Situs inversus totalis (SIT) is an extremely rare anomaly characterized by a left‐to‐right reversal of all the thoracic and abdominal organs. Only 11 cases of esophageal cancer with SIT have been reported worldwide, most of which underwent hybrid minimally invasive esophagectomy (MIE) but not total MIE. Here, we report a case of esophageal cancer with SIT successfully treated by total MIE, with a right lateral‐prone position adopted during the thoracic procedure. The relevant literature is also discussed and reviewed. Situs inversus totalis (SIT) is an extremely rare anomaly characterized by a left‐to‐right reversal of all the thoracic and abdominal organs. Only 11 cases of esophageal cancer with SIT have been reported worldwide, most of which underwent hybrid minimally invasive esophagectomy (MIE) but not total MIE. Here we report a case of esophageal cancer with SIT successfully treated by total MIE, with right lateral‐prone position adopted during the thoracic procedure. Situs inversus totalis (SIT), with an incidence of 1/8000 to 1/25000 live births, is a rare anomaly that refers to a mirror image reversal of the thoracic and abdominal organs.A 66‐year‐old male presented with a three‐month history of progressive dysphagia. The chest X‐rays, barium swallow and computed tomography (CT) confirmed the presence of SIT and middle third esophageal cancer Fig . Endosco2 insufflation was applied in both phase 1 and phase 2 with a pressure of 6 and 12 mmHg, respectively. In phase 1, the patient was placed in the right lateral‐prone position using four ports  the thoracoscopic operation phase to mobilize the esophagus and dissect the mediastinal lymph nodes; (ii) the laparoscopic operation phase to mobilize the stomach, dissect abdominal lymph nodes and create a gastric conduit; (iii) the cervical operation phase to perform anastomosis and dissect cervical lymph nodes through a right (not left) neck incision. COorts Fig . The camorts Fig . The thoorts Fig , d. In ports Fig . Five poorts Fig . A gastrA total of 48 lymph nodes were harvested during surgery. Total operative time from incision to closure was 480 minutes and the estimated blood loss was 80 mL. Postoperative examination was normal Fig , and theSitus inversus totalis (SIT) is a rare congenital anomaly of unknown etiology, and most people with SIT are asymptomatic and unaware of their unusual anatomy until they present for imaging examination of unrelated diseases. To the best of our knowledge, only 11 cases of esophagectomy for esophageal cancer with SIT have been previously reported globally  image reconstruction was a helpful adjunct as it provided the most graphic representation of the orientation of organs, especially for patients with SIT.Taken together, here we present a rare case of esophageal cancer with SIT that was successfully treated by total MIE. Although the surgical procedures of esophageal cancer with SIT are technically more challenging, the surgical procedure itself does not differ from routine surgery after careful recognition of the mirror‐image anatomy. Total MIE should therefore be considered as one of the feasible and safe procedures for esophageal cancer with SIT.The authors have no conflict of interest to disclose.


Arabidopsis thaliana; (b) provide summaries from recent reports submitted by MASC subcommittees, projects and resources associated with MASC and from MASC country representatives; and (c) initiate a call for ideas and foci for the “fourth decadal roadmap,” which will advise and coordinate the global activities of the Arabidopsis research community.The multinational Arabidopsis research community is highly collaborative and over the past thirty years these activities have been documented by the Multinational Arabidopsis Steering Committee (MASC). Here, we (a) highlight recent research advances made with the reference plant  The multinational Arabidopsis research community is highly collaborative. This has been demonstrated through global efforts to publish the Arabidopsis genome sequence in 2000; the long‐term support for the three international stock centres in the United States, United Kingdom and Japan; and in the development of community‐facing informatics resources such as The Arabidopsis Information Resource (TAIR), the Bio‐Analytic Resource for Plant Biology (BAR), the Munich Information Centre for Protein Sequences (MIPS), the International Arabidopsis Informatics Consortium (IAIC), and Araport.The Multinational Arabidopsis Steering Committee (MASC) has represented this global community through its joint oversight of the annual International Conference on Arabidopsis Research (ICAR); production of a MASC annual report1In 2019 according to the U.S. National Center for Biotechnology Information’s PubMed, the annual number of Arabidopsis publications increased after plateauing in 2013 Figure , which sThe publication and citation data point to the continued value of Arabidopsis research to plant sciences in general, but more importantly in the area of curiosity‐driven and discovery‐based science. In 2019, many high‐profile “Cell‐Nature‐Science” (CNS) publications featured Arabidopsis research and described several “firsts” in plant science. These included the discovery in plant nuclei of liquid–liquid phase separations of polyadenylation complexes  still enable breakthroughs to be made apace. We turn now to efforts to coordinate such resources, and highlight newly generated large datasets that will, in turn, enable further discoveries.2Plant Immunity” subcommittee and the activities of the “Phenotyping” subcommittee being subsumed within the International Phenotyping project reports. The other MASC subcommittees are “Bioinformatics,” “Clone‐based ORFeomics,” “Epigenetics and Epigenomes,” “Metabolomics,” “Natural Variation and Comparative Genomics,” “Proteomics” and “Systems and Synthetic Biology.” Below we highlight some important findings from each MASC subcommittee.The MASC Subcommittees were established in 2002, at the beginning of the second decadal roadmap, and serve to bring together international groups of scientists who work in a thematic research area. Over the past 18 years these subcommittees have contributed to the MASC annual report, led sessions at ICAR meetings and engaged with the wider community. There are currently eight MASC subcommittees; changes for 2019‐2020 include a new “2.1https://www.proteomicsdb.org) and ATHENA (http://athena.proteomics.wzw.tum.de:5002/master_arabidopsisshiny/).The Proteomics subcommittee reports that over the past year a significant development in Arabidopsis research came from a study by Mergner et al.  that quaThe Mergner et al.  manuscri2.2http://ipf.sustc.edu.cn/pub/athrna/). This resource will dovetail with a soon‐to‐be released resource of whole genome sodium bisulfite sequencing datasets.Arabidopsis has been the workhorse for elucidating mechanistic underpinnings of numerous epigenetic phenomena. These studies have both discovered and emphasized the importance of small RNAs, histone modifications, and DNA methylation during epigenome establishment and maintenance, in detection of self from non‐self, and in responding to various environmental challenges. This research is aided by a new community‐facing Arabidopsis RNA‐seq database, ARS, which contains gene expression data from more than 20,000 publicly available RNA‐seq libraries (2.3https://bit.ly/2Qhb5xC) and the Thalemine instance hosted by the BAR . The third component of the revamped Araport is a new Arabidopsis‐focused instance of the Genome Context Viewer (https://gcv‐arabidopsis.ncgr.org), developed and maintained by Andrew Farmer and Alan Cleary at the National Center for Genome Resources  platforms, that metabolomics be considered an equal partner to sister techniques in order to develop a “full picture of a plant.”2.5During the period of the current decadal roadmap the interrogation of natural variation has been an area of clear success. The hugely successful 1001 Genomes project has led to the development of software tools for further analysis of these publicly available datasets. These including the ViVa: Visualizing Variation  and in the TuxNet software that analyses RNA‐seq data to infer gene regulatory networks (https://omictools.com/tuxnet‐tool). The subcommittee has been active in the organization of external meetings with the inaugural International Plant Systems Biology Meeting taking place in France in September 2018, with a follow‐up event in Venice postponed until 2021 (https://meetings.embo.org/event/20‐plant‐systems).During 2019 there has been significant activity in the research area of the MASC Systems and Synthetic Biology subcommittee, particularly with the development of BAR‐integrated software tool to analyse protein‐protein and protein‐DNA interaction networks . Despite reporting a downward trend over the past decade, the ABRC distributes annually almost twice as many seed stocks as NASC (190K versus 100K). However, NASC has seen increases over the past few years that undoubtedly reflects that it is the go‐to stock centre for orders from China. The continued success of the stock centres relies on donations of plant material (mostly seeds) from the community and NASC reports that over the past few years German scientists have provided their largest number of donations.www.phylogenes.org), a new resource that facilitates inference of gene function based on phylogenetic relationships, and with the open access MicroPublications journal (https://www.micropublication.org/) that facilitates the publication of brief, novel findings, negative and/or reproduced results that may lack a broader scientific narrative. Each week TAIR loads 50–90 papers with the term “Arabidopsis” in the title or abstract into their curation queue. This includes a steady number of papers that report on new functions for previously characterized genes and an increase in the number of papers that describe high‐throughput experiments and contain large datasets. For a variety of reasons, curating from some papers can be challenging so TAIR have produced a document to advise researchers how to make the details of their research more “findable” (https://conf.arabidopsis.org/pages/viewpage.action?pageId=22807345).Despite losing government funding over 5 years ago, TAIR continues its excellent biocuration services via an innovative and successful subscription model . Fortunately, the implementation of these recommendations will be facilitated by a closely linked international community and will undoubtedly be a topic discussed for inclusion within the next roadmap.Oversight of the Arabidopsis informatics strategy has largely fallen to the International Arabidopsis Informatics Consortium (IAIC), which was funded by the NSF until 2020. In 2018 IAIC hosted a workshop in St Louis and its “take home” recommendation was for the establishment of a centralized “annotation authority” to advise on submissions from groups for new gene names across the Arabidopsis pangenome, to establish a consistent naming scheme, to distribute this format regularly and frequently, and to encourage its adoption .The International Plant Phenotyping community provides excellent links between Arabidopsis research and that conducted in other plant species. The MASC annual report includes brief updates from the International Plant Phenotyping Network (IPPN), European Plant Phenotyping Network (EPPN) and European Infrastructure for Plant Phenotyping (EMPHASIS). An important general feature of these networks is that they host regular calls for applications from researchers to access phenotyping infrastructures where they do not have them available at their home institutions. The European Infrastructure for Plant Phenotyping (EMPHASIS) project is arguably the most interesting development in this area. When it becomes fully operational in 2022 EMPHASIS will place plant phenotyping in a position to obtain centralized European funding through similar mechanisms to those that support other large European infrastructure projects such as the Square Kilometre Array for radio astronomy , and collaborators from the United States, Germany, and Japan. We also expect that there will be contributions toward decadal priorities from a broader group of MASC members, especially those representing countries with significant and increasing Arabidopsis research, such as China or India. The positive sense of East‐West collaboration that was felt by those who attended ICAR2019 in Wuhan was a promising beginning to these discussions.6What are the strategies that might be used to build globally sustainable digital infrastructures to support the integration of multi‐omic datasets?How can both the data and metadata from complex multi‐omic experiments be collated and shared for the benefit of the wider community in order to feed into translational pipelines?How can we integrate mechanistic and quantitative genetic insights to enable plant acclimation to vastly different climates, within a very short time period?How can the community build internationally cohesive and diverse collaborative teams of scientists to answer important questions in plant science?Over the next year, these ideas will be developed and will coalesce during discussions at a MASC‐supported discussion session at ICAR2021 in Seattle. The roadmap will be launched and published prior to ICAR2022 in Belfast and will hopefully lead in the planning of community‐driven projects over the coming decade.GP and NP conceived the manuscript, GP, NP, SB, and BU contributed to the manuscript. Members of Multinational Arabidopsis Steering Committee (MASC) provided background content.


Early cancer recurrence after oesophagectomy is a common problem, with an incidence of 20–30 per cent despite the widespread use of neoadjuvant treatment. Quantification of this risk is difficult and existing models perform poorly. This study aimed to develop a predictive model for early recurrence after surgery for oesophageal adenocarcinoma using a large multinational cohort and machine learning approaches.Consecutive patients who underwent oesophagectomy for adenocarcinoma and had neoadjuvant treatment in one Dutch and six UK oesophagogastric units were analysed. Using clinical characteristics and postoperative histopathology, models were generated using elastic net regression (ELR) and the machine learning methods random forest (RF) and extreme gradient boosting (XGB). Finally, a combined (ensemble) model of these was generated. The relative importance of factors to outcome was calculated as a percentage contribution to the model.A total of 812 patients were included. The recurrence rate at less than 1 year was 29·1 per cent. All of the models demonstrated good discrimination. Internally validated areas under the receiver operating characteristic (ROC) curve (AUCs) were similar, with the ensemble model performing best . Performance was similar when internal–external validation was used . In the final model, the most important variables were number of positive lymph nodes (25·7 per cent) and lymphovascular invasion (16·9 per cent).The model derived using machine learning approaches and an international data set provided excellent performance in quantifying the risk of early recurrence after surgery, and will be useful in prognostication for clinicians and patients. Early recurrence after surgery for adenocarcinoma of the oesophagus is common. A risk prediction model was derived using modern machine learning methods that accurately predicts risk of early recurrence using postoperative pathology.Machine learning may help Oesophageal adenocarcinoma carries a poor prognosis. Among the less than 40 per cent of patients who are candidates for curative treatmentEarly recurrence (less than 1 year) after surgery is a feared outcome, with rates of 20–30 per cent frequently reportedIn an ideal setting, prediction of early recurrence before embarking on a multimodal surgical pathway would provide useful information for patients and clinicians. However, staging information correlates poorly between preoperative and postoperative settingsNaive logistic regression has been the dominant approach to binary outcome prediction in clinical medicine for decades. Adoption of modern modified regression and machine learning techniques has been limited, in part owing to concerns over computational complexity and reliability. However, an increasing body of evidence has demonstrated that they outperform traditional techniques in predictive performanceThe Oesophageal Cancer Clinical and Molecular Stratification (OCCAMS) ConsortiumThe OCCAMS Consortium is a UK‐wide multicentre consortium set up to facilitate clinical and molecular stratification of oesophagogastric cancer. It has ethical approval for biological sample collection and analysis in conjunction with detailed clinical annotation (Research Ethics Committee number 10/H0305/1). Data collection and participation in research were approved by institutional ethics committees at each OCCAMS site and University Medical Centre (UMC) Utrecht.a priori. To include NACRT as a factor in the model, further patients were identified from University Hospitals Southampton and UMC Utrecht, where CROSS ‐type NACRTData were sourced from six tertiary oesophagogastric centres in the UK, as described previouslyThe primary outcome measure was early recurrence, defined as confirmed local, regional or distant recurrence at less than 1 year from the date of surgeryU and χ2 tests. The predictive models were generated on the whole data set. All available variables were included in the analysis. A circumferential resection margin (CRM) of less than 1 mm was considered to be involved (and hence R1), in accordance with Royal College of Pathologists (RCP) guidelinesUnivariable statistics were calculated using non‐parametric Mann–Whitney Elastic net regularized logistic regression (ELR)Appendix For ELR, the optimal α and λ hyperparameters  were selected by grid search using tenfold cross‐validation with five repeats during model generation and log loss as the metric for optimization. The RF model was derived from 1000 decision trees and hyperparameter tuning was conducted in a similar fashion . The XGB model was again derived by cross‐validation of hyperparameters , regularization penalty (η) and subsampling for regularization). Full details of hyperparameter tuning are available in Discrimination of the models was assessed using the area under the receiver operator characteristic (ROC) curve (AUC). In the context of this paper, if two random patients were selected, one with recurrence of cancer at less than 1 year and one disease‐free at 1 year, the AUC is equivalent to the probability the model will score the patient with recurrence higher than the patient without. Internal validation was performed using 0·632 bootstrapping, with 1000 resampled data sets. Bootstrapping was preferred for internal validation over splitting the cohort into derivation and validation sets, as this has been shown to reduce bias and improve overall model performance, particularly with moderately sized data setsUnadjusted tree models  and other maximum margin methods typically calibrate poorly as a consequence of their methodology, with predicted probabilities biased towards the centre. To allow meaningful interpretation of probability, isotonic regression was used to scale probabilities on the final model, as described previouslyIn contrast to logistic regression, assessing global variable importance is challenging using machine learning techniques and to an extent they are black boxes. As coefficients, as seen in logistic regression, are not used, an alternative method is required. The VarImp function of the caret R package was used, where ROC curves are generated for the outcome for each individual predictor, and the contribution to the global ROC curve calculated as a percentage. Owing to the nature of higher‐order interactions present in the model, variable importance in individual predictions must be calculated independently. The mean marginal contribution of each variable was calculated . Models were trained using the carethttps://uoscancer.shinyapps.io/EROC/). No data entered into the model were collected or stored.The calibrated final model was designed using R ShinyFig. versus 21·7 per cent), had worse ypT and ypN categories, R1 resection rate and grade of differentiation, and were more likely to have lymphovascular invasion   and internally–externally (across centres). All models demonstrated excellent discrimination on the training set (apparent discrimination). The RF model performed the best (AUC 0·980), followed by the ensemble model 0·902), XGB (0·849) and ELR (0·805) . Probabilities generated by the final model were scaled using isotonic regression. Calibration before and after scaling is shown in Fig. Table 2 value of 38·0 (P < 0·001) before and 4·5 (P = 0·806) after scaling. Similarly, the Brier score, a measure of overall model performance, also improved from 0·119 to 0·114.Calibration on the training set was visually best in the ELR, and worst in the RF and ensemble models  after surgery is the identification of patients who are at high risk of recurrence. The authors have purposefully avoided dichotomization/stratification based on outcome, and presented raw probability in preference to this. This will allow full discussions between surgeons/oncologists and patients regarding the benefits of adjuvant therapy and tailored to the individual patient's postoperative recovery and wishes. It may also allow stratification of adjuvant trials based on layered levels of risk.This cohort exhibited an early recurrence rate of 29·1 per cent, which is similar to that in previous reportsDiscrimination of the different models was similar, with minimal variability in AUC values between models on validation. However, the ensemble model consistently performed the best and is a suitable choice for the final model. The decline in performance from the training set to validation, which was particularly marked for the RF and ensemble models, is a consequence of the tuning process, whereby the optimum values are chosen from a grid of thousands after repeated tests . In this setting, the apparent performance of the model on the training set is overestimated and should be disregarded.P = 0·061 ). This suggests that, although postoperative pathology is more favourable with NACRT, this does not translate to better outcomeThere was marked heterogeneity in variable importance between models. This is interesting, particularly in the context of the models performing so similarly overall, and supports the idea of combining them to capture different patient information. The most important variables overall were number of positive lymph nodes and lymphovascular invasion, which accounted for 42·6 per cent of performance in the final model. This is not only biologically sensible, but the subject of several recent publicationsversus distant) would be informative. However, owing to the historical nature of the data for the majority of the patients (collected for the first study) it was not possible to ascertain this reliably for most of the cohort. The concern with NACRT is that improved locoregional control is at the expense of undertreatment of microscopic distant disease, particularly where the radiotherapy field is limited anatomically (for example with GOJ tumours). The expected consequence of this would be fewer locoregional recurrences and more distant recurrences, although this has not been demonstrated in other comparative studies and a recently published RCTTo explore this further, details of recurrence location  is, on the whole, more pronounced for GOJ tumours compared with oesophageal tumours (an example of a second‐order interaction), despite the recurrence rate being higher for oesophageal than GOJ tumours.Other risk factors for early recurrence, including perioperative blood transfusionFig. In that context, gains from mathematical and computer‐based techniques are key to precision in delivery of cancer care. Here, several modern approaches that produce viable models were demonstrated. This study used a data set that was relatively small and simple in a machine learning context, and the improvement in performance over a standard logistic regression was small . This is nonetheless important as such an improvement is in effect ‘free’. The strengths of this study lie in its multicentre nature and the heterogeneity of the cohort. This approach should maximize the utility of the model on external populations. All the data points used should be collected routinely at the majority of institutions, which should allow uptake without change in practice. The College of American Pathologists (CAP) definition of CRM positivity (CRM positive if there is tumour at the resection margin) was derivable for centre G, and performance was preserved in this subgroup if that definition was used instead of the RCP definition (AUC 0·813 with model generated on centres A–F (650 patients) and validated on centre G (162)) (The training set was limited to patients undergoing neoadjuvant therapy for adenocarcinoma of the oesophagus. No attempt was made to apply the model to a chemotherapy‐naive population, and it is unlikely to calibrate well in this group owing to the differing influence on survival of yp compared with p stagingA large multicentre cohort of patients who underwent oesophagectomy has been used to derive an accurate prediction model for early cancer recurrence, with excellent performance on validation. Machine learning techniques represent an attractive proposition for maximizing performance of predictive models.Appendix S1. Supporting InformationClick here for additional data file.


Spontaneous degeneration of an intervertebral disc is caused by inflammation that accompanies exposure of the avascular nucleus pulposus to circulation, triggering an autoimmune inflammatory reaction. Both intrinsic and extrinsic mechanisms of IVD regulation by various cytokines are involved in disc degeneration and spontaneous hernia resorption through inflammatory responses. The major goal of this narrative review was to assemble our past findings about the potential role of cytokines in disc diseases and to clarify directions for future research. A member of the tumor necrosis factor‐α (TNF‐α) superfamily, TNF‐like weak inducer of apoptosis (TWEAK) is constitutively expressed in the intervertebral disc, and induces a chronic, but relatively weak inflammatory response, thereby suppressing the formation of cartilage matrix and inducing production of matrix metalloproteinases (MMPs). Previously we indicated that TWEAK is involved in intervertebral disc degeneration by inhibiting the production of cartilage matrix in the intervertebral disc, and inducing the further expression of MMP‐3. Thymic stromal lymphopoietin (TSLP) is expressed primarily by epithelial cells, and induces inflammation at the time of tolerance failure in allergic disease. We found TSLP induced migration of immunocompetent cells to the disc in intervertebral disc disease by promoting the production of monocyte chemoattractant protein‐1 (MCP‐1) and macrophage inflammatory protein‐1 alpha (MIP‐1α) by the intervertebral disc and these cells may be involved in the resorption of herniated disc tissue. Considering the pivotal role of TWEAK and TSLP we review our current understanding of these factors and their involvement in disc degeneration. However, anti‐ or cytokine therapy for disc degeneration disease is not yet established or applied clinically. Therefore, a more detailed understanding of the role of cytokines in disc degeneration would be valuable. In intervertebral disc herniation, spontaneous retraction of the hernia mass has been confirmed,Major inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) may contribute not only to the natural regression of hernia masses,In previous reports, we described our established “mouse disc tissue culture,” that enabled us to extract discs from the caudal vertebrae of the mouse under a microscope, and culture and analyze the tissue under various stimulation by cytokines.We found that disc degeneration induces TNF‐like weak inducer of apoptosis (TWEAK) and Fn14 signaling.1.1We previously found that both TWEAK and Fn14 are expressed in the intervertebral disc Table ,10, 14, 1.2To date, many studies have focused on the mechanism by which major inflammatory cytokines such as TNF‐α and IL‐1β cause disc degeneration.However, although findings have been obtained in tissue culture of intervertebral discs from mouse and human surgical samples, how TWEAK and Fn14 contribute to intervertebral disc degeneration and the effect of their signal in vivo remains unknown. TWEAK neutralizing antibodies have been administered systemically in a mouse model of rheumatoid arthritis (RA), and as a consequence inflammation in the joints was suppressed, which holds promise for applications in vivo.1.32Thymic stromal lymphopoietin (TSLP) is an IL‐7‐related cytokine that is produced mainly by epithelial cells of trachea and skin, and is involved in regulating the function of immunocompetent cells that cause allergic diseases, such as bronchial asthma and atopic dermatitis.TSLP is highly expressed in diseased tissues of patients with allergic diseases such as asthma and atopic dermatitis, and analysis using genetically modified mice revealed that the action of TSLP is required to produce antigen‐specific allergic conditions. It is possible that TSLP produced by epithelial cells when exposed to exogenous stimuli acts on innate immune cells such as dendritic cells (DC), mast cells, eosinophils, and natural killer T (NKT) cells, which have an important role in the early stage of inflamation.Angiogenesis is a key pathophysiology in many inflammatory diseases such as tumors and traumas.2.1From our previous work, we propose that TSLP is expressed by the annulus fibrosus when exogenous stimulation applied to the intervertebral disc promotes the production of MCP‐1 and MIP‐1α by the intervertebral disc Table ,9, 30the2.2TSLP is highly expressed in diseased tissues of patients with allergic diseases such as asthma and atopic dermatitis, and analysis using genetically modified mice revealed that the action of TSLP is required to produce antigen‐specific allergic conditions.Further study in vivo and analyzing the presence of TWEAK and TSLP in healthy human tissues should be carried out to support our data obtained in vitro.3Taken together, our previous work illustrates the ability of inflammatory cytokines to induce intervertebral disc degeneration and spontaneous hernia resorption by various mechanisms.The authors declare no potential conflict of interest.T.O. analyzed and interpreted the data was a major contributor in writing the manuscript. H.H. was editorial supervisor of this study. Both authors read and approved the final manuscript.


We report utilization patterns and characteristics of patients treated with biologic anti‐inflammatory agents in a large commercially insured patient population in the United States. We identified adult (age ≥18 years) patients receiving biologic anti‐inflammatory agents between 1 January 2012 and 31 March 2019 across the five Research Partners in the Biologic and Biosimilars Collective Intelligence Consortium's Distributed Research Network. We examined the number of incident use episodes for each biologic, as well as patient demographic and clinical characteristics. Curated data and analytic tools from the Food and Drug Administration's Sentinel System were used to perform the analyses. We identified 90,360 incident episodes of tumor necrosis factor‐alpha inhibitors (TNFi) and 70,506 incident episodes of non‐TNFi medications. Adalimumab was the most common TNFi drug  and showed a steady increase in utilization during the study period compared to other TNFi agents. Rituximab was the most commonly initiated non‐TNFi medication (44% of non‐TNFi episodes). Other non‐TNFi agents, namely, ustekinumab, vedolizumab, and secukinumab, demonstrated notable increases in utilization over time. Biosimilar use was limited; we observed 653 incident episodes for infliximab‐dyyb and 39 incident episodes for infliximab‐abda. As more biologics enter the market, greater variation in the use of biologics with similar indications and between biologic originators and biosimilars is anticipated. Because information on efficacy and safety at the time of drug approval is limited, post‐marketing surveillance and research is needed to monitor medication safety and evaluate effectiveness between biologic drugs using real‐world data. BBCICBiologics and Biosimilars Collective Intelligence ConsortiumDRNdistributed research networkRArheumatoid arthritisTNFitumor necrosis factor‐alpha inhibitorWe analyzed utilization trends and patient characteristics for biologic anti‐inflammatory drugs available in the United States using a large healthcare claims database (Biologic and Biosimilars Collective Intelligence Consortium's Distributed Research Network) from January 2012 to March 2019.The total number of incident utilization episodes of biologic anti‐inflammatory agents steadily increased from 2012 to 2017 (the most recent year of complete data).Adalimumab and rituximab were the most commonly initiated tumor necrosis factor‐alpha inhibitor (TNFi) and non‐TNFi agent, respectively.1Since the first biologic agent, recombinant human insulin, was approved in 1982, biologics have accounted for one of the fastest growing segments of the prescription drug market in the United States (US).This Short Report presents usage patterns and characteristics of patients treated with biologic anti‐inflammatory agents in a large distributed network of commercially insured patients in the United States. Whereas previous utilization studies have generally focused on single or selected biologic agents ‐alpha inhibitors (TNFi) only) and have typically not considered indications across all therapeutic areas, our descriptive report provides a broad perspective of all relevant agents, indications, and all therapeutic areas.2www.bbcic.org). The BBCIC DRN consists of administrative claims data for approximately 95 million patient‐years from five Research Partners including two large national health insurers  and three regional health insurers or integrated healthcare delivery systems . These Research Partners participate in the Food and Drug Administration's (FDA) Sentinel System, enabling the BBCIC to leverage the curated data in the Sentinel Common Data Model, plus data standardization and analytic tools of the Sentinel System for conducting distributed analyses.We used data from the distributed research network (DRN) of the Biologics and Biosimilars Collective Intelligence Consortium (BBCIC), a non‐profit research consortium dedicated to investigation of the real‐world effectiveness and safety of biologics, including biosimilars in the US (https://www.bbcic.org/research/completed‐research).We identified adult age ≥18 years) patients receiving biologic anti‐inflammatory agents between 1 January 2012 and 31 March 2019 in the BBCIC DRN; some Research Partners provided data through only part of 2018. Medications of interest were currently available anti‐inflammatory biologic drugs in the US including both TNFi and non‐TNF‐alpha inhibitors (non‐TNFi); a full list of medications is provided in Table 8 years pAll patients were required to be continuously enrolled with medical and pharmacy coverage for a minimum of 365 days before the first observed administration/dispensing date for a medication of interest (index date). Coverage gaps of up to 45 days were bridged to account for potential administrative lapses in coverage. Incident use was defined as having no exposure to a given medication in the 365 days before the index date. Patients could contribute multiple “incident” episodes for the same or different drugs if they met the eligibility criteria, including the 365‐day washout period, each time. We examined the number of incident use episodes for each medication of interest, overall and according to patient demographic and clinical characteristics . Demographic characteristics were measured on the index date, and clinical characteristics were assessed during the 183‐day period before the index date (“baseline period”). The publicly available Sentinel System analytic toolkit  was used to perform the distributed analyses.Distributed analyses were conducted by each Research Partner, with results aggregated for final analysis. Cells with counts >0 and <10 are not presented to protect the anonymity of patients, per standard BBCIC practice; medications with <10 incident episodes are excluded from analysis. Institutional Review Boards for the participating Research Partners determined that this work does not meet the definition of human subjects research.3Over 30 million eligible health plan members who contributed more than 75 million person‐years met the study eligibility criteria. We identified 160,866 incident episodes across all study medications, including 90,360 incident episodes of TNFi products and 70,506 incident episodes for non‐TNFi medications , while the utilization for rituximab was relatively stable.The biologic agents with more than 10,000 incident use episodes were adalimumab, rituximab, etanercept, infliximab, and ustekinumab. All of these drugs were initially approved by the FDA before 2010, had expanded indications over time, and included indications across therapeutic areas. The biosimilar category included two drugs, infliximab‐adba (approved in 2017) and infliximab‐dyyb (approved in 2016). As these biosimilars are relatively new, it is still early to evaluate the trends of their utilization and the potential impact on the originator drug. The implications of biosimilars in the anti‐inflammatory biologics space in the US remain uncertain until further data on utilization become available.Several factors need to be considered in interpreting these results. Our data are generally representative of commercially insured individuals in the United States. Whether these findings would be applicable to other populations, for example, older patients with only Medicare insurance coverage, is uncertain. Also, as 2018 and 2019 data were partially complete, we were not able to fully analyze trends in utilization beyond 2017. Finally, there is the possibility of misclassification in identifying drug exposures and clinical characteristics through administrative claims data.As more biologics and biosimilars enter the market, it is anticipated that there will be a greater variation in the use across biologic products and more competition among products with similar indications and between biologic originators and biosimilars. Because information on rare but potentially serious adverse events at the time of drug approval is limited, post‐marketing surveillance will be important to monitor adverse drug events and evaluate outcomes between biologics and biosimilars in the real‐world environment. Comparative effectiveness and safety studies among these drugs are also important to inform clinical decisions for optimal treatment choices customized for individual patients. Given that biologic agents can be used for a variety of indications and patients may switch drugs, studies on indication‐specific benefits/risks of biologics and the impact of switching biologic agents on the treatment effectiveness and safety are warranted. Also, further research on potential factors associated with utilization patterns will be informative, including cost of treatments, route of administration, prevalence of indications, adverse drug events, step therapy, patient and provider preferences, and pharmacy benefit programs. Ongoing monitoring of biologics utilization patterns will help strategically identify future research priorities.5Institutional Review Boards for the participating Research Partners determined that this work does not meet the definition of human subjects research.Michael Kappelman is a consultant to Janssen, AbbVie and Takeda and receives research support from Janssen and AbbVie. All other authors have no competing interests.All authors contributed to the study conception and design for this work. Data analysis was led by James Marshall with input and direction from all other authors. Aaron Mendelsohn and Young Hee Nam prepared the first draft of the manuscript and all authors participated in the revision of the initial manuscript, and approved the final manuscript. All authors agree to be accountable for all aspects of the work.


Higher maternal anxiety scores were related to lower toddler Healthy Eating Index scores  with no significant variation over time. The relation between maternal diet quality and anxiety score varied over time . Higher maternal anxiety scores were associated with lower maternal diet quality at Time 1  and at Time 2 , but not at Time 3 . Findings suggest that mothers and toddlers exhibited similar low‐quality dietary patterns and that lower diet quality was associated with higher maternal anxiety scores. Approaches to enhance diet quality may consider incorporating anxiety‐reducing strategies into maternal and toddler care and feeding behaviour guidelines.We evaluated the association between maternal anxiety score and diet quality over time among mothers and toddlers in low‐income families. Longitudinal data were collected from 267 mother–toddler dyads in an obesity prevention trial. Participants were recruited from the Special Supplemental Nutrition Program for Women, Infants and Children and paediatric clinics between 2007 and 2010. Dyads were assessed at study enrolment (Time 1), 6‐month (Time 2), and 12‐month follow‐up (Time 3). On the basis of a 1‐day 24‐hr dietary recall, we estimated maternal and toddler diet quality using the Healthy Eating Index 2015. Anxiety, a time‐varying variable, was assessed via the State–Trait Anxiety Inventory. Associations between maternal anxiety score and maternal and toddler diet quality over time were assessed in adjusted mixed models. Maternal and toddler diet quality were positively correlated ( Maternal and toddler diet quality were positively associated.Mothers who reported higher anxiety scores had lower diet quality; this association was attenuated over time.Higher maternal anxiety scores were associated with lower toddler diet quality; the relationship was consistent over time.Toddler diet quality decreased over time, independent of maternal anxiety score.Toddlers who had, on average, mothers with higher anxiety scores relative to others tended to have lower diet quality. Future longitudinal studies with repeated measures of anxiety and diet should consider assessing the associations on a shorter timescale  to inform temporal specificity of anxiety–dietary intake relations.1In the United States, prevalence of high weight for length (>97th percentile) among infants and toddlers (<24 months old) reached 7.1% in 2011–2012  can be challenging for mothers, associated with toddlers' rapidly changing mood shifts, picky eating, food refusal, and food neophobia  due to additional concerns about food waste, time, and financial constraints  at different developmental stages of toddlerhood. This time‐varying relation is particularly important to study because anxiety may be time‐varying or constant Howard, . In addito examine the association between maternal state anxiety score and maternal diet quality over time;to examine the association between maternal state anxiety score and toddlers' diet quality over time; andto disaggregate the within‐person and between‐person associations between maternal state anxiety score and diet quality, if relation does not differ over time.This study had the following objectives:22.1Longitudinal data were obtained from a randomized obesity prevention trial of 277 low‐income mother–toddler dyads assigned to (a) safety (attention control), (b) maternal lifestyles , or (c) responsive parenting. Study subjects were recruited from a Women, Infants, and Children (WIC) clinic located in a semiurban community and a primary care paediatric clinic serving a low‐income urban community of the Mid‐Atlantic region of the United States between 2007 and 2010 , 187 mother–toddler dyads were interviewed; 181 and 171 mothers and toddlers had completed dietary recalls, respectively. At Time 3, we interviewed 224 mother–toddler dyads; 24‐hr dietary recalls were complete for 213 mothers and 202 toddlers. Figure 2.42.4.1We used an interviewer‐administered computerized multiple‐pass method (USDA AMPM) to collect data on intake of foods and beverages consumed within a 24‐hr period at three time points in mothers and toddlers . The HEI‐2015 was estimated on the basis of the 24‐hr diet recall, assessing the adherence to the 2015–2020 Dietary Guidelines for Americans (DGA) with high validity and reliability , and higher scores indicate higher consumption. In other four food components, lower intakes are desirable  and are coded such that higher scores indicate lower consumption. The 13 components were summed to generate the HEI total score for mothers and toddlers (0–100 points), where higher scores denote better diet quality. HEI components, range of possible scores, and average points over time are shown in Table 2.4.2not at all to very much so. Anxiety‐absent items were reverse coded, and the sum of the total score was calculated . Anxiety score in this study was treated in its continuous form.Maternal anxiety symptoms were measured using the six‐item short form of the state scale for the Spielberger State–Trait Anxiety Inventory (STAI)  for mothers. For toddlers, we used age‐ and sex‐specific World Health Organization growth charts to calculate BMI z‐scores  attributed to the longitudinal design of the study following the American Academy of Pediatrics recommendations  measured in centimetres using a Shorr measuring board  and weight (kg) using a Tanita 1584 Baby Scale  for toddlers and a Tanita 300GS for mothers measured in triplicate. All measures were assessed at three time points by trained research staff. BMI was calculated by dividing weight (kg) per height squared . A poverty ratio below 1 indicates that the average income for each respective family size is below the federal poverty threshold. The categorized income‐to‐poverty ratio variable was included as a covariate in all analyses.2.5p value of <.05.All variables were processed in QDS™ Warehouse Manager . All statistical analyses were conducted using Stata SE 15.1 for Windows software . For all analyses, the 95% confidence intervals (CIs) were reported. Statistical significance was defined by a Sociodemographic, anthropometric, dietary, and maternal anxiety variables were examined to describe the study population. Univariate distributions were examined to identify extreme values (outliers) and skewed continuous distributions; none were identified. Means, standard deviations (SDs), and prevalence (%) were estimated for key baseline descriptors. Multilevel growth models were used to assess change in mother and toddler's diet, and maternal anxiety score over time.Multilevel mixed‐effect models were performed to assess the association between maternal anxiety score in relation to maternal and toddler diet quality using maximum likelihood estimation, with random intercepts to account for the clustering of the repeated measures over time within the same individual. Our model specification checks, including assessment of model residuals, revealed that all normality assumptions were met when treating the outcomes in their continuous form. The intraclass correlation coefficients between repeated measures within each individual were .31 for mothers and .14 for toddlers, without including any predictor in the mixed‐effect models.First, we assessed the association between anxiety (time‐varying predictor) and diet quality over time. To assess whether the association varied over time, we included an interaction term between time of assessment and maternal anxiety score. On the basis of this model, we estimated and plotted the predicted mean diet quality over time by each level of maternal anxiety score. Because there is variation in the toddler's age at each time point, we conducted sensitivity analyses that assessed children's age (continuous) instead of the time point in the interaction.Then, if the association between diet and maternal anxiety score did not systematically change as a function of time, we disaggregated the within‐person and between‐person effects of maternal anxiety score and diet quality , and poverty rate (as time‐varying variables) and intervention group (time invariant). Variables were chosen on the basis of associations reported previously and significant associations with anxiety and diet quality. Sensitivity analyses were conducted additionally controlling for parity and age of the youngest child. For all the analyses, no significant effect by intervention group on diet quality was found in a three‐way interaction term ; thus, we combined the intervention and control groups, and the intervention group was treated as a covariate in the models.For the most parsimonious model, we selected the following covariates: maternal age (continuous), toddler age (continuous) and sex , maternal BMI (continuous) and toddler BMI . Mothers and toddlers exhibited similar dietary patterns  of low diet quality . Maternal anxiety scores were on average 10 points on the short‐STAI , meaning that most mothers reported not at all or somewhat anxious to each item. One third of mothers presented elevated anxiety scores (>12 points) at baseline. About half of the sample were first‐time mothers (48.1%), and anxiety scores did not differ between first‐time and other mothers (p = .77). There was no statistically significant change in either maternal/toddler diet or maternal anxiety score over time of assessment.On average, toddlers were 20.1 months (SD ±5.5), and mothers were 27.3 years old (SD ±6.2) at baseline. The majority of families lived below the poverty threshold (68.5%) and were enrolled in the WIC programme throughout the study. Mothers had an average of 2.4 children in the household, and living below the poverty line was positively associated with having more children in the household, compared with living above the poverty line . Higher maternal anxiety scores were associated with lower maternal diet quality at Time 1  and at Time 2 , but not at Time 3 . Therefore, we restricted the relation to be equal over time. Higher maternal anxiety score was associated with lower toddler diet quality , meaning that for each additional 6‐point increase in maternal anxiety score , there was a decrease in 1.6 HEI points for toddlers.The relation between maternal anxiety score and toddler diet did not vary over time , 95% CI , and was lower among toddlers who had mothers with higher anxiety scores compared with those with lower anxiety scores, although this difference did not vary statistically over time.Figure 4This study adds to our understanding of the relation between maternal anxiety and maternal and toddler's diet quality over time in a sample of low‐income WIC‐eligible families. Our findings suggest that maternal diet quality was lower among mothers who reported higher anxiety scores when their toddlers were between 12 and 30 months old, but differences were attenuated when children reached age 36 months.We found that toddlers' diets were of low quality, with slightly over 50 HEI points. Even though HEI scores are usually not calculated for children younger than 2 years (as this age group is not currently included in the DGA), similar studies have calculated the HEI score for this population . A nationally representative study reported diet quality at 64 HEI points among WIC toddlers between 13 and 24 months . In our sample of low‐income toddlers, HEI decreased for each additional month of age, as they transition from complementary feeding to family meals. This finding was not corroborated by a recent review that reported a positive association between family meals and child diet quality , possibly explaining the null association for the within‐persons effect. It is also possible that between‐ and within‐person levels of anxiety are associated with toddler diets through different mechanisms. Our study provides a first step towards disaggregating within‐ and between‐person sources of variance in the association between maternal anxiety score and toddler diet quality. Future longitudinal studies with repeated measures of anxiety and diet should consider assessing anxiety on a more frequent timescale  to inform temporal specificity of anxiety that is related to dietary intake. More intensive longitudinal data will not only be able to better estimate within‐ and between‐effects of anxiety and diet, but also be able to inform directionality of the association .Little attention has been given to health care services and policies among mothers of toddlers . Future longitudinal studies should include more intensive multiple assessment points with shorter “distance” between repeated measures taken on the same subject to explore reverse causality bias. Second, the assessment of diet quality might be susceptible to recall bias. Moreover, diet quality was based on 1 day of intake for mothers and toddlers for each time point. Although use of maternal report of child dietary behaviour for young children is standard, biased responses have been seen among mothers who experience mental health symptoms  and components of HEI for mother and toddler over time.Click here for additional data file.Table S2. Associations between maternal anxiety score and diet quality of mothers and toddlers across three time points.Click here for additional data file.


Successful development depends on the precise tissue‐specific regulation of genes by enhancers, genetic elements that act as switches to control when and where genes are expressed. Because enhancers are critical for development, and the majority of disease‐associated mutations reside within enhancers, it is essential to understand which sequences within enhancers are important for function. Advances in sequencing technology have enabled the rapid generation of genomic data that predict putative active enhancers, but functionally validating these sequences at scale remains a fundamental challenge. Herein, we discuss the power of genome‐wide strategies used to identify candidate enhancers, and also highlight limitations and misconceptions that have arisen from these data. We discuss the use of massively parallel reporter assays to test enhancers for function at scale. We also review recent advances in our ability to study gene regulation during development, including CRISPR‐based tools to manipulate genomes and single‐cell transcriptomics to finely map gene expression. Finally, we look ahead to a synthesis of complementary genomic approaches that will advance our understanding of enhancer function during development.Physiology > Mammalian Physiology in Health and DiseaseDevelopmental Biology > Developmental Processes in Health and DiseaseLaboratory Methods and Technologies > Genetic/Genomic MethodsThis article is categorized under: The correct spatiotemporal regulation of genes by enhancers is critical for successful development from a single fertilized egg to a complex multicellular organism. Herein, we discuss current genome‐wide approaches to identify enhancers and to dissect enhancer function. Figure created with BioRender. The correct spatiotemporal regulation of genes by enhancers is critical for successful development  Consortium  have been paired with next‐generation sequencing to generate genome‐wide maps of chromatin accessibility. DNase‐seq is based on DNase footprinting, in which DNase I hypersensitive sites are digested and DNA bound by TFs and other proteins is protected Figure c. While cis‐regulatory regions independent of any specific TF, cofactor, or histone modification. However, these methods identify promoters and insulators as well as enhancers. Furthermore, open chromatin does not necessarily mean that a given DNA sequence is active or functional. Enhancers located in open chromatin can be held in an inactive state by repressive TFs, which are common in development   Figure . This me2.6Enhancers direct gene expression in specific cell types, at specific times, and within specific developmental contexts, and thus it is important to use appropriate and homogeneous samples when performing genome‐wide techniques to identify putative enhancers. For example, TFs and other proteins can be expressed in a highly tissue‐specific manner, which could affect ChIP‐seq experiments as they rely on a specific protein of interest being present. Furthermore, the chromatin landscape assayed by ATAC‐seq or Hi‐C can depend on the gene regulatory programs active in a given cell type. Indeed, chromatin interactions are highly dynamic and can vary substantially between different cell types ‐like methyltransferase family   locus as a model system  promoter is known to be brought into contact with its distal T1 enhancer by a promoter‐proximal tethering element  to characterize the regulatory topology of Pitx1, which is required for hindlimb and mandibular arch development  data  have been developed. These methods are based on traditional reporter assays, in which a putative regulatory element is placed upstream of a reporter coding sequence  in a vector, transfected into cells, and assayed for reporter activity. Although the context is often heterologous, that is, using genetic elements from other organisms, these assays are a powerful tool with which to test the sufficiency of an element to regulate gene expression. MPRAs utilize advances in oligonucleotide synthesis  interrogate the activity of candidate enhancer sequences and (b) to investigate the functional consequences of mutations within known enhancers. Several studies have used MPRAs to functionally validate putative enhancers identified from genome‐wide assays  was developed in s Figure . This meCiona intestinalis, which belongs to the sister group of all vertebrates  method is able to test millions of enhancer variants for function in developing Ciona embryos  enhancer that contains GATA and ETS binding sites and directs specific expression in the neural plate  and the CRISPR‐associated protein 9 (Cas9)  along with Cas9 Figure b. The usPOU5F1 using human embryonic stem cells  can be fused to an effector domain that either activates (CRISPRa)  domain that represses gene expression  mutant zebrafish, which are defective in nodal signaling, no mutant‐specific transcriptomic states were identified, although wildtype states were significantly reduced or absent (Farrell et al., chordin locus in zebrafish, which is required for patterning the early dorsal ventral axis (Sasai et al., Tal1, a gene that is critical for hematopoiesis (Pijuan‐Sala et al., Tal1 mutant ESCs into wildtype blastocysts, and then dissociating and sorting cells from the resulting embryos prior to scRNA‐seq. Similar to findings in zebrafish, mutant cells did not form novel clusters. However, tdTomato‐positive, Tal1‐null cells from chimeric embryos were depleted of multiple blood cell clusters (Pijuan‐Sala et al., In maternal‐zygotic one‐eyed pinhead (MZMoving forward, comparing scRNA‐seq data from embryos bearing mutations within enhancers to stage‐matched wildtype transcriptomic atlases will be a valuable method for systematically assessing how enhancer sequences contribute to development on a global scale. This approach will provide unprecedented insight into the specific cell types that are affected by enhancer manipulations within the context of developing embryos.6.3When cells from embryos representing different developmental timepoints are subjected to scRNA‐seq, trajectories of differentiation can be reconstructed over time. Single‐cell trajectories are built by a computational approach termed pseudotime ordering (Trapnell et al., 6.4Schmidtea mediterranea, an immortal organism that continuously regenerates its tissues and has remarkable regenerative capabilities (Plass et al., Moving forward, we predict that methods to reconstruct developmental trajectories will be broadly applicable to the study of biological processes that share similarities with development, including cancer progression and tissue regeneration. Indeed, regeneration trajectories have been described in the regenerative planarian Xenopus, lineage topologies were found to be broadly conserved between species, while the observed abundances of different cell types were markedly different between species (Briggs et al., C. intestinalis provided new insight into the origins of the vertebrate telencephalon (C. Cao et al., The construction of developmental atlases may also be applicable to questions in evolutionary developmental biology. In a comparison of developmental trajectories from zebrafish and 6.5Several groups have demonstrated that these single‐cell methods can be performed in an efficient and cost‐effective manner, facilitating their application to the study of diverse developmental contexts. A current technical limitation is the inability to capture all of the different transcripts from a single cell, meaning that some transcripts will inevitably be undetected (J. Cao et al., cis‐regulatory dynamics in developing embryos. Further functional insight could come from scRNA‐seq of embryos bearing mutations in enhancer sequences. Because spatial information is lost when embryos are dissociated, construction of developmental atlases currently requires human annotation based on known marker genes. While human annotation can be semi‐automated and has been useful for validating these methodologies, in the future this limitation could be overcome by methods that couple spatial and transcriptomic data. For example, multiplexed fluorescence in situ hybridization techniques allow for the detection of thousands of RNA species to be imaged in single cells in tissue sections (K. H. Chen, Boettiger, Moffitt, Wang, & Zhuang, In future experiments, scRNA‐seq could be combined with single‐cell ATAC‐seq or Hi‐C to investigate the transcriptomic and chromatin landscapes of whole embryos. Although these combined approaches would not directly test enhancer function, they could identify putative developmental enhancers and provide insight into 7Since the discovery of enhancers over 30 years ago, technological advancements have greatly improved our ability to identify and study enhancers. Advances in high‐throughput sequencing, oligonucleotide synthesis, and genome editing technology have enabled new methodologies that have been critical in elucidating how enhancer sequences relate to function. The development of genome‐wide assays has enabled the identification of millions of putative enhancer sequences that are associated with particular chromatin signatures. Genome‐wide assays have also led to an increased interest in understanding enhancer function because it is now clear that the majority of disease‐causing genetic variants reside within putative enhancer sequences.Bottlenecks in our ability to functionally validate candidate enhancer sequences are starting to be overcome by MPRAs, although these are most valuable when done in a context‐specific way. Advantageously, MPRAs can be performed in whole embryos to give direct insight into tissue‐specific enhancer function during development. CRISPR screens are also useful because they allow enhancers to be identified and studied within the native genomic context, but such screens have not yet been done in developing embryos. The major challenge will be designing these in a way that is informative despite significant enhancer redundancy.Similarly, a recent wave of developmental atlases has given unprecedented insight into the transcriptomes of whole developing organisms with single‐cell resolution, but these methods are primarily descriptive in their present state. Given the current limitations of these methods, animal models are still important for their direct relevance to development and have proven to be a valuable tool with which to correct misconceptions that have arisen from genome‐wide data. Moving forward, technological advances in high‐throughput CRISPR screens and single‐cell transcriptomic analyses of developing embryos will likely allow for the study of enhancer function in developmental contexts. Together with MPRAs, these approaches will allow us to gain a broad understanding of which enhancer sequences are functional and important for successful development.The authors have declared no conflicts of interest for this article.Genevieve E. Ryan: Conceptualization; writing‐original draft; writing‐review and editing. Emma K. Farley: Conceptualization; writing‐original draft; writing‐review and editing.https://dx.doi.org/10.1002/wsbm.1390


EANPDB was combined with the previously published Northern African Natural Products Database (NANPDB), to form a merger African Natural Products Database (ANPDB), containing ∼6500 unique molecules isolated from about 1000 source species (freely available at http://african‐compounds.org). As a case study, latrunculins A and B isolated from the sponge Negombata magnifica (Podospongiidae) with previously reported antitumour activities, were identified via substructure searching as molecules to be explored as putative binders of histone deacetylases (HDACs).Medicinal plants have widely been used in the traditional treatment of ailments and have been proven effective. Their contribution still holds an important place in modern drug discovery due to their chemical, and biological diversities. However, the poor documentation of traditional medicine, in developing African countries for instance, can lead to the loss of knowledge related to such practices. In this study, we present the Eastern Africa Natural Products Database (EANPDB) containing the structural and bioactivity information of 1870 unique molecules isolated from about 300 source species from the Eastern African region. This represents the largest collection of natural products (NPs) from this geographical region, covering literature data of the period from 1962 to 2019. The computed physicochemical properties and toxicity profiles of each compound have been included. A comparative analysis of some physico‐chemical properties like molecular weight, H‐bond donor/acceptor, logP  This richness offers the African population diverse traditional means in treating ailments based on what nature has presented to them. However, due to poor documentation, some of this traditional information is being lost nowadays. This is one of the main factors behind the scientific exploration of the known traditional methods as well as the source species . Accordingly, several NPs have been identified and collected into several public and commercial databases and repositories.5, 6a Many of the medicinal plants from the Eastern African region have illustrated interesting values in traditional medicine, which makes use of indigenous knowledge to treat diseases.22.1The data was collected as part of our ongoing project, which is the development of chemical libraries of natural products from African medicinal plants, marine, fungal and bacterial sources.6e, 6f2.2inchified SMILES and the corresponding InChI are provided on our online platform.Data preparation was mainly done using PostgreSQL tables, as described in our previous publication.2.3The presence of certain structural features referred to as pan‐assay interference compounds (PAINS) have been established to certain behaviours . that could interfere in assay readouts all the way from target to cell without any common mechanism involved. The compounds of EANPDB were screened to estimate the proportion of molecules that are predicted to be PAINS. PAINS analysis was performed using PAINS1, PAINS2, and PAINS3 filters, as implemented in Schrödinger's Canvas program.2.4via a different mechanism.donor/accptHB, number of heavy atoms present, the number rotatable bonds, calculated molecular weight, predicted molar refractivity, the predicted total polar surface area and the computed octanol/water partition coefficient. Percentage variation based on counts as well as 2‐ and 3‐dimensional plots of PCA1, PCA2 and PCA3  were used to depict the diversity of molecules.Searching for novel compounds from a different chemical space with significant biological importance is currently vital in the field of drug discovery. This could be one approach towards facing the challenges of drug resistance. It is believed that such molecules could act 2.5In the field of small‐molecule drug discovery, scaffold novelty/diversity is an important issue for complicated (hard to treat) ailments.2.6mol_MW), the number of H‐bonds that would be donated/accepted by the solute to water molecules in an aqueous solution (donor/accptHB), the number of non‐trivial (not CX3), non‐hindered  rotatable bonds (#rotor), the computed octanol/water partition coefficient (QPlogPo/w), predicted IC50 value for the blockage of HERG potassium ion (K+) channels (QPlogHERG), predicted brain/blood partition coefficient (QPlogBB), predicted skin permeability (pQPlogK), the number of likely metabolic reactions (#metab), prediction of binding to human serum albumin (hsaQPlogK), the number of violations of Lipinski's “rule of five” (RuleOfFive) and the number of violations of Jorgensen's rule of three (RuleOfThree).Unique SMILES were used to generate the 3D models as well as the calculation of drug metabolism and pharmacokinetics (DMPK) properties of the collected molecules in our dataset using LigPrep and QikProp packages, respectively, of the Schrodinger software, as previously described.2.7in silico prediction of the toxicity was carried out on the freely accessible online pkCSM web server (Cambridge University) for all the EANPDB molecules.In a similar protocol.,2.8+‐dependent (Class III).Post‐translational modification of histone proteins by enzymes such as histone deacetylases  participate in several physiological processes and are considered potential drug targets for various diseases.A classical pharmacophore model for HDAC inhibitors as proposed by Jung et al.33.1via the manual curation of literature sources. The entire content of this database  can be accessed and downloaded from http://african‐compounds.org The current release of EANPDB is summarized in Table This study provides a simple to use and interactive online platform containing data for isolated NPs from sources of East African origin. For the sake of preserving the knowledge of the traditional application of medicinal plants in this region of Africa, this comprehensive database was developed Approximately 40 % of the molecules had no PubChem ID (CID). Additionally, >500 of the molecules at the time of isolation and characterization were being reported for the first time. Giving the chance, several other new molecules can still be isolated from some of the less explored plant families Figure . We obse1), synaptolepis factor K7 (2) and kirkinine (3)  Figure .[29, 1) was isolated by Rashid et al., from Toddalia asiatica (Rutaceae); a plant used traditionally in several communities in Kenya for the treatment of malaria and other ailments.1 is an anti‐HIV‐1 molecule capable of inhibiting HIV‐1 reverse transcriptase. Although this molecule showed interesting anti‐HIV activity, it was also cytotoxic to the host cells at approximately 15 μg/ml.1) could serve as a good starting point in the development of novel and potent anti‐HIV drugs.[2) and kirkinine (3), both reported as having anti‐tumoral and neurotrophic actions, acting via the modulation of protein kinase C.Synaptolepis kirkii (Thymelaeaceae), which is commonly used to manage epilepsy and snake bite.2 and 3 deviated from the local traditional usage of this plant species, this buttresses the point that NPs from medicinal plants have a broad spectrum/range of activities and can be chemically modified to suit our target of interest.[Nitidine  had scaffolds that could be predicted with an endpoint for the presence/manifestation as a PAINS. The list of matching PAINS and the number of scaffolds containing them are summarized in Table SI_1. In a similar way as previously reported by Baell,Additionally, comparative studies between EANPDB with other compound databases , logarithm of the octanol/water partition coefficient (QPlogPo/w), number of H‐bond donors (donorHB), number of H‐bond acceptors (accptHB), as well as an evaluation of the ratio of molecules violating the lead‐like rule of 3 (RuleOfThree) and the Lipinski rule of 5 (RuleOfFive) for orally available drug molecules. The variation in terms of percentages of these physicochemical properties among the selected datasets for comparison is shown in Figures A drug‐likeness study to show the distribution of the EANPDB molecules Table  was furtmol_MW generally followed a Gaussian‐like curve with maximum peaks around 300 Da. While molecules with mol_MW <500 Da dominated the EANPDB, NANPDB, DrugBank and NuBBE datasets, the StreptomeDB dataset rather demonstrated an almost level distribution in the number of molecules with ∼200 Da<mol_MW<∼550 Da. Approximately 85 % of the molecules contained in all the datasets analyzed had MW less than 500, thus meeting one of the Rules of Five for oral drugs. Similarly, the distribution of molecules concerning the predicted QPlogPo/w below 5 also dominated (∼85 %). All calculated QPlogPo/w for molecules in the different datasets used in this study for comparison followed a similar distribution pattern  has not been studied systematically and quantitatively and may vary otherwise from NPs from plant sources.Furthermore, analysis of the count of donor/accptHB Figure  content B Figure A while tB Figure B. HoweveAn evaluation of the computed physicochemical properties of the violation of the “Rule of Five” is summarized in Figure 3.4in silico methods such as virtual screening and toxicity prediction have continuously gained grounds to complement resource expensive wet laboratory experiments in the drug discovery pipeline. This is especially the case when it comes to NPs that are normally available only in low yields. A good proportion of the molecules were predicted as negative (complied) with the AMES mutagenic test in bacteria (Table +) channels (encoded by hERG I). About 85 % of the content of the EANPDB were predicted to have no hepatotoxic or skin sensitization effect. The human maximum tolerated dose (Max. tolerated dose in log mg/kg/day) extrapolated from animal data that gives an idea of the maximum recommended starting dose in phase I clinical trials  has been predicted for each molecule. The maximum (Max), minimum (Min) and Average (Avg) of the Max. tolerated dose (human) alongside the Oral Rat Acute Toxicity (LD50), Oral Rat Chronic Toxicity (LOAEL), Tetrahymena pyriformis toxicity and Minnow toxicity have been summarized in Table The application of ia Table . A negat3.5Molecules with ring system(s) were used in this analysis while those that do not have any rings were ignored. However, we observed that molecules without a ring system represented a very small minority of the complete datasets and therefore should not bias the overall scaffold analysis. The output file downloaded from the Scaffold (CSR curves) tap in PUMA shows that there is little scaffold diversity within the NANPDB and EANPDB Figure . A highl3.63SH and canonical SMILES; CS). The existence of several thiol based HDAC inhibitors such as romidepsin and largazole (both prodrugs that generate the thiol in vivo) supports our idea to find molecules that possess sulphur containing functional groups from our database that can be used as a ZBG in the development of HDAC inhibitors .A substructure search using CHs Figure , mostly s Figure . These m4in silico evaluation of these molecules show typical characteristics of good drug candidates.NPs, especially those from African sources deserve more attention as they have been proven to be underrepresented, unexplored and understudied for drug discovery. NPs have been reported to contribute strategically in the drug discovery process either as new drug molecules or the relevant scaffold for the synthesis of novel bioactive molecules. In this study, we are continuing our effort to provide an online free database of NPs from African sources.6f, 6gDetails on EANPDB content, molecular structures and their sources of the collection as well as computed descriptors can be accessed free of charge via the Internet at http://african‐compounds.org. The metadata is also available as additional spreadsheets.None declared.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


It is a central tenet of the clonal selection theory, that lymphocyte repertoires are tolerized to self‐antigens during their ontogeny. Germinal centres are the sites in secondary lymphoid tissues where B cells undergo affinity maturation and class‐switching to produce high‐affinity antibodies. This process is crucial, both in our ability to mount protective humoral responses to infections and to vaccinations, but it is also involved in untoward reactions to self‐antigens, which underlie autoimmunity. The process of affinity maturation poses a significant challenge to tolerance, as the random nature of somatic hypermutation can introduce novel reactivities. Therefore, it has been a long‐standing idea that mechanisms must exist which limit the emergence of autoreactivity at the germinal centre level. One of these mechanisms is the requirement for linked recognition, which imposes on B cells a dependence on centrally tolerant T follicular helper cells. However, as linked recognition can be bypassed by adduct formation of autoantigenic complexes, it has been an appealing notion that there should be an additional layer of dominant mechanisms regulating emergence of autoreactive specificities. About a decade ago, this notion was addressed by the discovery of a novel subset of T regulatory cells localizing to the germinal centre and regulating germinal centre B‐cell responses. Here, we detail the progress that has been made towards characterizing this T follicular regulatory cell subset and understanding the functions of these ‘guardians of the germinal centre’. A central aspect of the GC reaction is its regulation to protect against tissue damage and autoimmunity. This has long been thought to rely on the requirement for linked recognition, whereby T follicular helper cells (Tfhs) license B‐cell reactivities in T‐dependent responses. However, this mechanism is not fail‐safe. Indeed, many autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, are characterized by the presence of affinity‐matured, class‐switched antibodies to autoantigens.2Germinal centres are formed upon antigenic stimuli in all peripheral lymphatic organs, such as lymph nodes (LN), spleen and Peyer's patches. GCs induced in response to a transient pulse of antigen typically arise around day 5‐6, peak at about 12‐14 days and wane ~3 weeks.The first step in GC formation is the activation of B cells by soluble antigen or antigen retained by follicular dendritic cells in the follicle, and the concomitant activation of naïve CD4 T cells by antigen‐presenting dendritic cells, initiating their differentiation into Tfh Figure . The actWhile it was initially thought that B cells undergo isotype class‐switching in GCs, recent data have demonstrated that this most likely occurs in primary foci, prior to GC entry and seeding.3+ T cells is initiated through initial contact with antigen‐presenting dendritic cells. The pre‐Tfhs migrate to the T‐B border and contact cognate B cells, eliciting further signals that fix the Tfh transcriptional programme. The Tfhs subsequently localize to the GC and make transient contacts with B cellsTfhs have been extensively studied in the context of foreign antigen responses and are thought to control GC B‐cell cycling between LZ and DZ in a peptide‐major histocompatibility complex II (MHCII)–directed manneret al found that peptide‐MHCII density impacted the ability of B cells to enter the GC reaction, but once in the GC, B cells with a 50% reduction in peptide‐MHCII density competed efficiently with wild‐type B cells. This notion fits well with prior observations by us, that once tolerance is broken and autoreactive GCs are initiated, they evolve clonally in a manner comparable to foreign antigen–elicited GCs.The notion that Tfhs directly control GC B‐cell cycling between the DZ and LZ was recently challenged by observations in a foreign immunogen setting suggesting that the fitness of GC B cells does not titrate with peptide‐MHCII density.Taken together, these observations imply the necessity of auxiliary tolerogenic mechanisms controlling physiological GC responses and preventing break of tolerance at the GC stage. Indeed, it has become clear that an additional T follicular cell subset is centrally involved in regulating GCs, namely the Tfr.4Although it had been previously noted that Tregs have the capacity to enter follicles and suppress GC responses,Induction of Tfr requires co‐stimulation through CD28 and ICOSAs can be seen in Table 5Tfr development is regulated by the Tfh‐produced cytokine IL‐21, which inhibits the proliferation of Tfr through Bcl‐6‐mediated suppression of IL‐2 responsiveness via downregulation of CD25.6A central aspect of immune function is the phenomenon of ‘memory’, based on the persistence of specialized memory lymphocytes after resolution of an immune response, which can be reactivated upon repeated exposure to the same challenge.7Deletion of Tfr leads to loss of control of GCs, which expand due to an increase in total numbers of both GC B cells and Tfh.8+ CD25+ Tregs are capable of directly killing B lymphocytes in vitro in a perforin‐dependent manner, it has also been suggested that Tfr may directly kill B cells, although it was noted that Tfrs express lower levels of granzyme B than conventional Treg.Tfr may restrict GC reactions either indirectly, through effects on Tfh or directly through effects on B cells Figure . It has 9Relatively little data have emerged regarding the specific behaviour of Tfr in autoimmune GCs, but numerous studies have linked Tfr deficiency with autoimmunity. A priori, one would expect a major role for Tfr‐mediated GC control specifically in autoimmune disorders characterized by high levels of circulating autoantibodies. Indeed, in SLE, the frequency of circulating Tfr has been shown to be reduced, while the Tfh/Tfr ratio was increased. Interestingly, there was a negative correlation of the frequency of Tfr to disease activity and the titre of anti‐dsDNA antibody. The Tfh/Tfr ratio was positively correlated with disease activity, indicating that the Tfr suppressive effect upon Tfh is lowered in SLE. In successfully treated patients, the frequency of Tfr was increased and the Tfh/Tfr ratio decreased.The role of Tfr in controlling autoimmunity also extends to autoimmune disorders traditionally considered T cell–driven, but where B cells have recently emerged as important contributors to initiation of disease by virtue of their antigen‐presenting capabilities. Hence, Tfrs were recently associated with the development of type 1 diabetes mellitus.The roles of Tfr cells in autoimmune disease still remain to be fully elucidated, but a more comprehensive overview of the current state of knowledge has been provided elsewhere.10Many outstanding questions remain regarding the function of Tfr in regulation of GC homeostasis in health and disease. A deeper understanding of the exact molecular mechanisms by which Tfrs regulate the humoral immune response, both indirectly through Tfh and directly through effects on B cells, may begin to unravel this further. Considering the rapid progress that has been made since their discovery only a decade ago, there can be no doubt that the coming years of research into Tfrs will be highly fruitful.The authors declare no conflict of interest.Both authors contributed to drafting and finalizing the manuscript.


Diel vertical migration (DVM) is common in aquatic organisms. The trade‐off between reduced predation risk in deeper, darker waters during the day and increased foraging opportunities closer to the surface at night is a leading hypothesis for DVM behaviour.Mysis, an omnivorous mid‐trophic level macroinvertebrate that exhibits strong DVM between benthic and pelagic habitats and plays key roles in many deep lake ecosystems. However, some historical literature and more recent evidence indicate that mysids also remain on the bottom at night, counter to expectations of DVM.Diel vertical migration behaviour has dominated research and assessment frameworks for Mysis literature using Web of Science  to quantify the frequency of studies on demographics, diets, and feeding experiments that considered, assessed, or included Mysis that did not migrate vertically but remained in benthic habitats. We supplemented our WoS survey with literature searches for relevant papers published prior to 1945, journal articles and theses not listed in WoS, and additional references known to the authors but missing from WoS .We surveyed the freshwater Mysis ecology. Moreover, the literature suggests that reliance on Mysis sampling protocols using pelagic gear at night provides an incomplete picture of Mysis populations and their role in ecosystem structure and function.Results from the survey suggest that relatively little attention has been paid to the benthic components of Mysis DVM and provide an expanded framework that more fully considers the role of benthic habitat. Acknowledging benthic habitat as an integral part of Mysis ecology will enable research to better understand the role of Mysis in food web processes.We summarise current knowledge of  For demographics, we surveyed WoS using the terms “Mysis” AND “abundance OR densit* OR biomass* OR growth OR size OR length OR distribution” under the field “Topic”. We used the terms “Mysis” AND “diet OR gut OR stomach” under the field “Topic” to survey WoS for in situ diets. For experiments on Mysis feeding behaviour, we used two sets of terms under the field “Topic”. The first included “Mysis” AND “feeding OR predation” AND “experiment” under the field “Topic”, and the second included “Mysis” AND “contaminant* OR toxi*” under the field “Topic”.We conducted literature surveys for three aspects of freshwater Mysis taxonomy was revised in the mid‐2000s, with new names given to four sibling species formerly known as M. relicta , journal articles and theses that were not listed in WoS, and additional references known to the authors. We did not include papers that developed new sampling devices  included benthic‐caught individuals in a minority of studies, ranging from 15% of the studies that estimated biomass (n = 40) to 40% of studies that assessed life‐stages  used benthic‐ or combined benthic‐ and pelagic‐caught individuals in only 21% of the studies, whereas 43% of the studies used only pelagic‐caught individuals and 36% did not report the habitat from which Mysis were captured for the experiments .We also found that ts Table . PelagicMysis ecology and the role of Mysis in ecosystems. As evidenced by findings from studies that have examined benthic and pelagic Mysis at night , get caught on obstructions, or not hold tight to bottom with sharp bathymetric relief or complex substrate. Towing a net through the water column is far easier and safer, despite the necessity of working at night. A number of early studies concluded that pelagic vertical net tows at night were the best method for quantitative estimates of Mysis because they yielded the highest areal density estimates among methods , provide an incomplete picture of Mysis populations and their role in ecosystem structure and function.Overall, the literature suggests that assessments of 3.2Mysis energy dynamics. Mysis consume and grow on a variety of benthic foods including detritus, zooplankton eggs, amphipods, and benthic zooplankton , with growth rates similar to those observed in situ  detritus  capture success rate for Mysis off bottom was nearly double that when Mysis were on bottom  and other sculpin species , the primary demersal fishes that prey upon mysids in the Laurentian Great Lakes  what drives decisions to migrate or not; (2) the importance of benthic habitat for Mysis assessment; and (3) how benthic resources may impact Mysis dynamics in a broader ecosystem context.Below, we identify a series of important questions and hypotheses that we believe will advance our understanding of the role of benthic habitat to 4.1Conditions in benthic and pelagic habitats vary seasonally Figure , as do m(1a) Is partial DVM a result of fixed or plastic behaviours at the individual or group level? Whether a Mysis population can be divided into sub‐groups that each behave consistently within groups but differently among groups, or Mysis behaviour is an outcome of individual choice in response to external conditions or internal states, remains unclear. Several early studies proposed such alternative behaviours  What is the relative importance of pelagic and benthic prey resources for Mysis growth? How is growth influenced by prey availability and nutritional quality between benthic and pelagic habitats and across seasons? Do individuals need to migrate to survive, grow, and reproduce? Our synthesis suggests that a more diverse perspective is needed to better quantify the absolute and relative roles of benthic and pelagic prey resources for Mysis growth. However, observations that individuals captured in pelagic habitat during the day tend to be smaller, that smaller individuals move higher up in the water column at night, and larger individuals tend to dominate night benthic habitat  Are predation risks higher in benthic or pelagic habitats? Are DVM patterns dependent on Mysis density and the presumably closer proximity to predators when on the bottom (2‐D habitat) than in the water column (3‐D habitat)? To our knowledge, studies on Mysis predation risk to fish have been primarily focused on pelagic settings  What proportion of Mysis populations remain benthic at night? What proportion remain pelagic during the day? The proportion of Mysis that remains on the bottom at night probably varies among sites, lakes, and seasons, as does the proportion that remains suspended in the pelagic zone during the day  What are the demographic differences between benthic‐ and pelagic‐caught mysids, and how do such differences influence population production estimates? Assessing demographic differences between benthic and pelagic habitats across bathymetric depths, time of day, and seasons will provide more accurate population assessments, as well as contribute to questions and hypotheses about the mechanisms driving partial DVM (see questions 1a–c above) and to fill in missing biomass and production in food web models (see Food web and ecosystem effects above).4.3Mysis can be intense and alter zooplankton community structure and function (Lasenby et al., Mysis abundance or growth because they can exploit benthic resources (Chipps & Bennett, Mysis may also be able to resist declines in system productivity as a result of oligotrophication (Barbiero, Lesht, & Warren, Zooplanktivory by 5Mysis DVM in the published literature and the likely, but perhaps under‐appreciated, role that benthic habitat plays in Mysis ecology suggests a complementary lens through which we should approach Mysis research. Partial DVM in freshwater mysids appears to be the norm rather than the exception. Thus, instead of the seemingly implicit assumption that the pelagic habitat is the most important habitat for Mysis and the benthic environment is simply a hiding place during daylight hours, we propose an alternative and perhaps provocative perspective that benthic habitat is equally, if not more, important than pelagic habitat to understanding Mysis ecology. Such a shift in perspective requires testing that focuses research more equally on the two habitats. A more‐balanced perspective will result in a better understanding of the drivers of Mysis DVM behaviour and yield new insights into the ecosystem effects of animals, such as mysids, that rely on both benthic and pelagic habitats.The disproportionate focus on the pelagic phase of Tables S1–S3Click here for additional data file.