI RDC infrastructure as one part of Plant Data Commons."}
+{"text": "Staphylococcus aureus (S. aureus) is a worldwide leading versatile pathogen that causes a wide range of serious infections. The emergence of antimicrobial resistance against S. aureus resulted in an urgent need to develop new antimicrobials in the new era. The methicillin-resistant S. aureus (MRSA) prevalence in hospital and community settings necessitates the discovery of novel anti-pathogenic agents. Staphyloxanthin (STX) is a key virulence factor for the survival of MRSA against host innate immunity. The current work aimed to demonstrate the anti-virulence properties of meloxicam (MXM) as compared to diclofenac (DC), which was previously reported to mitigate the virulence of multidrug-resistant Staphylococcus aureus and test their activities in STX production. A total of 80 S. aureus clinical isolates were included, wherein a qualitative and quantitative assessment of STX inhibition by diclofenac and meloxicam was performed. The quantitative gene expression of STX biosynthetic genes  and hla (coded for \u03b1-hemolysin) as a virulence gene with and without DC and MXM was conducted, followed by molecular docking analysis for further confirmation. DC and MXM potently inhibited the synthesis of STX at 47 and 59 \u00b5g/mL to reach 79.3\u201398% and 80.6\u201396.7% inhibition, respectively. Treated cells also revealed a significant downregulation of virulence genes responsible for STX synthesis, such as crtM, crtN and global transcriptional regulator sigB along with the hla gene. Furthermore, computational studies unveiled strong interactions between the CrtM binding site and DC/MXM. In conclusion, this study highlights the potential role and repurposing of DC and MXM as adjuvants to conventional antimicrobials and as an anti-virulent to combat MRSA infections. Staphylococcus aureus (S. aureus) is one of the most predominant Gram-positive bacteria responsible for a wide range of bacterial infections, including bacteremia, pneumonia, osteomyelitis, skin infections, and heart valve infections  \u00d7 100 [The STX extraction and quantification was performed according to Al-kazaz et al. . The mosm] \u00d7 100 .v/v [The change in proteolytic activity of MRSA isolates was evaluated using skimmed milk agar. The skimmed milk agar was prepared by adding fresh skimmed milk to sterile molten nutrient agar  to a final concentration of 5% v/v . Overnigv/v . This asThe nuclease activity alterations were detected using DNase agar  according to Lagac\u00e9-Wiens et al. with some modifications. Overnight cultures of treated (with sub-MICs of the tested drugs) and untreated MRSA isolates in TSB were centrifuged at 6000 rpm for 10 min, then the supernatants  were injected into cups made in DNase agar plates. The DNase agar plates were then incubated for 24 h at 37 \u00b0C . The clehttp://insilico.ehu.es/mini_tools/discriminatory_power/) (accessed on 10 December 2022). The similarity index (Jaccard/Tanimoto Coefficient and number of intersecting elements) between all samples was calculated using an online tool  (accessed on 10 December 2022) [Genotyping of the MRSA isolates was performed by Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) using a pair of published primers (F: 5\u2032-ATG TAA GCT CCT GGG GAT TCA C-3\u2032) (R: 5\u2032-AAG TAA GTG ACT GGG GTG AGC-3\u2032) . The PCRer 2022) .mecA gene using a pair of primers (F: 5\u2032-GTA GAA ATG ACT GAA CGT CCG ATA-3\u2032) (R: 5\u2032-CCA ATT CCA CAT TGT TTC GGT CTA A-3\u2032) [The genotypic identification of the MRSA isolates was conducted by conventional PCR for detection of TA A-3\u2032) . The PCRTA A-3\u2032) .crtM and crtN, and those involved in virulence, such as hla  and sigB . Total RNA was extracted from DC/MXM treated and untreated isolates after 24 h of incubation by the Trizol method. Conversion into cDNA and qPCR analysis was performed in a 1-step reaction using GoTaq\u00ae 1-Step RT-qPCR System A6020  as per the manufacturer\u2019s protocol. The qPCR data were analyzed using QuantStudio 5 . Expression of the selected genes was quantified by 2(\u2013\u0394\u0394Ct) method after the normalization of cycle threshold (Ct) values of all tested genes to that of the housekeeping gene 16srRNA [The qPCR analysis was performed to assess the effect of DC/MXM treatment on selected genes encoding for STX biosyntheis in MRSA, such as  16srRNA . The priMolecular docking was performed to assess the binding energy and interactions of DC and MXM with CrtM of MRSA. The 3D crystal structure of CrtM (ID: 2ZCQ) was downloaded from the Protein Data Bank. The 3D structures of the target compounds used in the study were sketched using the BIOVIA Discovery Studio v 4.5 program . The docking step was performed using the cDOCKER protocol, in which the scoring system was based on the cDOCKER energy. The 3D and 2D structures were visualized through BIOVIA Discovery studio visualizer 2016 v16.1.0.15350 [p \u2264 0.05. Statistical analysis was performed with IBM SPSS Statistics for Windows, Version 23.0. Armonk, NY, USA: IBM Corp.Numerical data were explored for normality by checking the distribution of data and by using tests of normality (Kolmogorov\u2013Smirnov and Shapiro\u2013Wilk tests). All the experiments were carried out in triplicates. Data were presented as median, range, mean and standard deviation (SD) values. The repeated measures ANOVA test and Mann\u2013Whitney U test were implemented. The significance level was set at sigB and other virulence genes such as the hla gene. Genotypic analysis was in accordance with phenotypic analysis, with no significant difference between the anti-virulence potential of both drugs. Thus, the present study proposes the repurposing of DC/MXM as adjuvants to conventional antimicrobial treatments against serious MRSA infections. The current study unveiled the anti-virulence potential of DC/MXM treatment against MRSA. Both drugs revealed significant STX inhibition that was further confirmed by molecular docking analysis, emphasizing the strong interaction between the CrtM binding site and DC/MXM. Additionally, real-time PCR analysis highlighted the efficiency of the proposed treatment in downregulating the expression of genes responsible for STX synthesis, along with the transcriptional regulator gene"}
+{"text": "The development of Fricke gel (FG) dosimeters based on poly (PVA) as the gelling agent and glutaraldehyde (GTA) as the cross-linker has enabled significant improvements in the dose response and the stability over time of spatial radiation dose distributions. However, a standard procedure for preparing FG in terms of reagent concentrations is still missing in the literature. This study aims to investigate, by means of spectrophotometric analyses, how the sensitivity to the radiation dose and the range of linearity of the dose\u2013response curve of PVA-GTA-FG dosimeters loaded with xylenol orange sodium salt (XO) are influenced by ferrous ammonium sulphate (FAS) and XO concentrations. Moreover, the effect of different concentrations of such compounds on self-oxidation phenomena in the dosimeters was evaluated. PVA-GTA-FG dosimeters were prepared using XO concentrations in the range 0.04\u20130.80 mM and FAS in the range 0.05\u20135.00 mM. The optical absorbance properties and the dose response of FG were investigated in the interval 0.0\u201342.0 Gy. The results demonstrate that the amount of FAS and XO determines both the sensitivity to the absorbed dose and the interval of linearity of the dose\u2013response curve. The study suggests that the best performances of FG dosimeters for spectrophotometric analyses can be obtained using 1.00\u20130.40 mM and 0.200\u20130.166 mM concentrations of FAS and XO, respectively. The final concentration of radiation-induced Fe3+ ions is proportional to the energy deposited by ionizing radiation in the dosimeter, i.e., the absorbed dose. Three-dimensional (3D) spatial information on the absorbed dose is obtainable within the gel volume, and it can be captured and retrieved by a suitable readout technique ..3+)-(XO)3+ and XO concentrations [3+ ions or XO favors the formation of the (Fe3+)2-(XO) complex or the (Fe3+)-(XO)2 complex, respectively. Upon Fe3+ binding, the yellow-orange color of FG dosimeters loaded with XO changes to violet, allowing us to point out the formation of the complex in the visible range. In fact, the (Fe3+)2-(XO) and (Fe3+)-(XO) complexes present an absorption band in the range of approximatively 500\u2013620 nm, while the (Fe3+)-(XO)2 complex absorbs light at a shorter wavelength in the spectral region overlapping the tail of the main absorption peak of the free XO at 430 nm [The probability of each complex\u2019s formation depends on the Fetrations . For exatrations ,62 that t 430 nm .2+ in the dosimeters.Examples of \u0394OA spectra of XO-PVA-GTA-FG dosimeters prepared with an XO concentration of 0.200 mM and two different FAS concentrations , independently of the employed gelling matrix see .3+ ions produced by self-oxidation phenomena and the formation of (Fe3+)2-(XO) and (Fe3+)-(XO) complexes. Indeed, such complexes are characterized by a main OA peak at 585 nm [3+-(XO)2 complexes are expected to be the major species. Such complexes absorb light at a shorter wavelength [An absorption band centered at approximately 585 nm can be also observed in the OA spectra of the dosimeters prepared with very low XO concentrations . This peak can be explained by the presence of Fet 585 nm . For higvelength , i.e., iExamples of \u0394OA spectra of XO-PVA-GTA-FG dosimeters prepared with an FAS concentration of 0.40 mM and different XO concentrations and irradiated at various doses are shown in 3+)-(XO)2 is predominant, presenting an absorption peak under 500 nm. When decreasing the concentration, the main complex becomes the 1:1 (Fe3+)-(XO) complex with a peak at about 585 nm in an acidic medium. However, the complex (Fe3+)2-(XO), prevailing when the iron concentration is higher than the XO concentration, also shows an absorption peak at the same wavelength [Only the spectral region of interest for dosimetric purposes  was considered. It is worth noting that the boundary of the absorption region strictly depends on the XO concentration. In fact, the shape of the \u0394OA spectra of the dosimeters prepared with the highest XO concentration of 0.800 mM a was difvelength ,63. Thus3+)-(XO)2 complexes make a greater contribution than the other ones due to the effect of the availability of XO molecules that can be bounded with radiation-induced ferric ions. Consequently, in the samples with the highest XO concentration the absorption band related to (Fe3+)2-(XO) and (Fe3+)-(XO) that peaked at 585 nm appeared to only be a shoulder of the main absorption band that peaked at a lower wavelength and was related to the (Fe3+)-(XO)2 complexes [Actually, the shape of the \u0394OA spectra of omplexes .A similar shape was observed for the \u0394OA spectra of the samples prepared with a XO concentration of 0.400 mM, but only for doses \u226414 Gy b.Actually, the relative ratio between the concentration of xylenol orange and the concentration of ferric ions in complexes with different stoichiometric ratios (and consequently their absorption bands) depends on the dose, i.e., on the concentration of ferric ions produced in the dosimeters after exposure to ionizing radiation ,59.The samples with XO concentrations of 0.200 mM and 0.166 mM c,d were The observed saturation effects of the response of these dosimeters were attributable to the low concentration of XO molecules that can be bounded with the radiation-induced ferric ions.A thorough analysis of the dose\u2013response curve of the XO-PVA-GTA-FG dosimeters prepared with an FAS concentration of 0.40 mM and different XO concentrations is shown in For the samples with an XO concentration ranging from 0.080 mM to 0.800 mM, straight lines were fitted to the experimental data. The results of the fit parameters, together with details of the dose interval considered for the fitting procedure, are given in The slope values of In addition to the use of the cumulative \u0394OA, dose\u2013response curves similar to those of A straight line was fitted to each dose\u2013response curve and the sensitivity to the radiation dose  for each sample at each individual wavelength was accordingly obtained.The complete results of the wavelength-dependence of the sensitivity to the radiation dose for XO-PVA-GTA-FG dosimeters prepared with different XO concentrations are shown in The results of the analysis of the dose\u2013response curves of XO-PVA-GTA-FG dosimeters prepared with different FAS and XO concentrations indicate that the use of an FAS concentration in the interval 0.40\u20130.60 mM, coupled with the use of an XO concentration in the interval 0.133\u20130.200 mM, guaranteed satisfactory dosimetric properties of the samples both in terms of sensitivity and linearity .The dose\u2013response curves of these samples are plotted in i and that obtained at the time t0.Besides the optimization of FAS and XO concentrations to guarantee an adequate level of sensitivity and a wide range of linearity, the effects of such compounds on the self-oxidation features of XO-PVA-GTA-FG dosimeters were investigated. The results suggest that the self-oxidation rate did not significantly depend on the XO concentration when an FAS concentration of 0.40 mM was used b. SimilaA systematic study on the effects of variation in ferrous ammonium sulfate (FAS) and xylenol orange (XO) concentrations on the dosimetric properties of Fricke gel dosimeters prepared with poly (PVA) cross-linked by glutaraldehyde (GTA) was carried out. The investigated properties concerned the dose\u2013response curves , the self-oxidation rate, and the level of self-oxidation.From the outcomes achieved in this study, some conclusions can be drawn about the behavior of the tested XO-PVA-GTA-FG dosimeters. Firstly, increasing the FAS concentration does not significantly increase the absorbed dose\u2013optical response range, nor does it increase the optical sensitivity. However, a more pronounced level of self-oxidation was noticed; thus, an increase in the FAS concentration tends to decrease the temporal stability. On the other hand, lower FAS concentrations reduce the dosimeter\u2019s response range. However, there was no evidence of variations for the optical sensitivity. Furthermore, it was found that the XO concentration is the main factor responsible for the limited absorbed dose response.Starting from these considerations, the experimental data were in line with the literature data on traditional and natural gel matrices. In particular, 1.00\u20130.40 mM and 0.200\u20130.166 mM are the optimal intervals of FAS and XO concentrations, respectively, to be used in the preparation of dosimeters in order to maximize their performances in the case of spectrophotometric analyses.The results obtained in this paper allow us to begin a new investigation on the possibility of improving the dosimetric stability of the FG by adding alternative chelating agents and/or antioxidants, such as sulfosalicylic acid (SSA), methylthymool blue sodium salt (MTB), ethylenediaminetetraacetic acid (EDTA), and dimethylsulfoxide (DMSO)."}
+{"text": "In December 2019, coronavirus disease 2019 spread worldwide, causing acute respiratory distress syndrome. Coronavirus disease 2019 presents from an asymptomatic infection to severe disease causing multiorgan failure. Neurological manifestations were observed in some patients, including intracerebral hemorrhage. Bilateral basal ganglia hemorrhage is rare due to trauma.Our patient was a 14-year-old\u00a0Iranian boy with multiple trauma and loss of consciousness who tested positive for coronavirus disease 2019. The brain computed tomography scan reported bilateral basal ganglia hemorrhage. Bilateral ground glass opacity was reported through a chest computed tomography scan.In this study, we reported a 14-year-old boy referred to the emergency room due to multiple trauma. Through the medical interventions, bilateral basal ganglia hemorrhage was discovered incidentally. Coronavirus disease 2019 was detected in this patient on the basis of findings in chest computed tomography scan and positive real reverse transcription polymerase chain reaction test. Several clinical reports and series exploring the relationship between coronavirus disease 2019 and ischemic strokes have been published. Coronavirus disease 2019, like other acute respiratory syndromes, can invade the central nervous system through hematogenous and neuronal dissemination or it can be an immune response to the cytokine storm. In conclusion, it is vital to know the pathophysiology of the neurological manifestations of coronavirus disease 2019 and prevent the mild neurological manifestations leading to severe conditions. Severe acute respiratory syndrome\u2013coronavirus-2 (SARS\u2013CoV-2) caused coronavirus disease 2019 (COVID-19) in Wuhan, China in December 2019. COVID-19 is a contagious respiratory disease spreading worldwide . Intracr2 saturation was 93%, temperature was 38.5\u00a0\u00b0C, and the other vital signs were stable. In the ER, his Glasgow Coma Scale (GCS) score was 3/15 (E1V1M1). No free fluid in the abdomen and pelvic cavity in focused assessment with sonography for trauma was reported. A right bundle branch block was observed in his electrocardiogram (ECG). Echocardiography was performed and reported as normal. He was then referred to the neurosurgery ward and was hospitalized for almost 40\u00a0days.The case is a 14-year-old Iranian boy with no past medical history presenting with multiple trauma from a motorcycle accident who was transferred to the Poursina hospital emergency room (ER) in Rasht on 9 June 2020. In addition, he had an extensive laceration on his right knee and a star-shaped laceration on his parietal region. OHe had no signs and symptoms related to COVID-19. However, during hospitalization, spiral high-resolution computed tomography (HRCT) scan of his lung without contrast revealed patchy ground-glass opacity in both lungs (mostly in the right lung) .In this study, we reported a 14-year-old boy referred to the ER due to multiple trauma. He had no history of coagulopathy or anticoagulant consumption. In this case, trauma precipitated parietal contusion. Through medical interventions, bilateral basal ganglia hemorrhage was discovered incidentally.Several clinical reports and series exploring the relationship between COVID-19 and ischemic strokes have been published .Neurologic symptoms in COVID-19 can be divided into several categories, ranging from nonspecific symptoms such as a headache to severe forms such as cerebrovascular disease .Basal ganglia hemorrhage due to traumatic events is rare .Systemic and metabolic disease, neurodegenerative disease, and vascular disease can involve basal ganglia and thalamus , 9.COVID-19, like other acute respiratory syndromes, can invade the central nervous system (CNS) through hematogenous and neuronal dissemination, or it can be an immune response to the cytokine storm , 11.There is not enough data about the underlying mechanisms of neurological disorders caused by COVID-19. A total of 2% of patients have neurological symptoms. One of the ways in which COVID-19 can invade the neurological system is through the mucous layer of the nose that contains the olfactory receptor cells. Olfactory nerves might deliver viruses and infections to the brain. On the other hand, COVID-19 causes respiratory infection and the level of oxygen in blood may decrease. Brain function can be impaired through hypoxemia Fig.\u00a0 12].Fig.Fig12].FSome other pathophysiology of ischemic stroke in COVID-19 are hypoperfusion of the brain, hypertension leading to posterior reversible encephalopathy syndrome, and septic embolization in patients with bacterial superinfection Fig.\u00a0 13]..13].ACE2 serves important roles in various parts of the body, such as the lung, kidney, brain, liver, and endothelial cells. COVID-19 decreases the expression of ACE2. Due to reduced ACE2 levels, the renin\u2013angiotensin\u2013aldosterone system (RAAS) will deteriorate and cause multiorgan damage. Diminished ACE2 can cause intracerebral hemorrhage through several mechanisms. For instance, increasing the blood pressure, endothelial dysfunction, damage to the brain vessels, and finally depression of angiotensin-(1-7) and Mas receptor [Ang (1-7)/MasR] signaling, which has a neuroprotective, antifibrotic, and vasodilatory effect , 15.Immune response to the cytokine storm and chemokines can disrupt the blood\u2013brain barrier and neuroinflammation. The breakdown of the blood\u2013brain barrier and inflammation both have a main role in ischemic stroke , 17.Intracranial hemorrhage was documented in some cases as a complication of COVID-19. Some risk factors are arterial hypertension and anticoagulation therapy (pretreatment of unrelated disease to COVID-19 thromboprophylaxis) .Hyperinflammatory state may be the main cause of complication in patients with diabetes mellitus (DM) as well as COVID-19. A case series was reported in October 2020 that included three patients with COVID-19 and ICH. All three patients had risk factors for ICH such as hypertension and DM. DM and ICH are associated through some mechanisms. High blood glucose can cause endothelial dysfunction. Dysfunction of the brain\u2019s small vessel endothelial can lead to ICH .There is a hypothesis that the state of hyperinflammation in diabetic patients is related to higher mortality in these patients . High C-We reported a 14-year-old boy with bilateral basal ganglia hemorrhage and documented COVID-19. Our patient had no special past medical history of hypertension, DM, and other diseases. There is no evidence that he consumed anticoagulant agents before. There is some research that ICH is propounded as a complication of COVID-19. Basal ganglia hemorrhage related to trauma is rare .The underlying pathophysiology for COVID-19 related ICH can involve hyperinflammatory state due to increased cytokine production. Although a few case series and case reports were published on some patients COVID-19 as well as ICH, more investigation and research regarding the association between COVID-19 and ICH are needed.It is essential to discover the pathophysiology of neurological manifestations of COVID-19. Neurological findings of COVID-19 range from mild conditions such as headache to severe conditions such as stroke. Therefore, acknowledgement as to the causes and prevention of the situations leading to critical events should be considered in treatments of COVID-19."}
+{"text": "The idea of EAE  is put forward on the basis of the mature development of ecological aesthetics and AE theory. Starting with EAE, establishing people's aesthetic attitude and improving people's spiritual realm will help to reverse people's hostile attitude towards nature and rebuild the harmonious relationship between man and nature. This paper studies the application optimization of AE (aesthetic education) resources in universities based on ML (machine learning) from the perspective of resource development. The recommendation algorithm based on ML is the main idea of the classification of AE resources, and the classification model of AE resources is constructed. Through deep learning, we can learn the effective features of items from the content data in advance and then transform the learned features into CF (Collaborative Filtering) target learning task. Optimize the voting mechanism of the algorithm, and compare the RF_VM (random forest with optimized voting mechanism) algorithm with the traditional RF (random forest) algorithm. Experiments show that the algorithm proposed in this paper can effectively classify texts and has high feasibility. The core of basic education reform is curriculum reform, and AE is an aspect that cannot be ignored in curriculum reform. The development and utilization of AE curriculum resources is an important part of AE. The essence of EAE  is AE with vivid ecological beauty, which exerts a subtle influence on college students, which is unmatched by other ecological education and AE forms . It shouThe smooth implementation of education requires a well-designed school curriculum. Process is a means and tool for achieving educational and teaching objectives, as well as an important link in determining educational quality . AE  \u201317 is thStein et al. conducted a study to eliminate heterogeneous influence by removing sensitive attributes from the decision-making process and adding fairness constraints to avoid heterogeneous processing. Observe that the standard fairness constraint is nonconvex; then use covariance to turn the nonconvex problem into a convex problem. Finally, measure the output results and sensitive attributes of parameters to investigate the sensitive attributes of multiple classification and analysis. Some scholars looked at the fairness metrics of a variety of algorithms to see how they correlated with different definitions and discovered that they were very closely related. The transformed data retains most of the characteristic signals of insensitive attributes, intersection-sensitive attributes are proposed, and the effects of two sensitive attributes do not overlap . Nguyen Ecological problems are closely related to people, and ecological imbalance is the deviation of people's ideas and consciousness. To solve ecological problems, we must first solve the problem of people's ideology, which is actually a problem of people's education. AE aims to establish people's aesthetic attitude and create aesthetic living conditions, as an education to improve people's living standards. Starting with AE, it is of great significance to establish people's aesthetic attitude for solving ecological problems and improving people's living conditions, and EAE is still a new field of AE in China.EAE's mission is to educate and infect people through art and various forms of EAE so that they can achieve true enlightenment, good education, and emotional edification in a pleasant spirit and gradually develop people's ecological aesthetic sensibility and consciousness. Realize the survival of ecology and aesthetics and perform a one-of-a-kind and irreplaceable role. On the one hand, we should focus on infecting and educating people about the aesthetic method of perceptual freedom so that they can consciously educate beauty or \u201cgive freedom with freedom.\u201d On the other hand, understanding the natural laws of the ecosystem should be a priority in the implementation of EAE so that people can correctly apply the natural laws and realize the harmonious unity between man and nature. EAE imbues educated people with a sense of beauty and encourages them to pursue and yearn for ecological beauty. Colorful ecological landscapes, endless back and forth, and endless phenomena make people feel a new rhythm of life, ideal living conditions, and a stirring sense of life, which makes them indulge and miss. Ecological beauty education will have a positive impact if we can make conscious use of these resources and educate people about ecological beauty.The difference of EAE resources in schools is caused by the imbalance of social economy, the difference of management system and supply mode, and the information asymmetry of social demand for talents. The difference of school EAE resources constitutes the difference of school EAE process and effect. The diversity and complexity of school EAE resources determine the instability of education resources, among them are human factors, material factors, policy orientation, development and changes of social and economic conditions, and other factors. Its mobility is mainly manifested in the flow of teachers' resources and students' resources and funds.The aesthetic course of university is mainly realized through aesthetic activities, so the appreciation and creation of various kinds of beauty is an important part of the course content. If you want to appreciate various forms of beauty, you must know and understand the basic knowledge of various forms of beauty. Through the study of various meanings, features, forms, elements, and other knowledge of beauty, we can accumulate certain theoretical knowledge and become the basis of aesthetic practice. The forms of beauty can be divided into natural beauty, social beauty, and artistic beauty, and the aesthetic practice part of the application of university AE resources is also selected from these four parts. It can be concluded that the basic framework of university AE resource application is shown in The frame diagram only shows what the AE courses in universities should have, which can be emphasized or supplemented in the implementation of specific courses. For example, in the part of aesthetic practice, you can appreciate and create natural beauty, artistic beauty, and social beauty at the same time, or you can choose one kind of beauty as the main part of the course and another form. Even in the part of basic theory, theoretical study can be appropriately increased or decreased according to class time, students' aesthetic quality, classroom content arrangement, and so on.Social ecological beauty is the beauty reflected in the social ecosystem, which widely exists in all fields of people's production and life, is reflected in the harmonious and orderly social life, and embodies people's ecological aesthetic ideal. Therefore, we can cultivate people's sensibility and ecological aesthetic consciousness through various social and ecological aesthetic forms. In the implementation of EAE in schools, the development of material resources such as educational environment resources should reflect the spirit of the times, and the architectural style should fully reflect the characteristics of vitality and simplicity. At the same time, we should also pay attention to the nationalized and localized material resources, which contain our precious culture and fine traditions, and embody the harmonious unity between man and nature, tradition and modernity, and ideas and modernity.The world, including humans, is an interconnected life ecosystem that appears as an organic whole. The concept of ecological science highlights the inseparable relationship between man and nature, breaking the existing concept of separation and opposition between man and nature. Everything in this place is connected. Life and life, life and environment, life and environment, and life and environment, communicate and coordinate with one another, cooperate with one another, displaying a beautiful form. The use of a recommendation system  is one oThe HHR_L model is proposed to solve the data sparseness, cold start, and scalability problems in recommendation system. HHR_L constructs a recommendation algorithm based on hash, which enables the recommendation system to handle large-scale data recommendation, improves the recommendation efficiency, and provides an effective method for scalable recommendation. The model framework as an example of HHR_L is shown in k suggestions are given by Hamming distance sorting.At first, we initialize the hidden layer feature representation of the project by DAE (Denoising Autoencoder); then we fine-tune the DAE network by combining the paired sorting objective function based on CF, and finally get the hash codes and items of users, and the top-The basic idea of hashing algorithm is to compress input data of arbitrary length and map it to data output of fixed length. A key skill is dimension reduction. For test samples, if the information from adjacent samples and related markers is known, the maximum posterior criterion is used to predict a group of markers in the test samples.ith mark of sample x isThen, through the maximum posterior probability criterion, it can be shown that the yx(i) represents the posterior probability of the label corresponding to sample x. Because the posterior probability is difficult to be directly calculated, it can be converted into prior probability and conditional probability according to Bayesian theorem. Therefore, if the label of the sample is unknown, the labeled sample can be expressed asP(Hib) represents the prior probability of Hib, P(x|Hib) represents the conditional probability that the tag belongs to sample x when Hib is known, and P(Hib) and P(x|Hib) can be obtained from a given data set.Among them, D > 0, the hash function Rk\u27f6Z satisfies the formula\u03b1 \u2208 Rk represents a K-dimensional feature vector that obeys N standard normal distribution, \u03b2 \u2208 R represents a feature vector randomly selected from the range of , and D represents the width of the bucket.And change the distribution of hashed points on the machine by selecting a specific hash function. First, given the parameter k groups s={s1, \u22ef, sk}. For example, the ith group will have a cluster center point \u03bci. We distinguish data points based on the median points of two neighboring groups, and the median plane between them is hyperplane. The definition of the vertical plane is as follows:From the above steps, we can get the quantization result from B, D, Y, \u0398, when the intermediate variable X is updated, the items unrelated to X in formula equation . In particular, the update rules of Ub, Vb is a stack of left singular vector and right singular vector of matrix Vb, which can be determined by A collection of natural languages is referred to as a document set. To classify and organize the document set, all that is required is to extract elements from the document set that can express the content of each document. Because \u201cword\u201d is a fully expressible semantic object in Chinese documents, it is frequently chosen as the metadata of text features. The metadata of text features must first be extracted before text mining can begin. Currently, all types of text representation methods suffer from the same issue. There are many dimensions that can be used to represent a document, and many documents that are not useful for text clustering. As a result, text features must be extracted twice. After the text feature vectors have been dimension reduced, they can be clustered, and the clustering results can be analyzed in light of the current situation.M isP is the adjacent co-occurrence probability of X, Y, and X, Y represent two Chinese characters. P(X), P(Y) are the probability of X, Y appearing separately in the text. This can indicate the tightness between Chinese characters. When the value of M is greater than a certain threshold, it can be considered that X, Y can form a word.\u201cThesaurus,\u201d or machine dictionary, has as many entries as possible. Word segmentation based on Thesaurus is the most commonly used, the simplest operation method and the most effective method. Match the item to be processed with Thesaurus, and if the match is successful, display the item string as a word. If they appear more at the same time, they are most likely to form a word. Therefore, the confidence of two adjacent words can usually be obtained by calculating their co-occurrence frequency. The mutual information CI  is a simple and effective dimension reduction method. For supervised learning, CI constructs CI subspaces, takes the cyclic vector composed of these CI subspaces as the base vector, and projects the text cyclic vector onto this subspace.ith classification is Centeri, its calculation method is as follows:Ni is the number of texts in category Ci and Docij represents the j prototype vector in category Ci.Classified prototype vector: assuming that the prototype vector of the PCA  is a widely used method of pattern recognition and signal processing, which is especially effective for data compression and feature extraction. In this paper, we apply PCA method to text classification, hoping to obtain low-dimensional representation of text vectors.M categories in the training set sample, the following is how to calculate the covariance matrix of each internal document category separately:Assuming that there are Xi is the covariance matrix of the internal document classification in the ith classification, x is the text vector in the ith classification, and \u03bci represents the prototype vector in the ith classification.Among them, M-dimensional space to D-dimensional space. This is because the sum of error squares of the data obtained by the algorithm can always be kept to a minimum. At the same time, the mutual information between the original vector x and the mapping vector y is the largest.The essence of PCA method is that you can always get the best linear mapping from When dealing with multiclassifier combinations in classification problems, the voting mechanism is frequently used in machine learning. In the above rules, the relative majority voting rule, which selects the class with the highest output among all machine classification algorithms, is a commonly used voting rule. The RF (random forest) algorithm must aggregate the classification results of each DT in the forest and vote on classification categories when determining the classification results. The DT (decision tree) model's similarity can be calculated from two perspectives: DT semantics and DT structure. The concrete implementation idea for improving the voting mechanism is to combine the DT classification effect with the weighted voting method of class probability to improve the RF voting mechanism. The accuracy of data classification outside the bag reflects the classification effect of DT. The DT classification effect improves as classification accuracy improves.X and the number of correctly classified samples is Xcorrect, then the accuracy rate of DT's out-of-bag data is the ratio of the two. The accuracy of DT data outside the bag is used to weight DT, and the weight of DT in RF algorithm isBefore making the classification decision, it is necessary to calculate the accuracy rate of each DT's out-of-bag data. If the total number of samples is Pi, the probability that the sample belongs to class y is Pi(y), the weight of DT is wi, and the number of DTs in RF is k. The weighted value z(y) of the sample belonging to class y is obtained by the formulaRemember that the probability that DT outputs that the sample belongs to each class is The probability that the sample belongs to each class is calculated by the above method, and the class with the largest weighted value is selected as the classification result.The RF_VM (Random Forest Classification Using Optimized Voting Mechanism) model can be divided into three modules: learning module, weight calculation module, and decision module. The algorithm flow is shown in First, we implement a training module, which is almost the same as the traditional RF algorithm. Next, the weight calculation module calculates the weight of each DT according to formula . FinallyThe computer used in the experiment is running Microsoft Windows 10. In this article, we choose Python as the development language of our experiment. Try PyCharm software to make Python language development more efficient.As an excellent Python integrated development software, PyCharm can provide intelligent Python support through intelligent code completion, code inspection, and navigation. PyCharm supports project and file management and various scientific packages, such as Anaconda, matplotlib, and NumPy. In order to verify the effectiveness of the proposed algorithm in cold start and sparse scenarios, we choose to experiment on two public datasets (Amazon dataset and Yelp 9th round dataset), and the sparsity is as high as 99.9%. For content data, first, delete punctuation marks and numbers. And words with stop words and length less than 2 usually have no clear meaning, so the remaining words are processed by English word segmentation.In this paper, Accuracy and MRR  are used to evaluate the recommendation performance of HHR_L in cold start environment. The experimental results are shown in It can be seen that the recommendation accuracy of HHR_L is higher than that of comparison method. This is because  and 20]20] are hIn addition, although the recommendation accuracy of HHR_L increases steadily with the increase of data density, the performance of  is unstaK-Nearest Neighbors) algorithm is used for classification, where K=10 is shown in Keep more commonly used words in text documents to improve classification accuracy. CI_PCA and DF (Document frequency) are compared in dimension reduction effect, so KNN  for the superparameters of the algorithms, which also proved the importance of value optimization.EAE is extremely important for environmental protection and improving human living conditions. The EAE educational method should focus on aesthetics, psychological discipline, and infection, and its practical implementation should be integrated with all aspects of school operations. Teachers' human resources are the main body, curriculum resources are the foundation, and material resources are the carrier in the development of school EAE resources, in order to create a humanized environment, build an information platform, and carry out various activities. When designing an ML algorithm, try to avoid data sparsity and a cold start. Data scarcity is not a big deal because of the curriculum limitations, so we pay attention to the new curriculum. By projecting the high-dimensional prototype text onto a low-dimensional subspace and then reducing the dimension according to the classification covariance matrix, the CI PCA algorithm significantly reduces the classification cost. When the proposed RF algorithm is combined with the optimized voting mechanism, the algorithm's classification ability can be greatly improved."}
+{"text": "Endocytosis is a fundamental mechanism by which cells perform housekeeping functions. It occurs via a variety of mechanisms and involves many regulatory proteins. The GTPase dynamin acts as a \u201cmolecular scissor\u201d to form endocytic vesicles and is a critical regulator among the proteins involved in endocytosis. Some GTPases , membrane proteins , and secondary messengers  mediate dynamin-independent endocytosis. These pathways may be convergent, as multiple pathways exist in a single cell. However, what determines the specific path of endocytosis is complex and challenging to comprehend. This review summarizes the mechanisms of dynamin-independent endocytosis, the involvement of microRNAs, and factors that contribute to the cellular decision about the specific route of endocytosis. Endocytosis is an evolutionarily conserved cellular process in all eukaryotic cell types . BiologiThough dynamin\u2019s role in cellular endocytosis has been well studied at the molecular level, recent studies shed new light on dynamin-independent internalization and trafficking of endosomes ,19. The In this review, we discuss dynamin-independent endocytosis mechanisms, the role of microRNAs (miRNAs), and factors that determine the endocytosis (dynamin-dependent/independent) mechanisms.Endocytosis of GPI-APs reflects a unique internalization route. It is clathrin- and dynamin-independent, and the cargo is routed into GPI-AP-enriched early endosomal compartments (GEECs) by integration of uncoated tubulovesicular clathrin-independent carriers (CLICs) emanating directly from the plasma membrane . Small GADP-ribosylation factors (Arfs) are Ras family GTPases that are classified into three families . Class IFlotillins are evolutionarily conserved membrane-associated proteins and include flotillin-1 and flotillin-2. They are ubiquitously expressed in mammalian tissues and regulate many cellular functions ranging from cell signaling, regulation of the cortical cytoskeleton, endocytosis, and protein trafficking to gene expression ,50. The 2). These results suggest that CaSR-mediated ERK1/2 signaling occurs in a dynamin-independent manner [2+ influx, respectively. In neuronal cells, the neuronal activity and cell signaling pathways such as PKA activation are regulated by intracellular calcium [Calcium plays a vital role in synaptic transmission and internalization of membrane vesicles following neurotransmitter release via exocytosis . The keyt manner . Studies calcium . A disti calcium ,72,73. L calcium . Taken tPinocytosis is a process of cell drinking where the cell internalizes extracellular fluid via endocytosis . PinocytMicropinocytosis takes part in several cellular functions. The uptake of thyroglobulin (Tg) occurs through micropinocytosis, where Tg rapidly fuses with primary lysosomes and releases iodinated compounds upon subsequent maturation . It is aMacropinocytosis is an actin-driven endocytic process and it can be either a signal-dependent process (occurs in response to growth factor stimulation) ,84 or a Entosis is a non-apoptotic cell death program with a cell-in-cell (CIC) cytological feature. It is often referred to as a cannibalistic quality of cells, where those detached from adherent junctions are engulfed by other healthy cells . In contUnder physiological conditions, entosis prevents the accumulation of unhealthy cells. For instance, entosis prevents aneuploidy in a p53-dependent manner during mitosis. Generation of an abnormal cell or DNA damage during prolonged metaphase in mitosis triggers the activation of tumor suppressor p53, which drives cell-in-cell formation via Rnd3-compartmentalized RhoA activity . FurtherThere are distinct yet-to-be-characterized endocytic mechanisms that are regulated by a dynamin-independent mechanism. For example, a pinocytic chaperone pincher is involved in the internalization of myelin-associated inhibitors for axonal growth Nogo-A in neurites. Nogo-A internalization is associated with neuronal growth arrest via the cAMP pathway . AnotherMicroRNAs (miRNAs), also known as small non-coding RNAs, are widely recognized for regulating gene expression, which modulates physiological and pathological processes. However, their role in endocytosis and intracellular trafficking remains largely unknown. Over the past decade, research on miRNAs has shown their involvement in endocytosis processes ,104,105.Dynamin-independent endocytosis mechanisms are widely implied to regulate physiological functions and pathological processes. Several nutrients and cellular components are internalized through endocytosis and affect cellular physiology. For example, Arf6-mediated endocytosis is crucial for nutrient and ion transport, cell\u2013cell interactions , and immunological functions  . Folate Endocytosis is a complex biological trafficking process within cells that attracts considerable therapeutic interest, as dysregulation of endocytosis has been linked to the pathogenesis of several diseases. In astrocytes, the dynamin-independent endocytosis by Rab5 significantly upregulated an amyloid-\u03b2-peptide and thus can be further explored as a therapeutic target in Alzheimer\u2019s disease . SimilarThe dynamic plasma membrane hosts numerous endocytic pathways. While existing data suggest distinct molecular machinery and their cargo specificity in internalization and trafficking during endocytosis, several questions remain unanswered regarding its diversity, complexity in molecular machinery, and choice of internalization route in a divergent cellular context. The endocytosis fate of cargo cannot be generalized, and it can be varied depending on the type of internalization, extracellular environment of the cell, pathophysiological status, cell type, receptor subtype, etc. In the context of receptor trafficking, there have been several instances where a single receptor can undergo internalization via both dynamin-dependent and independent pathways. For example, internalization of EGFR through a clathrin-dynamin-dependent mechanism recycles the receptor to the plasma membrane and prolongs EGFR activity, which may have a role in cell proliferation and cancer cell growth . In contCollectively, dynamin-independent endocytic mechanisms play important roles in multiple processes ranging from physiological functions to pathological conditions. Of note, unraveling the pathways that intersect dynamin-dependent and -independent endocytosis is challenging and often leads to a common molecular outcome. The use of advanced molecular tools involving single-cell genomic studies and characterization of the endocytic pathway in the context of specific cargo being routed and their trafficking could provide helpful insights in dissecting the dynamin-independent endocytic biology at the molecular level. Furthermore, miR\u2019s role in endocytic regulation is emerging, and determining their role in signaling and the functional outcome has therapeutic importance. Taken together, understanding these mechanisms of endocytosis might offer disease-modifying strategies and serve as a valuable tool for designing novel therapeutic candidates for such diseases."}
+{"text": "Mental disorders among adolescents represent a high burden and early onset. They compromise their physical health, survival, and future potential. On the other hand, young people have inadequate access to essential health services in sub-Saharan Africa. We aimed to review school-based psychological interventions, contents, delivery, and evidence of effectiveness designed to treat depression, anxiety, or posttraumatic stress symptoms among adolescents and young adults aged 10\u201324.We searched articles on the following databases: PubMed, Scopus, Embase, and Science Direct from 17/10/2022 to 30/12/2022. Furthermore, relevant studies were searched from advanced google scholar, google and identified reference lists. We used MeSH browser for key words: psychological interventions, depression, anxiety, posttraumatic stress disorder and lists of Sub-Saharan Africa countries. We combined words using standard Boolean operators . The quality of studies was evaluated using the Cochrane Collaboration\u2019s risk of bias tool and the results were presented as a narrative synthesis since the interventions were very heterogenous.Fourteen randomized controlled trials were included for systematic review and more than half (57.14%) were from Kenya and Nigeria. Common school-based psychological interventions were cognitive behavioral therapy and Shamiri interventions . More than half (57.14%) of the interventions were delivered by non-specialists like teachers, lay providers and community health workers. Nearly one-fifth of the interventions were used individual modality. School-based psychological interventions provided by non-specialists also produced a greater reduction in adolescents\u2019 depressive, anxiety, and post-traumatic stress symptoms compared to the control groups.Cognitive behavioral therapy and Shamiri interventions were the common treatment delivered in school settings. The range of interventions could be effectively delivered by non- professionals that promote task-shifting of psychological interventions from very scarce mental health specialists in these countries.Trial Registration: ProsperoCRD42022378372. https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022378372. Mental disorders are responsible for a large proportion of burden of disease among adolescents in all societies. Most mental health conditions begin early in life (12\u201324 years of age): 50% arising before age 14 and 75% by 24, even though they are often first identified later in life [According to the Lancet Child and Adolescent Health published in 2018, the definition of adolescent age is more expanded and inclusive from 10\u201319 years to 10\u201324 years. This is the delayed role transitions that include completion of education, marriage, and parenthood shifts familiar perceptions of when adulthood begins. The age of 10\u201324 years more closely to adolescent growth and understandings of the life phases and facilitate extended investments across a wide range of settings. It is essential for developmentally appropriate framing of laws, social policies, and service systems .The Sub-Saharan Africa (SSA) has the highest rate of disability-adjusted life years and the fastest-growing adolescent population in the world . HoweverMental disorders among adolescents represent the main threat to their health, survival, and future potential worldwide . The mosDespite the high burden and impacts, most mental health conditions remain unrecognized and untreated . Many AfIntervention development and adaptation have been primarily focused on HIV/AIDS service delivery in school or community programs by non-specialist health workers. There is a critical need for child and adolescent mental specialist, integration of those services into primary healthcare, coordinated cross-sectoral cooperation, and established referral networks . EvidencPsychological interventions in school enhance academic achievement by addressing self-esteem and social well-being . AdolescThere are trials of school-based psychological interventions carried out in SSA countries among adolescents with the need for summarized relevant studies to provide a better treatment appropriate to the culture. The studies were too heterogenous to pooled using meta-analysis in terms of psychological intervention in principles, duration of therapy, session duration, delivery mode , content, different approaches, and outcome measurements. One of the recommendations by Cochrane Handbook, the results will not be pooled using meta-analysis if there are substantial heterogeneity found because it may produce misleading results . TherefoThis systematic review focused only on randomized controlled trials (RCTs) since they have a lower risk of bias compared to other study designs. Therefore, the findings from this review provide the basis for practitioners working to improve the mental health of adolescents, policy planners, and researchers involved in developing and delivering school-based mental health initiatives.The review aimed to assess school-based psychological interventions\u2019 contents, delivery and evidence of effectiveness designed to treat depression, anxiety, or PTSD among adolescents aged 10\u201324.We used the PICO framework for the systematic review question: are school-based psychological interventions (intervention) effective for adolescents (population) compared to usual or other care (comparison) on depressive, anxiety, or post-traumatic stress symptoms (outcome) in SSA)?We used the Cochrane Handbook for the systematic review of interventions version 6.3 and reported according to the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) 2020 guidelines  See S1 . We usedArticles eligible for inclusion were required to demonstrate the following characteristics:Population: participants were adolescents (10\u201324 years). This is a broader and more encompassing definition of adolescence to frame laws, social policies, and service systems in a manner that is developmentally appropriate [ropriate .Intervention: school-based psychological interventions conducted in SSA and designed to reduce depressive, anxiety and PTSD symptomsComparison: control group that include no intervention, usual care, alternative intervention, or waitlistOutcome: The psychological treatment outcomes included depression, anxiety, or post-traumatic stress disorder (PTSD)Study design: Randomized controlled trials conducted in SSA. The restriction of SSA intended to narrow down the scope of review to countries that share similar socio-economic, cultural, and political situations.Context: Published in English language (due to limitation of others language by the team) from 2010\u20132022. The year 2010 was also selected because WHO launched the Mental Health Gap Action Program (mhGAP) to scale up care and services using evidence-based interventions for preventing and managing mental illness, including child and adolescents mental health problems [problems .There were exclusion criteria:Health institutions-based interventionsStudies that did not measure depression, anxiety or PTSD as an outcomeStudies that included adults and adolescents without presenting results separately by ageStudies evaluated mixed interventions, such as both psychological and pharmacological interventions.Trials of interventions delivered in universities or colleges. We believed that all college/university students in SSA not under the age of 24 years and the nature of stressors somewhat different from secondary school students.In the beginning, index terms and keywords were identified based on the participants, intervention, comparator and outcome (PICO) framework . We systWe used MeSH browser for key words: psychological interventions, depression, anxiety, posttraumatic stress disorder and lists of Sub-Saharan Africa countries. We combined words using standard Boolean operators . The key terms used to retrieve primary articles were adolescent* OR youth* OR teen* OR \u201cyoung people\u201d AND depression OR \u201cdepressive symptoms\u201d OR \u201cdepressive disorder\u201d OR \u201caffective disorder\u201d OR \u201cmajor depression\u201d OR \u201cmajor depressive disorder\u201d OR \u201cprobable depression\u201d OR \u201canxiety disorders\u201d OR \u201csocial anxiety\u201d OR anxiousness OR anxious OR anxiety OR \u201cinternalized problems\u201d OR \u201cemotional problems\u201d OR \u201cpost-traumatic\u201d OR posttraumatic OR PTSD OR \u201cpsychological distress\u201d AND \u201cgroup psychological interventions\u201d OR \u201cbrief psychosocial interventions\u201d OR \u201cpsychosocial interventions\u201d OR \u201cpsychological interventions\u201d OR \u201cpsychological treatment\u201d OR \u201cpsychological therapy\u201d OR \u201cpsycho-supportive interventions\u201d OR psychoeducation OR \u201cself-help intervention\u201d OR \u201cstress management\u201d OR \u201cemotional support\u201d OR \u201cinterpersonal therapy\u201d OR \u201cbehavioral intervention\u201d OR \u201cbehavioural activations\u201d OR \u201ccognitive behavioural therapy\u201d OR \u201ccognitive behavioral therapy\u201d OR \u201cproblem solving\u201d OR \u201cgroup therapy\u201d OR \u201csocial skill interventions\u201d OR psychotherapy OR counseling OR mindfulness AND lists of Sub-Saharan Africa countries see .We removed duplicate articles using endnote (version 20.0.0.14672) before starting the two-stage selection process. Two independent authors (MT and BA) read study titles and abstracts based on eligibility criteria and classified references: yes (included), maybe (included) and no(excluded). Any disagreement was resolved via consensus or by involving a third author (TW). Abstracts in which eligibility was unclear underwent full-text review. Then, the remaining full-text articles were assessed for inclusion by two authors (MT and BA). Furthermore, relevant studies were searched from identified reference lists manually.Two reviewers critically appraised the internal validity of eligible studies using the Cochrane Collaboration\u2019s risk of bias tool (RoB 2) . The RoBWe used data extracted based on recommendations by the Cochrane Handbook for Systematic Reviews of Interventions. Two reviewers (MT and BA) extracted data independently using a data extraction form that includes: the name of the first author, year of publication, country of study, age of participants, sample size, type of intervention, number of sessions, length, comparison group and study outcomes, the effect of the interventions. Data were extracted into a Microsoft Excel spreadsheet See .Search results were summarized in the PRISMA 2020 flow diagram. Out of 3033 articles retrieved, 66 articles were removed due to duplication through EndNote 20 citation manager. Then, 2967 papers were excluded after the title and abstract screening based on inclusion criteria. The remaining forty-one full texts were checked in detail for inclusion criteria again and only fourteen RCTs were included for systematic review. The main reasons for twenty-seven full text exclusions were: not school-based  studies, out of SSA, out of age range, not randomized controlled trials, including pharmacological intervention and not reported depression, anxiety, or PTSD as an outcome .From the included RCTs, only three were found to have a low risk of bias \u201333. The Overall, 4262 individuals were represented from seven countries in SSA. From fourteen included studies, the highest number reported from Kenya (four) ,33,37\u201339The details of intervention characteristics include content, mode of delivery, duration, number of sessions, who delivered the intervention, and the control group found in the More than half (n = 8) of the interventions were delivered by non-specialists such as teachers ,40, lay The number of intervention sessions among fourteen RCTs ranging from single to 16 sessions. One session length ranged from the shortest 30 minutes to a maximum of 90 minutes. Follow-up durations of interventions across the included studies ranged from one week ,40,43 toBased on pre- and post-intervention assessments, the effectiveness of psychological interventions in reducing the symptoms of depression, anxiety, or PTSD was determined. Post-intervention results showed a significant reduction in symptoms for depression ,38,40\u201342Fourteen RCTs were conducted on the effectiveness of school-based psychological interventions after contextual adaptations from seven countries in this region, the majority (71.4%) were from Kenya, Nigeria, and South Africa. The most common psychological interventions were CBT  and Shamiri intervention . In general, psychological interventions provided in school settings were effective in reducing depressive, anxiety and posttraumatic stress symptoms when compared to the controlled groups based on pre-and post- interventions measures.There was considerable variation in the school-based psychological interventions, modality, intensity of the session, facilitators expertise, duration of interventions and follow-up period to address the commonest mental health problem of adolescents. In our review, the psychological interventions were delivered by different professionals such as psychiatrists, social workers, guidance counselors, Nurses, and teachers. Interventions delivered by trained non-specialists also found a reduction in depressive, anxiety or traumatic stress symptoms. This might be non-specialists can be easily accessible, cost-effective and reduce stigma for majority of adolescents. A meta-analysis revealed that teacher-delivered interventions were more effective than controls in reducing depressive, anxiety, and posttraumatic stress symptoms in students ,46. In sSessions numbers used range from single session to 16 sessions, while the follow-up period is from one week to two years. This is consistent with a systematic review among adolescents suggesting that school-based psychological interventions were successful in reducing mental health problems  and sympIn this review, the most frequently utilized intervention was CBT. Studies that utilized CBT in this review, found significant reductions in depressive or traumatic stress symptoms. This is consistent with previous study findings that reported that CBT delivered to young people in schools can reduce symptoms of depression and anxiety ,50,51. AIn this review, Shamiri (a Swahili word for thrive) was the second most common school-based intervention. The Shamiri intervention teaches youths to cultivate a growth mindset, practice gratitude, and take value-aligned action. Four RCTs in Kenya have shown the intervention effectively lowers youth mental health issues like depression and anxiety while enhancing social interactions, academic performance, school atmosphere, and other functioning outcomes ,33,37,38The majority of interventions in these reviewed articles used a group-based modality. This might be because group therapy became effective for interpersonal and intrapersonal psychological problems. Group therapy can provide many possible advantages that individual therapy does not: understanding of the universality of the problem, improved communication skills, forming relationships, social skills, improved self-esteem, and increased coping skills . A meta-The improvement was significantly better than control group and depressive, anxiety, or PTSD symptoms were significantly lower within a month of completing psychological therapy . The difThis review can be used as baseline evidence for the Ministry of Health and Education, school leaders, teachers, health professionals, and psychologists when selecting evidence-based interventions to support students\u2019 mental health and wellbeing in Sub-Saharan countries and in LMICs with similar socio-economic status. Psychological interventions in school settings are effective at reducing depression, anxiety, and trauma symptoms. This emphasizes the significance of providing mental health services in educational settings since many students experience a range of mental health problems that may affect their achievement and wellness in terms of their academic, relational, social, and physical well-being. Of note, teacher-delivered interventions in school could be less stigmatized, easily accessible, cost-effective, and affordable for most adolescents to fill the large mental health care gap. This in turn, alleviate the four main barriers of adolescents not seeking and accessing help for their mental health problems identified in the previous systematic review. These include, individual factors , social factors , perceptions of the therapeutic relationship , and systemic and structural barriers  . In addiOur reviews showed that school-based psychological interventions significantly reduce depressive, anxiety, or PTSD symptoms among adolescents. However, there were several limitations in this review. First, we advise the generalizability of this finding to SSA in caution since there was the over-representation of two countries, under-representation of others, and no representation for most of the countries in SSA. Second, the intervention delivery format, duration of interventions, sessions intensity, and follow up length varied among the included studies. Therefore, to bring a single conclusion on the above-mentioned variability of interventions could not be drawn. Future reviews better examine how these variabilities of interventions may influence their effectiveness. In addition, a few trials found a low risk of bias and nearly half of them were not registered during the quality assessment.This systematic review provides evidence on school-based psychological interventions for managing common adolescent mental health problems such as depression, anxiety, and posttraumatic stress disorders in SSA. The review showed school-based psychological interventions had a stronger impact on symptom reduction. The most commonly used interventions from the included studies were CBT and non-CBT approaches. The range of interventions could be effectively delivered by non-professionals such as teachers, per-lead or community leaders that promote task-shifting of psychological interventions from very scarce mental health specialists in these countries. Particularly, psychological interventions provided in schools by teachers or peer-led facilitators can be more affordable, less stigmatized, and easily accessible for the vast majority of adolescents. This makes the interventions could be more scalable as well as sustainable to address the mental health need of adolescents. This, in turn, narrows the very large mental health treatment gaps in SSA, helps the students improve functioning at school, and reduces the distress of the student, teachers, and parents.S1 Checklist(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file."}
+{"text": "Details of the catalytic mechanism haveremained under debate and, to date, synthetic model complexes of theCuZ*/CuZ sites are extremely rare due to thedifficulty in building the unique {Cu4(\u03bc4-S)} core structure. Herein, we report the synthesis and characterizationof [Cu4(\u03bc4-S)]n+  clusters,supported by a macrocyclic {py2NHC4} ligand, inboth their 0-hole (2) and 1-hole (3) states,thus mimicking the two active states of the CuZ* site duringenzymatic N2O reduction. Structural and electronic propertiesof these {Cu4(\u03bc4-S)} clusters are elucidatedby employing multiple methods, including X-ray diffraction (XRD),nuclear magnetic resonance (NMR), UV/vis, electron paramagnetic resonance(EPR), Cu/S K-edge X-ray emission spectroscopy (XES), and Cu K-edgeX-ray absorption spectroscopy (XAS) in combination with time-dependentdensity functional theory (TD-DFT) calculations. A significant geometrychange of the {Cu4(\u03bc4-S)} core occursupon oxidation from 2 (\u03c44(S) = 0.46,seesaw) to 3 (\u03c44(S) = 0.03, square planar),which has not been observed so far for the biological CuZ(*) site and is unprecedented for known model complexes. The singleelectron of the 1-hole species 3 is predominantly delocalizedover two opposite Cu ions via the central S atom, mediated by a \u03c0/\u03c0superexchange pathway. Cu K-edge XAS and Cu/S K-edge XES corroboratea mixed Cu/S-based oxidation event in which the lowest unoccupiedmolecular orbital (LUMO) has a significant S-character. Furthermore,preliminary reactivity studies evidence a nucleophilic character ofthe central \u03bc4-S in the fully reduced 0-hole state.The active site ofnitrous oxide reductase (N The rtransfer1a.16\u221223 activity1a.214(\u03bc4-S)} coresof both restingCuZ* and CuZ sites adopt a seesaw geometry witha large CuI\u2013S\u2013CuII angle of \u223c160\u00b0 dx2\u2212y2 and the S 3py orbitals,thus leading to a CuI\u2013S\u2013CuII \u03c3/\u03c3 superexchangepathway for electron delocalization in the 1-hole CuZ*form.18 Electron paramagnetic resonance (EPR) andmagnetic circular dichroism (MCD) spectroscopies evidenced that thespin density of 1-hole CuZ* is delocalized unevenly overthe two Cu ions (\u223c5:2),18 whereas the spin populationis evenly localized on two and three Cu ions for the 1-hole CuZ\u00b0 and CuZ, respectively,24 indicating significant structural differences between these states.Spectroscopic methods in combination with theoretical calculationshave shown that the additional coordination site at the CuI\u2013CuIVedge may be vacant (for 0-hole CuZ*) or may be occupiedby a \u03bc2\u2013OH (for resting CuZ*),25 terminal CuIV\u2013OH (for CuZ\u00b0),23 \u03bc2-S2\u2013 (for CuZ),15 and \u03bc2-SH\u2013 (for 1-hole CuZ).24 Despitethe apparent distinctions in the edging ligands, the {Cu4(\u03bc4-S)} core structures were so far assumed to belargely unchanged, mostly on the basis of computational models thatused truncated structures derived from the parental resting species,which also satisfies the requirement for a minimal reorganizationenergy during ET processes.24 Nevertheless, due tothe lack of crystallographic data of N2ORs where the CuZ*/CuZ site is in a nonresting state, the structuralproperties of CuZ* (1-hole), CuZ\u00b0, andCuZ (1-hole), especially their {Cu4(\u03bc4-S)} core structures, are yet to be investigated. Gainingexperimental information about geometric and electronic structurevariations during redox interconversion of the {Cu4(\u03bc4-S)} unit would thus be valuable and might contribute to theunderstanding of the catalytic mechanism of N2O reduction.The {Cuof \u223c160\u00b0 1a,13,15 4(\u03bc4-S)} core structureand the rich redox chemistry of the CuZ*/CuZ site have stimulated many efforts to synthesize model complexeswith such a motif, not only as an alternative way to study the short-livedbiological intermediates but also to exploit the use of such metalclusters in, e.g., photochemistry and catalysis.28 However, buildinga tetranuclear copper core bearing a single S-cap has proven extremelychallenging due to the propensity to form copper clusters with multipleS atoms.32 So far, only very few model complexes containing the {Cu4(\u03bc4-S)} motif have been reported, and their supportingligands are limited to bidentate phosphine ligands  or amidinate ligands (NCHN\u2013). The first synthetic example of a {Cu4(\u03bc4-S)} cluster was reported by Yam et al. in 1993 before the structureof N2OR was elucidated; hence, it was not discussed inthe context of the biological site.34 Its {Cu4(\u03bc4-S)} core, which was isolated solely inthe [4CuI] state, was supported by a diphosphine ligand(dppm) and featured a square pyramidal geometry (A). After Yam\u2019swork, some di- and tricopper clusters bridged by a single S atom werereported,37 but the second example of a synthetic {Cu4(\u03bc4-S)} cluster appeared only in 2014.38 Modifying the dppm ligand, Mankad et al. isolateda {Cu4(\u03bc4-S)} cluster with NH-bridgedbidentate phosphorus ligands , yet again only in the[4CuI] state. Interestingly, this complex showed stoichiometricN2O reduction reactivity under very specific reaction conditions,which was proposed to be supported by the secondary coordination sphereN\u2013H groups that could serve as hydrogen bond donors.39 A seminal breakthrough was made by the samegroup when the neutral diphosphine ligands were replaced by anionicamidinate ligands (NCHN\u2013), and a redox pair of {Cu4(\u03bc4-S)} clusters in the [2CuI:2CuII] 40 and [3CuI:CuII] 41 states was obtained. The {Cu4(\u03bc4-S)}cores in both states adopt square pyramidal geometries with ratherbent Cu\u2013S\u2013Cu (opposite Cu ions) angles of up to 126\u00b0.EPR spectroscopy showed that the unpaired electron of the 1-hole speciesis delocalized evenly over the four Cu ions. At low temperature (\u221278\u00b0C), N2O can be stoichiometrically reduced by the1-hole species to N2 and O2\u2013 along withthe formation of the 2-hole species.42The unique {Cugeometry 1b, A. Afds dppa; 1b, B, ye 2-hole; 1b, C40 a 1-hole, 1b, D41 sZ siteis still very limited, with an obvious scarcity of ligand scaffoldsthat can stabilize {Cu4(\u03bc4-S)} clustersin different states. In particular, a system that allows for the isolationand investigation of a pair of {Cu4(\u03bc4-S)} complexes in the biologically relevant [3CuI:CuII]  and [4CuI]  states is still lacking. N-Heterocycle carbenes (NHCs) are increasingly employedas alternative ligands and as surrogates of histidine residues inbioinspired model chemistry, facilitating the stabilization of reactiveintermediates and unusual oxidation states.49 In our recent work, we found that the macrocycles {py2NHC2}  and {py2NHC4} (L), featuring combinations of pyridine and NHC donors,show great flexibility and can support mononuclear complexes [L\u2019Cu]1/2/3+ in various Cu oxidation states 51 as well as binuclear complexes [LCu2]2/3+ inthe CuICuI and mixed-valent Cu1.5Cu1.5 states,52 respectively.The mixed-valent complex [LCu1.5Cu1.5]3+ features a large spin delocalization energy and fast electron self-exchangerate, which structurally and functionally mimics the CuA site. In the present study, novel {Cu4(\u03bc4-S)} clusters scaffolded by the macrocycle L were isolated and fullycharacterized in both the 0-hole and 1-hole states, i.e., in the activestates of the CuZ* site during the N2O reductionprocess (4(\u03bc4-S)} clusters reported so far, and a significant redox-inducedstructural change in the {Cu4(\u03bc4-S)} corehas now been evidenced by X-ray diffraction (XRD) analyses. The correspondingelectronic structure changes have been analyzed by a range of spectroscopictechniques, including UV\u2013vis, EPR, Cu K-edge X-ray absorptionspectroscopy (XAS), and Cu/S X-ray emission spectroscopy (XES), andthe experimental results were correlated with DFT calculations.In spiteof the significant achievements made by the Yam and Mankadgroups, the modeling chemistry for the unique Cuprocess 1a,c.The2-S-bridged neutral dicopper species [LCu2(\u03bc2-S)] that could mimic a fragment of thecore structure ofthe CuZ*/CuZ site. However, treating the dicoppercomplex [LCu2](PF6)2 (1) with 1 equiv of sodium sulfide (Na2S) in acetonitrile(MeCN) gave, after workup and slow diffusion of diethyl ether (Et2O) into the MeCN solution, the \u03bc4-S-cappedtetranuclear copper complex, [L2Cu4(\u03bc4-S)](PF6)2 (2) as yellowcrystals (2 has a {Cu4(\u03bc4-S)} core supported by two macrocyclic ligands L (1. Two Cu ions(Cu2 and Cu4) in opposite positions are coordinated by NHCs from thesame ligand (L) but different {pyNHC} cavities, while the other twoopposite Cu ions (Cu1 and Cu3) are coordinated by NHCs from differentligands L, thus rendering two pairs of Cu ions in distinct coordinationenvironment (Cu2 and Cu4 vs Cu1 and Cu3). In contrast, in all otherCuZ* model complexes reported so far, neighboring Cu ionsof the {Cu4(\u03bc4-S)} core are spanned bybidentate phosphine or amidinate ligands in the same coordinationmode (A\u2013D).41 The Cu\u2013S bonddistances in 2 are found in the narrow range 2.3021(6)\u20132.3499(5)\u00c5 and are similar to the Cu\u2013S distances in the CuZ*/CuZ sites of N2OR.15 The projection of the Cu4 base is an irregular quadrilateral,with Cu\u00b7\u00b7\u00b7Cu distances varying from 2.972(1) to 3.370(1)\u00c5 (4-S1)\u2013Cu3 angle of 160.80(3)o is comparable to that in the CuZ*/CuZ sites (\u223c160\u00b0), while the Cu2\u2013(\u03bc4-S1)\u2013Cu4 angle of 134.71(3)o is larger than thatin the biological systems (\u223c90 o). The \u03c44 value is 0.46 for the \u03bc4-sulfide ligandin 2, indicating a seesaw shape of the {Cu4(\u03bc4-S)} core ,53 which issmaller than \u03c44(\u03bc4-S) in the CuZ*/CuZ sites (0.74/0.71).15 Notably, this \u03c44(\u03bc4-S) value isthe smallest among the reported CuZ*/CuZ modelcomplexes (0.59\u20130.90), in which the {Cu4(\u03bc4-S)} core is best described as a square pyramid with morebent Cu\u2212\u03bc4-S\u2013Cu angles . Interestingly, the molecular structureof the cation of 2 in the solid state has an approximate C2 local symmetry. However, 1H NMRspectra in different solvents  show only one set of signals in the temperature range studied with equivalent protons of both halves of the macrocyclicligand (L), implying a higher level of symmetry in solution. Thismight be caused by the fast conformational inversion of the {Cu4(\u03bc4-S)} core as well as the ligand macrocycle,leading to an averaged overall C2v symmetryon the NMR time scale.Initially, we were aiming at the synthesisof a \u03bccrystals 1.X-ray igands L 2a. All Cionmode 1b, A\u2013D.3370(1)\u00c5 2b.The C2 isstable both in solidstate and in common solvents under an inert atmosphere. In MeCN solution under aerobic conditions,however, it decomposes slowly to give precursor complex 1, which likely proceeds through the oxidation of 2 byO2 and subsequent decomposition of the oxidized species(vide infra). The reformation of 1 showsthat the Cu\u2013CNHC bonds are labile and that the rearrangementof L, interconverting the binding modes seen in 2 and 1, is reversible. The identity of 2 was furtherconfirmed by combustion analysis and by electrospray ionization massspectrometry (ESI-MS); the latter shows a dominant peak at m/z = 645.1 amu (in MeCN) with an isotopepattern that is characteristic of the dication [L2Cu4(\u03bc4-S)]2+ , indicating that the {Cu4(\u03bc4-S)} core structure of 2 is retained in solution.At room temperature (rt), 4(\u03bc4-S)} cluster canbe accessed, which would emulateboth activestates of the CuZ* site that are passed through in thecatalytic N2O reduction cycle, the redox properties of 2 were examined electrochemically. Cyclic voltammetry (CV)of 2 in MeCN (100 mV/s) at rt reveals one reversibleredox event at a potential of \u22120.65 V , assignedto the [L2Cu4(\u03bc4-S)]3+/2+ redox couple when a small potential range from \u22120.98 to \u22120.28V (vs Fc+/0) was scanned. This redox potential is significantlycathodically shifted compared to the 0-hole model complexes [(\u03bc2-dppm)4Cu4(\u03bc4-S)](PF6)2 and [(\u03bc2-dppa)4Cu4(\u03bc4-S)](PF6)2  for which onlyirreversible oxidation events were observed.54 This differencecan likely be attributed to the strong \u03c3-donating ability ofthe NHC ligands; at the same time, the NHC-based scaffold L is capableof stabilizing the {Cu4(\u03bc4-S)} core inboth relevant oxidation states. However, the redox event at E1/2 = \u22120.65 V becomes irreversible whenthe scan range is extended anodically to +0.42 V (vs Fc+/0), and a quasi-reversible redox event at E1/2 = \u22120.06 V appears (1ox/1 (E1/2 = \u22120.09 V).52 Thiscan be attributed to the rapid decomposition of the species resultingafter 2-fold oxidation of 2, giving two equiv of 1. In line with this interpretation, the current of the redoxevent at E1/2 = \u22120.06 V is approximatelytwice the current of the initial oxidation of 2 at \u22120.65V.To assess if the 1-holestate of this {Cu appears 3a that i2 55 at \u221280 \u00b0C, both absorptionsof 2 vanish and an intense broad band at \u03bbmax = 790 nm as well as a band with lower intensity at \u03bbmax = 530 nm emerge, reaching a plateau in intensity afterthe addition of 1 equiv of oxidant (2Cu4(\u03bc4-S)](PF6)3 (3). The observation of an isosbesticpoint at 470 nm indicates a clean conversion of 2 to 3 without intermediate step(s) 55 and subsequent reoxidation with 1equiv of [Cp*2Fe]PF6 . In situ-formed 3 is stable at \u221235\u00b0C for several hours without noticeable decay according to UV\u2013visspectroscopy, suggesting that the isolation of 3 shouldbe feasible at such low temperature.While the oxidized species (1-holestate) is metastableat rt,UV\u2013vis titration experiments showed that it is relatively stableat low temperatures. The UV\u2013vis spectrum of complex 2 3b in ace oxidant 3b. The c step(s) 3b. The i2 with [Cp*2Fe]PF6 was carried out at\u221235 \u00b0C in acetone } core to be establishedby X-ray diffraction analysis . The cation of 3 is structurally similar to the cationof 2 consisting of a {Cu4(\u03bc4-S)} core, yet in an unprecedented square planar geometry where the\u03bc4-S resides in the center of the square with a \u03c44(S) value of 0.03. This is in remarkable contrast to 2 and all of the other model complexes (A\u2013D)as well as CuZ*/CuZ sites where the {Cu4(\u03bc4-S)} cores have either seesaw shapes orsquare pyramidal shapes. To improve the crystallization behavior,counteranion exchange of 3 with NaBF4 wassubsequently performed, which resulted in high-quality purple crystalsof 3-BF4.Bulk oxidation of  acetone 1. After analysis S2. The c3-BF4 (4(\u03bc4-S)} core as thatin 3 with a slightly larger \u03c44(S) valueof 0.11, which might originate from the collective packing effectsof anions (PF6\u2013 vs BF4\u2013) and solvents (acetone vs MeCN). Upon oxidation tothe 1-hole state, the Cu4 base in 3-BF4 is less asymmetric than in 2, withCu\u00b7\u00b7\u00b7Cu distances in a narrower range (3.095(1)\u20133.265(1)\u00c5; 2, become pairwise unequalin 3-BF4 with two long Cu\u2013Sbonds  \u00c5 and Cu3\u2013S1, 2.3191(9)\u00c5) and two short Cu\u2013S bonds \u00c5 and Cu4\u2013S1, 2.2005(9) \u00c5). Careful inspection ofthe structure reveals that each Cu ion adopts a trigonal planar geometry;however, the {C2NHCCuS} planes centered on Cu2and Cu4 are approximately perpendicular to the Cu4 baseplane with a dihedral angle of 86.3 and 89.0\u00b0, respectively,while the {C2NHCCuS} planes centered on Cu1and Cu3 are bisected by the Cu4 base plane with dihedralangles of 45.3 and 40.8\u00b0, respectively. Such a spatial configurationmight affect the interaction of the Cu 3d orbitals with the \u03bc4-S 3p orbitals, resulting in the inequality of Cu\u2013Sbond lengths in 3-BF4. Notably,the two short Cu\u2013S bonds  are significantly contracted comparedto the corresponding Cu\u2013S bonds in 2, suggestingthat the two opposite metal ions Cu2 and Cu4 are predominantly involvedin the oxidation process (vide infra).The structureof the cation of 3-BF4 2a featur1H NMR spectrum of 3 at 263 K displaysbroad singlets in the range of 0\u201318 ppm due to its paramagneticnature . The S = 1/2 ground state of 1-hole 3 was confirmed by SQUIDmagnetometry where a constant \u03c7MT value of 0.40 cm\u20133 mol\u20131 K (corresponding to \u03bceff = 1.79 \u03bcB) over the entire temperature range from2 to 300 K was observed and best-fitted as an S =1/2 system with giso = 2.07 . The continuous-wave X-band (9.63GHz) EPR spectrum of 3 in acetone recorded at 50 K ispseudoaxial with a hyperfine pattern on the low field side attributedto the 63/65Cu (I = 3/2) nuclei, withrelatively small splittings and an intensity pattern that suggeststhe unpaired electron is delocalized over more than one Cu ion  and intensitypattern resulting from two equivalent copper centers.52 To further resolve the EPR parameters, a two-pulse echo-detectedQ-band (34.0 GHz) EPR spectrum of 3 in acetone was collectedat 15 K. This higher-frequency spectrum resolved the rhombic splittingof the g-tensor, particularly the narrow g3 feature . The overall linewidth of the Q-band spectrum is larger than the X-band spectrum, acommon occurrence for Cu centers measured at higher microwave frequencies,resulting in only a broad g1 feature withoutany well-resolved copper hyperfine coupling.56 Both the X- and Q-band spectra are best simulated with a rhombic g-tensor g =  and two equivalent Cu hyperfine tensors A(Cu) =  MHz. The giso of 2.06 also agrees well with the value derived from the magneticsusceptibility measurements. Most importantly, the model and simulationwith two equivalent Cu ions reproduce best both the position and breadthof the g1(A1) features. Attempts to reproduce both the X-and Q-band EPR spectraby simulation with only a single Cu hyperfine interaction were unsuccessfuland unable to reproduce the multifrequency EPR data . Therefore, the multifrequency EPR analysis evidencesthat the unpaired electron in 1-hole 3 is delocalizedover two equivalent Cu ions.The cnature S14. The Z\u00b0 site, which is the active intermediate speciesduring the N2O catalytic cycle.23 In addition, the maximum Cu hyperfine coupling in 3 (128 MHz) is comparable to that in CuZ\u00b0 (118 MHz),indicating that the spin population on the Cu ions in these two speciesis similar (4(\u03bc4-S)} sites or model complex D (Z* site) or equally over three(for the reduced CuZ site) or four Cu ions (for model complex D).It is worth noting that spin delocalization equallyover two oppositelypositioned Cu ions has also been observed for the biological 1-holeCu similar 1. In conomplex D 1 show a 4(\u03bc4-S)} clusters (2 and 3-BF4) was further investigatedby density functional theory(DFT)calculations at the B3LYP/def2-TZVP level. The seesaw and square planar{Cu4(\u03bc4-S)} core structures of the cationsof 2 and 3-BF4,respectively, as well as relevant bond lengths/angles, were well reproducedin the optimized structures (\u03c44(S) = 0.44 and 0.10; Table S3). The singly occupied molecular orbital 328) of 3-BF4 is primarily composed of Cu 3dyz /3dxz  orbitals and the \u03bc4-S 3pz (39%) orbital featuring \u03c0-antibondinginteractions . Such \u03c0*interactions in the ground state of 3-BF4 contrast the situation in the 1-hole CuZ* site18 and model complex D(2NHCCuS} planes (centered on Cu2 and Cu4) are almostperpendicular to the Cu4 base plane (vide supra). Such a configuration, combined with the large Cu\u2013(\u03bc4-S)\u2013Cu angles, enables the effective \u03c0/\u03c0overlap of Cu 3dyz/3dxz orbitals and the \u03bc4-S 3pz orbital in 2/3-BF4 . Removing one electronfrom the \u03c0* HOMO of 2 upon oxidation strengthensthe Cu\u2013S bonds, especially the Cu2\u2013S1 and Cu4\u2013S1bonds, which accordingly leads to the planarization of the {Cu4(\u03bc4-S)} core structure. The Loewdin spindensity was found to be delocalized over the four Cu ions via these\u03c0* interactions between the Cu and S atoms, constituting anexcellent superexchange pathway (4-S center (42%) and moderately locatedon the Cu2 and Cu4 ions (each 18%), while the Cu1 and Cu3 ions featureonly minor spin populations (each 5%) (4-S in 3-BF4 (42%) is obviously larger than that in the CuZ*site (14%)17 and model complex D (32%),41 indicating a higher extent ofS participation in the redox process. The large spin delocalizationon the \u03bc4-S and high covalency of the Cu\u2013Sand Cu\u2212NHC bonds might also account for the rather small anisotropyof the EPR spectrum that adopts a small g1 value.57The electronic structureof these {Cucomplex D,42 where2Cu4(\u03bc4-S)]3+/2+ redox couple, Cu K-edge X-ray absorption (XAS), and Cu and S valenceto core (VtC) X-ray emission spectra (XES) of 2 and 3 were measured. These data, together with the correspondingcalculated spectra, are shown in Figures S34\u2013S36. The Cu K-edge XAS  clearlyevidences an increase in the rising edge position of 3 as compared to that of 2 (by \u223c1 eV) consistentwith a significant metal-based contribution to the oxidation eventin 3. The Cu and S VtC XES probe the transitions fromthe filled valence levels to the 1s core holes on the Cu and S, respectively;59 hence, taken together, the relative contributions of copper andsulfur to the filled bonding levels can be experimentally assessed.The Cu VtC XES  shows verysimilar spectra for 2 and 3, highlightingthat the Cu K-edge XAS, which primarily probes the lowest unoccupiedmolecular orbital (LUMO), is a more sensitive electronic structureprobe in the present case. It is of interest to note that the S VtCXES  shows clear changes between 2 and 3, suggesting a modulation of the sulfurinvolvement in bonding, which may be attributed to either a substantialligand-based oxidation or increased sulfur covalency. Time-dependent(TD)-density functional theory (DFT) and one-electron DFT calculationswere utilized to calculate the XAS and XES spectra, respectively.The general trends in the data are well-reproduced by the calculations,supporting that the DFT electronic structures of 2 and 3 are reasonable descriptions of the electronic ground states. Complex 2 is best described as a fully reduced cluster, while 3 has a hole character with a LUMO that is \u223c50% copper-basedand \u223c50% ligand-based, reflecting the high covalency of thecluster .To obtain further insight into theelectronic structure variationsof the I2 . The large steric hindrance aroundthe metal ions enforced by the two macrocyclic ligands may accountfor the inertness of 2 toward these substrates. Thoughthe four Cu ions are shielded by the macrocyclic ligands, however,an open cleft allowing for access to the central \u03bc4-S is present on one side of the Cu4S cluster in the 0-holestate, which could make an attack on the S atom possible; this contraststhe situation in the planar 1-hole state Cu4S cluster,where this cleft is mostly closed . The large contribution of the \u03bc4-S 3pz orbital (39%), which is perpendicular to the Cu4S plane, to the HOMO of 2 was expected to imparta nucleophilic character to the S center. Indeed, 2 reactsrapidly with electrophilic [Me3O]BF4 via S-alkylation, giving the dicopper(I) complex 1 and its thiolato-bridged congener [LCuI2(\u03bc2-SMe)]+ . A distinct N2O activation modewhere the S atom is involved as one binding site has been proposed.42 Attack at the S atom  in 2 is an indication of the potentialability of 2 to functionally mimic the CuZ* site. Modification of the macrocyclic ligand to provide a largerring, increased metal ion access, and more electron-rich Cu ions areundergoing.Given that the fully reduced (0-hole) state andthe 1-hole stateare proposed as active states of Cu4(\u03bc4-S)}cluster found in the active CuZ* or CuZ siteof the metalloenzyme N2O reductase, which mediates thefinal step of bacterial denitrification,can be isolated by using a flexible hexadentate macrocyclic ligandsystem L that provides two binding pockets and NHC ligation for theCu ions. This new {Cu4(\u03bc4-S)} clusteris distinct from the very few reported CuZ*/CuZ models that are based on bidentate {PXP} and {NCN} ligands. [L2Cu4(\u03bc4-S)]2+/3+ couldbe isolated in the fully reduced 0-hole state  and in the singly oxidized 1-hole state (S = 1/2), viz. in both states that have been proposed forthe relevant intermediates of CuZ* in the catalytic N2O reduction cycle. Comprehensive structural and spectroscopiccharacterization of the [L2Cu4(\u03bc4-S)]2+/3+ complexes has provided novel insights into howthe {Cu4(\u03bc4-S)} core structure of CuZ* might adapt to the 1e\u2013 oxidation statechange, revealing a planarization upon oxidation that gives rise topronounced \u03c0* interactions between Cu 3dyz/3dxz orbitals and the \u03bc4-S 3pz orbital. The unpaired electronspin in the 1-hole state is almost equally located on the \u03bc4-S center and on the Cu ions , with metal contributionspredominantly from two Cu ions located opposite each other in the{Cu4(\u03bc4-S)} core. This is similar to whathas been proposed for the active 1-hole intermediate (CuZ\u00b0) of CuZ*, and it is also reflected in a significantcontraction of the corresponding Cu\u2013S bonds upon oxidationof the present {Cu4(\u03bc4-S)} model clusterfrom its 0-hole state to its 1-hole state. Cu K-edge XAS and Cu/SK-edge XES spectroscopies, combined with DFT calculations, corroborateda mixed metal- and sulfur-based hole character with a highly covalentCu4S core of the oxidized species.The presentstudy demonstratesthat a model of the unique {Cu4(\u03bc4-S)} core structures of the CuZ(*) site to be largelyunchanged in both resting and active states, structural constraintsimposed by the protein scaffold were considered by fixing the positionof the distal nitrogen or the C\u03b3 atoms of each histidineligand in the DFT geometry optimizations.24 However, theoretical work also indicatedthat, if all constraints are removed, the original seesaw {Cu4(\u03bc4-S)} cluster rearranges to a trigonalpyramidal geometry with three Cu and S atoms at its base and thatthe protein strain energies are higher for the 1-hole species.20 The latter is reminiscent of our 1-hole complex,which indeed undergoes a structural rearrangement from a seesaw toa square planar core geometry. Though the protein environment wassuggested to be strained, the flipping of some histidine rings61 as well as a conformational switch of the activesite relevant to its function has indeed been observed for these N2OR enzymes63 and has been suggested for theirCuA site by theoretical calculations as well.64 Furthermore, a complex network of hydrogen bondsinvolving the protein environment has been suggested to determinethe orientation of the histidine side chains.65 Therefore, a change in local pH or a shift in hydrogen bonding couldlead to a change in the orientation of the histidine ligands, resultingin a corresponding change in the geometry of the {Cu4(\u03bc4-S)} core. Based on this, geometric and electronic structurevariations of the CuZ(*) sites are conceivable when shuttlingthrough different states during N2O reduction; the presentpair of bioinspired {Cu4(\u03bc4-S)} clusters 2/3 clearly illustrates possible variations inresponse to the 1e\u2013 oxidation state change.How can thesefindings be related to the enzyme active site? Inprevious computational models that predicted the {Cu"}
+{"text": "Rousettus aegyptiacus). Bats were anesthetized with a combination of alfaxalone, midazolam, and morphine administered subcutaneously. Incisional line infiltration using bupivacaine was administered in all bats, and additional bilateral intratesticular injection was administered in the males. Ovariectomy was performed via a dorsal approach, by bilateral midline skin incisions at the paralumbar fossa level. Orchiectomy was performed via a ventral approach, by bilateral midline incisions of scrotal skin above the testes. Following surgery, all bats were administered flumazenil for midazolam reversal, and meloxicam for postoperative analgesia, subcutaneously. All bats recovered from anesthesia uneventfully. Bats were monitored for complications up to 10 days following surgery, when skin sutures were removed. No morbidities or mortalities occurred during this period in any of the bats. In conclusion, ventral approach orchiectomy and dorsal approach ovariectomy using the injectable combination alfaxalone-midazolam-morphine in conjunction with local anesthesia and meloxicam are feasible procedures in Egyptian fruit bats and can be performed with relative ease. However, further studies using these techniques in a larger group of bats should be performed to establish their safety.The purpose of this report is to describe the anesthetic and analgesic management and the surgical procedures of gonadectomy in six  healthy adult Egyptian fruit bats ( Rousettus aegyptiacus (R. aegyptiacus), are a common display species in zoological parks, and due to traits like longevity, robust immune system, echolocation and high brain to body ratio, this species is studied in laboratory settings  agonist. Alfaxalone has a high therapeutic index, short half-life, and minimal cardiovascular effects at clinical dose, thus, it can be used for injured or compromised patients  is a synthetic neurosteroid anesthetic agent acting as a \u03b3-aminobutyric acid receptor A  for ovariectomy and orchiectomy, respectively. Bats were housed at a bat colony at the Zoological Garden of Tel-Aviv University and were brought to KSVM-VTH via bat carriers shortly prior to surgery. Acclimatization was not performed, as it was a short procedure and bats were returned to the bat facility following recovery from anesthesia. Due to lack of an appropriate fine weight scale, bats' body weight was estimated to be 130 g  and 150 g  based on the authors' experience working with this species.Six healthy adult (age unknown) Anesthesia was induced with a combination of 15 mg/kg alfaxalone , 1.5 mg/kg midazolam  and 1.5 mg/kg morphine  combined in an insulin syringe  administered subcutaneously between the shoulder blades while bats were gently restrained covered with a drape. Following injection, bats were monitored every minute for relaxation signs (head relaxation and decreased wing muscle tone), while held under the drape. First effect appeared 4 \u00b1 1  min following injection. Mean time to recumbency without movement  was 7 \u00b1 1 (5\u20138) min, although 10 min from injection, one female still showed an occasional ears and wings movement. As anesthetic depth was considered light, the anesthetist decided to administer an extra dose of alfaxalone (5 mg/kg) at 11 min after the initial injection. Mobility then ceased within 2 min. During anesthetic induction, four of the six bats displayed muscle twitching, that subsided after several minutes as anesthetic depth increased.Following aseptic preparation, bupivacaine 0.5% solution  diluted in sodium chloride 0.9%  was infiltrated using an insulin syringe  at the planned incision line prior to surgery . In the fR) was measured by observation of thoracic movements. Body temperature was measured using rectal thermometer. Anesthetic depth was considered appropriate in all bats , and no movement or elevation in heart or respiratory rates in response to surgical stimulation occurred. Physiologic variables and anesthetic complications are presented in During the procedure, anesthetic depth and physiologic variables were monitored every 5 min. Isoflurane was available for rescue anesthesia in case anesthetic depth was considered inadequate. Heart rate (HR) and rhythm were monitored using a doppler ultrasonic flow detector  placed and taped on the sternum at the heart level. Respiratory frequency  were administered subcutaneously, and eye lubrication was applied . During surgery, bats were actively warmed with a forced-air warming system .Bats were placed on an insulated operating table and oxygen was supplemented (1 L/min) A dorsal approach was used to prevent post-operative self-mutilation. Bats were placed in ventral recumbency, and the dorsal skin was shaved and aseptically prepared over the thoraco-paralumbar region and draped. A skin incision (1\u20131.5 cm) was made ~1 cm lateral to the midline, at the paralumbar fossa region, in a cranial to caudal direction . The fatIn two females, moderate non-specific local hemorrhage of the tissues around the ovary occurred. Local pressure was applied using sterile wooden cotton swabs until the bleeding subsided. The muscle layer was sutured using absorbable ligatures  with a simple continuous pattern, following replacement of the fat pad. Simple continuous subcutaneous sutures , and intradermal continuous sutures  were placed. Finally, for extra protection against mutilation, three to four nylon sutures were placed in the skin in a simple interrupted pattern . Mean ovariectomy time  was 31 \u00b1 5 (26\u221237) min.Scrotal skin was aseptically prepared and draped. The testis was manually held slightly distal to the body in order to prevent its retraction into the abdomen. A 5\u20138-mm longitudinal ventral scrotal incision was made through the skin, subcutaneous tissue and vaginal tunics, using number 15 scalpel blade. The testis was exteriorized and the epididymis, ductus deferens, testicular artery and vein (pampiniform plexus) identified. The ductus deferens and testicular vessels were clamped using mosquito hemostats, and three circumferential ligatures  were placed 0.5 cm apart around them. The ductus deferens and testicular vessels were dissected distal to the distal ligature, to disconnect the testis and epididymis from the body. Scrotal skin was sutured using nylon ligatures . Orchiectomy time  was 7\u201310 min, and bleeding was minimal.Two female bats began flickering their ears during placement of the last skin suture (at 49 and 50 min following anesthetics injection). In order to facilitate recovery, at the end of surgery bats were administered 0.3 mg/kg flumazenil  subcutaneously to antagonize the midazolam .Total anesthesia time (from anesthetics injection to antagonist administration) was 59 \u00b1 7 (52\u201368) min for ovariectomies and 28\u201337 min for orchiectomies. Recovery time (from flumazenil administration until climbing to the carrier roof) was 30 \u00b1 5 (22\u221235) min. Recovery time in the present report was longer than reported for dexmedetomidine-based protocols  . This diFor postoperative analgesia, bats were administered a subcutaneous injection of 1 mg/kg meloxicam  at the end of surgery, and then continued with 1 mg/kg oral meloxicam  every 24 h for two more days.Following complete recovery from anesthesia, bats were taken back to the bat facility and housed in dark, temperature and humidity-controlled conditions. Fruits and water were provided in accordance with recommendations for the species. Bats were individually housed in order to prevent picking and tearing the sutures by other bats. Bats underwent veterinary exam daily and skin sutures were removed after 10 days. No morbidities or mortalities occurred during this period in any of the bats.R. aegyptiacus using a surgical approach and anesthesia and analgesia regime, including local anesthesia, not previously reported in this species.The present report describes the feasibility of performing gonadectomy in R. aegyptiacus undergoing gonadectomy. Both protocols provided quicker induction compared with the present report , although, both protocols required isoflurane supplementation during surgery (mean inspired fraction of 0.7%) , which result in personnel exposure , 9. Seveof 0.7%) .R. aegyptiacus by the authors   . Althougyptiacus . This emR. aegyptiacus under alfaxalone-midazolam , was carried out using a ventral midline incision and all bats recovered uneventfully. Surgical time was longer than the time in the current report and lasted 45\u201356 min . It is possible that due to the pecking nature of fruit bats they are prone to postoperative self-mutilation compared with vampire bats. The potential surgical complications posed when using the ventral ovariectomy approach, make the dorsal approach potentially superior in R. aegyptiacus, as in the present report surgical time was shorter, and complications seemed to be minimal. However, this technique should be tested in a larger group of bats to validate its safety.Ovariectomy using a ventral, lateral or dorsal approach was described in numerous species including equine, canine, feline and rodents \u201342. Ovar5\u201356 min . Anotherreported . The maiVentral approach orchiectomy technique as described in the present report, can be easily used in this species. Testes were retrieved with ease from the abdominal and pre-scrotal positions, vessels were ligated in a simple manner and no potential complications during or following surgery were observed.Rousettus lanosus), and orchiectomy with radiosurgery was performed in 125 Jamaican fruit-eating bats (Artibeus jamaicensis), and one Ruwenzori bat, under general anesthesia. Total surgery time for open orchiectomy was much longer than the total surgery time in the present report (26\u201338 min compared with 7\u201310 min). Postoperative complication (prolonged recovery) occurred in 1/14 bats. Radiosurgeries were short, lasting 5\u20137 min, however, major complications occurred; 4.8% morbidity rate , and 1.6% mortality rate (2 bats)  . ComplicR. aegyptiacus males. The authors reported that it was difficult to identify the mesoductus deferens, and postoperatively 27% of the testes appeared to adhere to the scrotal sac for at least 14 months. Otherwise, the technique appeared quick (Another study described vasectomy performed in nine ed quick . Althoug2, and continuous rectal temperature. Postoperative pain evaluation was not performed, and to the authors' knowledge, there are no specific pain scales for bat species. For this reason, meloxicam was administered postoperatively regardless of pain evaluation. Additionally, the inability to accurately weigh the bats is a major limitation of this report. In very little animals where weight estimation is challenging, small errors can lead to large differences in drug doses.Limitations to this case report include the small number of animals, and lacking the ability to monitor blood pressure, hemoglobin oxygen saturation, electrocardiogram, end tidal COR. aegyptiacus bats and can be performed with relative ease. However, further studies comparing this anesthesia regimen with other protocols and assessing the reported surgical procedures in a larger group of bats should be performed to establish their safety.In summary, ventral approach orchiectomy and dorsal approach ovariectomy using the injectable combination alfaxalone-midazolam-morphine in conjunction with local anesthesia and meloxicam are feasible procedures in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.This case series was carried out as a standard treatment in accordance with good clinical practice. Written informed consent was obtained for including the animal in this case series.ST and YS-B contributed to the data acquisition and interpretation, drafted and revised the manuscript, and approved the final version to be submitted."}
+{"text": "Rib fractures are the most common injuries in chest trauma. Compared with younger patients, elderly patients with rib fracture have a higher incidence of complications and mortality. A retrospective study was conducted to investigate the effect of internal fixation compared with conservative treatment on the outcome of rib fracture in elderly patients.We used a 1:1 propensity score matching method to perform a retrospective analysis of 703 elderly patients with rib fractures treated in the Thoracic Surgery Department of Beijing Jishuitan Hospital between 2013 and 2020. After matching, the length of hospital stay, death, symptom relief and rib fracture healing were compared between the surgery and the control group.p\u2009=\u20090.000). After 9 months of follow-up, the fracture healing rate in the surgery group was significantly higher than that in the control group . The fracture healing time (p\u2009=\u20090.000), improvement in pain score (p\u2009=\u20090.000) and duration of pain medication use (p\u2009=\u20090.000) were also significantly better in the surgery group than in the control group.The study included 121 patients receiving SSRF in the surgery group and 121 patients receiving conservative treatment in the control group. The length of hospital stay in the surgery group was significantly longer than that in the conservative group (11.39d vs. 9.48d, Compared with conservative treatment, surgical treatment can prolong hospital stay to some extent. However, it has the advantages of more rapid healing and lessened pain. For rib fractures in elderly individuals, surgical treatment is a safe and effective option under strict surgical indications and is recommended. At present, there is no complete Chinese database of chest trauma patients, and it is difficult to obtain accurate data on chest trauma. It is conservatively estimated that the number of patients with rib fracture is 1.5 million to 2 million per year  and the control group (435 cases) according to the surgical stabilization of rib fractures. The inclusion criteria were as follows: (1) age \u226560 years and (2) diagnosis of acute rib fracture based on medical history and imaging evidence. The exclusion criteria were as follows: (1) patients lost to follow-up ; (2) patients with severe injuries other than chest, that is, patients with abbreviated injury scale (AIS) score \u22653 for the head and neck, face, abdomen, limbs or body surface .Due to significant differences in the basic characteristics of the two groups after study inclusion , they co2.3.Surgical treatment: The surgical treatment was surgical stabilization of rib fractures (SSRF), performed under general anesthesia, with all patients in supine or decubitus position. preoperative ultrasonography was used to locate the broken end of the rib fracture and select the appropriate incision approaches according to the rib fracture site and the anatomical characteristics of the chest wall. The incision length is usually between 5\u2005cm and 15\u2005cm. The surgical steps were as follows: cutting the skin, separating the muscle layer along the muscle space, exposing the broken ends. The fracture was fixed by placing a new pure titanium U-shaped plate  at the broken end and ensured that there were 2\u20133 fixation nails on both sides of the broken end . The wouConservative treatment: Conservative treatment included the usage of rib straps or chest plates to immobilize the thorax, oxygen supplementation, sputum atomization, pain reliever, and management of fracture complications. Patients with pulmonary infection were given anti-inflammatory therapy. Closed thoracic drainage was performed for patients with massive hemothorax and/or pneumothorax. Patients with severe lung injury were treated with mechanical ventilation. Long-term oral administration of Imericoxib tablets 0.1GM BID for analgesia.2.4.(1) The admission status of the patient: length of hospital stay, length of ICU stay, and duration of mechanical ventilation; (2) The number of hospital deaths; (3) Delayed symptoms after treatment: fracture healing rate (follow-up for 9 months), fracture healing time, reduction of pain score, and duration of pain medication use. The evaluation of pain relief and the fracture healing index included the pain score and follow-up fracture healing. All patients were evaluated for the pain score on admission and at discharge. The usage and duration of pain medications were followed up after discharge.Fracture healing was followed up after discharge, mainly assessed by imaging examination, physical examination and clinical symptoms. In this study, we selected a CT scan to examine the healing of the rib fracture because we could clearly see the fracture line on the axis of CT . In both2.5.This was a cohort study. Baseline information of the surgery and control groups included patient age, sex, underlying diseases , smoking, number of fractures, flail chest, fracture complications  judged by imaging changes, AIS score, injury severity score (ISS) score and pain score upon admission. With these basic characteristics as predictive variables, the calliper value was set as 0.005, and the ratio was 1:1. We calculated the bias matching score of each case using the logistic regression equation, and cases with similar scores in the two groups were matched. After matching, we compared the observation indices between the 242 patients in the surgery and control groups to analyse differences in the efficacies of these two treatment methods for acute rib fracture in elderly individuals.2.6.X\u2009\u00b1\u2009SD), and an independent sample t test was used for comparisons between groups. Quantitative data with a nonnormal distribution were analysed as medians and quartiles [M (p25\u2013p75)], and the Mann\u2012Whitney U-test was used for comparisons between groups. The Wilcoxon signed-rank test was used for comparison of paired quantitative data. Qualitative data were expressed as the frequency, and the \u03c72 test was used for comparisons between groups. p\u2009<\u20090.05 was considered to be statistically significant.SPSS version 26  was used for data analysis. Quantitative data with a normal distribution are expressed as the mean\u2009\u00b1\u2009standard deviation (3.p\u2009>\u20090.050). However, they were significantly different in terms of age, sex, diabetes, number of fractures, flail chest, AIS scores for the chest, limbs and body surface, ISS, and admission pain score (p\u2009<\u20090.050). After matching, none of the baseline conditions were significantly different between the two groups (p\u2009>\u20090.050).Of the 242 patients after matching, 98.8% had multiple rib fractures (239 cases), 36.0% had flail chest (87 cases), 52.9% had minor injuries (128 cases), 35.9% had serious injuries (87 cases), and 11.2% had severe injuries (27 cases). According to the visual analogue scale (VAS) score, 88.4% (214) of the patients had severe pain.To reduce the impact of underlying diseases and injury severity as confounding factors on the evaluation of therapeutic efficacy, we performed a 1:1 matching of age, sex, underlying diseases, smoking, fracture severity and complications in the two groups of patients. After matching, there were no significant differences in these basic conditions between the two treatment groups or within a group before treatment . The mat3.1.p\u2009>\u20090.050); however, the length of hospital stay was significantly longer in the surgery group than in the control group (p\u2009<\u20090.050) (p\u2009<\u20090.050), except that women, those \u226575 years old, and those without pleural effusion did not show significant differences (p\u2009>\u20090.050).There was no significant difference in the length of ICU stay or mechanical ventilation duration between the surgery group and the control group (<\u20090.050) . Table\u00a033.2.p\u2009>\u20090.050)  .3.3.p\u2009<\u20090.050). The fracture healing rate in the surgery group was significantly higher than in the control group (p\u2009<\u20090.050). Additionally, the healing time in the surgery group was significantly shorter than in the control group (p\u2009<\u20090.050).After 9 months of follow-up, the fracture healing rate was 96.67% in the surgery group and 88.89% in the control group. There was a significant difference in the healing rate , 6 and hp\u2009=\u20090.000), and the pain score was lower after surgical treatment. There were significant differences in the improvement of pain score and the duration of pain medication use between the surgery group and the control group (p\u2009<\u20090.050). Pain relief was significantly better in the surgery group than in the control group, and the duration of pain medication use was significantly shorter in the surgery group  . AdditioHowever, as a special population, there is still a lack of specific research on the evaluation of surgical treatment of rib fractures in patients over 60 years old. Rib fracture is the most common chest trauma and is often associated with adverse outcomes such as lung infection, ARDS, respiratory failure, and death , 22. TheBased on the diagnosis and treatment experience of our center and previous literature reports, there are significant differences in the characteristics of trauma between the elderly and young people. The main cause of injury in the elderly is blunt trauma . ElderlyThis study showed that compared with conservative treatment, surgical treatment can extend the length of hospital stay to some extent, but it has advantages in terms of the fracture healing rate, fracture healing time and fracture pain relief.Slobogean GP et al.  conducteWe found that there was no significant difference in the length of ICU stay or mechanical ventilation between the two groups. However, the length of hospitalization was significantly longer in the surgery group than in the control group. We analysed the baseline factors that may have affected the length of hospital stay. Except for the pleural effusion group, the length of stay in the surgery group was significantly longer than in the control group regardless of whether the patients had flail chest, pneumothorax or atelectasis before surgery. This may be related to the poor recovery ability of elderly individuals after surgery. A recent meta-analysis by Choi J  showed tn\u2009=\u2009123) and therefore the correlation analysis was not reliable. In our study, the mortality rate decreased significantly after sample matching, indicating that the mortality results are indeed affected by the sample size. Mortality in both groups was very low, especially in the surgery group, which had a lower mortality than the control group, but the difference was not statistically significant.Few datasets on rib fractures in elderly individuals have been available for previous studies. Nevertheless, previous studies have found that the overall mortality for patients with rib fracture ranges from 0.0% to 33.3%, compared with 0.0% to 30% for patients who received SSRF and 0.0% to 57.9% for those who received nonsurgical treatment , 37, 38.Marasco S et al.  found thPatients in the surgery group were significantly better than those in the control group in terms of the improvement in pain score and the duration of pain medication use . This stAs for the cost of treatment, given the advantages of the surgical group, pain improvement with fewer drugs, less ICU and mechanical ventilation need to make this treatment option more cost-effective. But on the other hand, the cost of surgical implants, surgical procedures, anesthesia, occupational therapy services and initial specialized equipment, as well as consumables, may be a major barrier for acceptance of this technique.Based on a large amount of previous clinical experience, our centre has developed indications for the selection of surgery to treat elderly patients with rib fracture. At present, our centre recommends SSRF for elderly patients with flail chest and who have no contraindications to surgery. For non-flail chest patients with two or more rib fractures and obvious displacement of the broken ends in more than half of the fractures, surgery is recommended. For patients with non-flail chest with complications, conservative analgesia and respiratory management should be routinely used. If not effective, patients with early severe pain and well-aligned broken fracture ends without obvious surgical contraindications are recommended for surgical treatment. Patients with severe complications should be treated with positive pressure ventilation by an endotracheal intubation ventilator. For patients with poor conservative treatment, respiratory deterioration and long-term chest floating, if there is no obvious contraindication, physicians should consider SSRF.There are some limitations to this study. The number of patients was greatly reduced after matching, which may have influenced the results. This study was a single-centre study, and the results showed that the length of hospital stay, length of ICU stay, mechanical ventilation duration, and mortality were all lower than in previous studies. As a primary trauma and burn treatment centre located in Beijing, we receive critical chest trauma patients from all over the country, and our institute is a leader in surgical quality, ICU quality and nursing quality in China. Therefore, the results may not be generalisable to other treatment centres. To more objectively evaluate the curative effect of SSRF in China, studies are needed with larger sample sizes and that include more centres.With the development of surgical techniques and the improvement of quality of life, age is no longer an absolute contraindication for surgical treatment. We hope that surgical treatment can provide better options for elderly patients and improve patients' quality of life. Of course, surgery itself is traumatic, especially for the elderly, so the length of hospital stay after surgery will be prolonged, but the mortality rate of patients does not increase, and the long-term recovery of patients also shows a better effect, we hope that through our efforts can provide more excellent treatment methods for the treatment of patients with rib fracture.5.In conclusion, we studied the efficacy of two treatment methods for elderly rib fracture patients using the propensity score matching method. We found that surgical stabilization of rib fractures extended the length of hospital stay compared with conservative treatment, but it had advantages in terms of the fracture healing rate, healing time, and pain relief. For rib fractures in elderly individuals, surgical treatment does not significantly increase mortality when applied under strict surgical indications."}
+{"text": "Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by a constant accumulation of lipids in the liver. This hepatic lipotoxicity is associated with a dysregulation of the first step in lipid catabolism, known as beta oxidation, which occurs in the mitochondrial matrix. Eventually, this dysregulation will lead to mitochondrial dysfunction. To evaluate the possible involvement of mitochondrial DNA methylation in this lipid metabolic dysfunction, we investigated the functional metabolic effects of mitochondrial overexpression of CpG (MSssI) and GpC (MCviPI) DNA methyltransferases in relation to gene expression and (mito)epigenetic signatures. Overall, the results show that mitochondrial GpC and, to a lesser extent, CpG methylation increase bile acid metabolic gene expression, inducing the onset of cholestasis through mito-nuclear epigenetic reprogramming. Moreover, both increase the expression of metabolic nuclear receptors and thereby induce basal overactivation of mitochondrial respiration. The latter promotes mitochondrial swelling, favoring lipid accumulation and metabolic-stress-induced mitophagy and autophagy stress responses. In conclusion, both mitochondrial GpC and CpG methylation create a metabolically challenging environment that induces mitochondrial dysfunction, which may contribute to the progression of MASLD. Metabolic dysfunction-associated steatotic liver disease (MASLD) is a growing epidemic, which mirrors the increased trend of obesity in Western diet-consuming countries. It has an estimated prevalence of 20\u201330% in Europe and is the most common cause of chronic liver disease worldwide ,2. MASLDPNPLA3 gene being one of the main genetic risk factors . In. In27]. \u03b1, HNF4) ,43 was id stress . Interesd stress . Hepatic bile acid synthesis is the major catabolic mechanism for cholesterol elimination from the body and is strictly regulated. Interestingly, recent studies identified bile acid metabolic dysregulation-induced cholestasis as a key factor in steatohepatitis disease etiology in MASLD ,49,50,51Furthermore, new evidence links disturbed bile acid metabolism to mitochondrial dysfunctions, including changes in respiration, mitochondrial swelling, and a decrease in mitochondrial transmembrane potential ,53,54. IAltogether, these findings suggest that mitochondrial GpC/CpG methylation elicits metabolic stress damage, which needs to be overcome by a fast turnover of dysfunctional mitochondria through the process of mitophagy. Consequently, mitochondrial methylation could represent another risk factor in MASLD, given the mounting body of evidence demonstrating a robust correlation between impaired mitophagy\u2013autophagy and the progression of MASLD ,59,60,61NR5a2 (LRH1)), known to be involved in upregulation of mitochondrial respiration, as a direct consequence of GpC/CpG mtDNA hypermethylation , which reflects the \u2018length-to-width ratio), and surface area in Fiji [p-value < 0.05 was considered statistically significant.Cells were seeded in a T25 culture flask in a humidified atmosphere (37 \u00b0C and 5% CO in Fiji , as desc in Fiji . Statist"}