Drowning is a leading cause of child death in the coastal Sundarbans region of India due to the presence of open water, lack of supervision and poor infrastructure, but no prevention programs are currently implemented. The World Health Organization has identified interventions that may prevent child drowning in rural low-and middle-income country contexts, including the provision of home-based barriers, supervised childcare, swim and rescue training and first responder training. Child health programs should consider the local context and identify barriers for implementation. To ensure the sustainability of any drowning prevention programs implemented, we conducted a qualitative study to identify the considerations for the implementation of these interventions, and to understand how existing government programs could be leveraged. We also identified key stakeholders for involvement. We found that contextual factors such as geography, cultural beliefs around drowning, as well as skillsets of local people, would influence program delivery. Government programs such as accredited social health activists (ASHAs) and self-help groups could be leveraged for program implementation, while Anganwadi centres would require additional support due to poor resourcing. Gaining government permissions to change Anganwadi processes to provide childcare services may be challenging. The results showed that adapting drowning programs to the Sundarbans context presents unique challenges and program customisation. Drowning is a leading cause of morbidity and mortality in low-and middle-income countries (LMICs) . Of thesThe World Health Organization  recommends the implementation of four effective community-based interventions in rural LMIC settings to reduce drowning in young children. These interventions are activities that may be feasibly implemented in low-resource contexts and have been shown to reduce drowning burden . These iPrevious research and experience in the sustainable program design has shown that it is essential to understand the context, local perceptions and possible implementation-related challenges before designing and implementing community-based programs ,9,10. ThA key strategy that improves program sustainability is linking program goals with government priorities and leveraging existing programs ,14. A coWe conducted the formative contextual analysis required to design a sustainable drowning reduction program for the Sundarbans, as guided by WHO recommendations. The objectives were as follows: (1) identify community perceptions and preferences towards the recommended drowning interventions; (2) explore the feasibility of leveraging ICDS, ASHA or SHG programs for the delivery of drowning reduction interventions; (3) identify contextual challenges and considerations for the design and delivery of the program; and (4) identify key stakeholders who should be engaged during the development and implementation of the program.We applied qualitative methods to understand the micro context in which drowning reduction interventions could be delivered in the Sundarbans. In-depth interviews (IDIs), focus group discussions (FGDs) and observations were conducted and triangulated to develop this understanding. IDIs gave insights into individual-level perspectives, and FGDs were used to identify community norms and perceptions. Observations allowed for the better understanding of government program operations and systems. Qualitative methodology was guided by the Consolidated Criteria for Reporting Qualitative Research (COREQ) see  20]..20].Data collection was conducted in partnership with a local non-governmental organization (NGO), the Child in Need Institute (CINI). CINI has operated child and maternal health programs in rural West Bengal for the past 46 years and has extensive connections with communities and local government in the Sundarbans region. The data collection of community-based participants was completed by two male data collectors recruited by CINI, managed by S.R. and R.P. who work as the programs’ manager and director, respectively. The data collectors had previous experience in qualitative research in West Bengal and were trained by the researchers in the study aims and tools. One of the data collectors had experience conducting qualitative data collection in the Sundarbans and was familiar with the community. M.G. conducted English-language interviews, such as with grassroots organisations.All data collection occurred face-to-face. IDIs and FGDs were held in locations that best suited participants, such as in community schools or Anganwadi centres. In addition to the data collectors and participants, NGO partner facilitators were present for some IDIs, FGDs and observations to lend logistical support. All IDIs and FGDs were audio recorded and lasted between 30 and 90 min. Field notes were also taken by one data collector and collated to make key point summaries of each IDI, FGD and observation on a daily basis, which was shared with the research team.All Bengali transcripts were translated into English for analysis. No interviews were repeated. Transcripts were not returned to participants for comment due to the logistical and literacy barriers.All participants were adults over the age of 18. A minimum of three IDIs and two FGDs were conducted for each stakeholder type to ensure the capture of varying responses. Stakeholder types interviewed included (1) community-level participants including men, women and leaders, (2) government program participants and delivery staff, and (3) grassroots organisations. These stakeholder types are described in detail below. This range of stakeholders enabled for the identification of contextual considerations from the perspectives of program implementers and beneficiaries and ensured that individuals from different levels of the social hierarchy were heard. Data collection ceased once saturation in each type was reached.Community men, women and leader participants were recruited through convenience sampling. The partner NGO first approached local government bodies for permission to conduct the interviews. Data collectors then entered the communities as recommended by the Gram Panchayats  and engaged local leaders such as ASHA and Anganwadi workers, who introduced the data collectors to possible participants. Participants were required to be parents and usual residents of the community living there for the past three years. Participants were recruited across all 19 blocks of the Sundarbans to ensure a range of perspectives.These participants provided insights into community acceptance and perceptions towards drowning interventions, as well as possible barriers and enablers to implementation in the local context. A total of ten IDIs and nine FGDs were conducted with community-level participants, with men and women equally represented.Anganwadi workers, SHG members and ASHA workers were approached through purposive sampling after entering communities in which permission for data collection had been granted by local government officials. Communities had one or two ASHA and Anganwadi workers each, so whoever was available upon contact was scheduled for interview. As self-help group members were found in many households, community members would lead data collectors to the closest home of a member. These participants were included if they were active in their respective programs for a minimum of 6 months within the Sundarbans region.Observations of SHG meetings and Anganwadi centres were conducted to understand their operations. By policy, one Anganwadi centre is required to serve a population of 1000 people, providing any children aged 3–6 years old with early childhood education activities for two hours each day along with a nutritious meal. Each centre should have one Anganwadi worker and one helper, and usually operates between 7 and 10 a.m. We observed the children who came to the Anganwadi Centres and provided insights into how children interacted with the Anganwadi workers, as well as the ground realities of the program delivery. Observations of SHGs identified decision-making methods and revealed the role of SHGs in the community. Anganwadi centres and SHGs for observations were purposively selected in partnership with a local NGO working with these programmes to cover a range of performance levels. Nine government program participants  were interviewed. Two FGDs with SHG members were also held and three observations each were conducted at Anganwadi centres and SHG meetings.Interviews were conducted with the individuals from organisations working in the child health, education, safety or nutrition in the Sundarbans or other similar rural contexts in West Bengal. This provided insights into the considerations and challenges related to delivering grassroots programs in the Sundarbans. Potential participants were introduced to the researchers by our partner NGO and were required to have oversight over program delivery for at least one year. Three representatives from grassroots organisations were interviewed.Tools for all IDIs, FGDs and Observations were developed before the commencement of data collection and translated into Bengali. The data collection guides were semi- structured to ensure all domains relevant to research questions were covered.All participants were also shown a pictorial presenting the WHO-recommended drowning interventions. Barriers were described as any physical object preventing children’s access to water such as playpens, door barriers or fencing. Childcare was described as any group-based supervision in an enclosed space. Swimming lessons encompassed both swim and rescue training skills, and first responder training was described as training adults on how to save children if they fall into water or start drowning.All participants provided verbal or written informed consent depending on their literacy level. Ethical approval was granted by the University of New South Wales Human Research Ethics Committee (HC 190274) in Sydney, Australia and The George Institute for Global Health (India) Ethics Committee (06/2019) in New Dealhi, India.Analysis of the transcripts was completed using NVivo 12 . NarratiStakeholders were identified and then allocated to level of power and interest as based on Mendelow’s Matrix . The levRefusal to participate in the study was less than 10%. Below we discuss the overall and intervention-specific considerations for program implementation in the Sundarbans, the feasibility of using government programs and identify the stakeholders who must be involved in program design and delivery.The A range of considerations were identified that applied to all drowning reduction interventions.Participants showed heterogeneity in preferences between the interventions. All interventions were generally considered acceptable. Some participants recognised that each of the interventions targeted different age groups and expressed a need for an age-targeted and comprehensive approach  members were primarily interested in the delivery of childcare services and suggested they may provide pick up and drop off services for children to and from swimming and childcare interventions. Some SHGs were already involved in delivering government programs, such as the mid-day meal scheme in schools. They expected to be paid for involvement  are responsible for monitoring program performance. Although Gram Panchayats are responsible for the program implementation of ICDS, they do not have the permission to make operational changes as their targets and delivery requirements are set by State policy and enforced by block-level supervisors. Grassroots organisation and community leader participants noted that engaging block-level representatives may be challenging without higher state-level permissions which may take months to obtain. Grassroot participants had experienced that government departments were cautious about giving permissions when liabilities were not clear. These participants stated that running a parallel program for childcare may be easier than using the ICDS  of tying children indoors to the ends of rope, there is some evidence that walking school bus programs can prevent injury in children . While tTo ensure the community ownership and development of an acceptable and feasible program, the next step of program design should involve community participatory approaches ,47. The Due to ethical constraints, we were not able to gather information on participants’ caste or religion. It is unclear if the perspectives found are representative across a range of religious groups. In addition, some government program workers were recruited with assistance from Gram Panchayats. These may have been the more active and well performing workers and may not be fully representative of typical programs. This was particularly mitigated by ensuring at least one poor performing worker of each type was purposively recruited.The Sundarbans are a high-risk region for child drowning, and we aimed to identify the mechanisms and considerations for the implementation of drowning reduction programs in the region. We found that program design should consider contextual factors such as geography, cultural beliefs around drowning, skillsets of local people and household-level needs. It was found feasible to leverage government programs such as ASHA workers and SHGs for program recruitment and implementation, while the optimisation of Anganwadi centres for the provision of childcare may be challenging due to poor resourcing and permissions required. Community-based young clubs were also possible implementers of programs. Program development and implementation should involve a range of stakeholders such as local government members, block and district-level health and development officials, community leaders and residents. The results show that the development of drowning reduction programs in rural LMIC contexts should be catered to the local social and environmental context to ensure acceptability and feasibility.


Chlamydia screening in high schools offers a way to reach adolescents outside of a traditional clinic setting. Using transmission dynamic modeling, we examined the potential impact of high-school-based chlamydia screening programs on the burden of infection within intervention schools and surrounding communities, under varying epidemiological and programmatic conditions.A chlamydia transmission model was calibrated to epidemiological data from three different settings. Philadelphia and Chicago are two high-burden cities with existing school-based screening programs. Rural Iowa does not have an existing program but represents a low-burden setting. We modeled the effects of the two existing programs to analyze the potential influence of program coverage and student participation. All three settings were used to examine a broader set of hypothetical programs with varying coverage levels and time trends in participation.In the modeled Philadelphia program, prevalence among the intervention schools’ sexually active 15–18 years old population was 4.34% after 12 program years compared to 5.03% (4.39–5.43%) in absence of the program. In the modeled Chicago program, prevalence was estimated as 5.97% (2.60–7.88%) after 4 program years compared to 7.00% (3.08–9.29%) without the program. In the broader hypothetical scenarios including both high-burden and low-burden settings, impact of school-based screening programs was greater in absolute terms in the higher-prevalence settings, and benefits in the community were approximately proportional to population coverage of intervention schools. Most benefits were garnered if the student participation did not decline over time.Sustained high student participation in school-based screening programs and broad coverage of schools within a target community are likely needed to maximize program benefits in terms of reduced burden of chlamydia in the adolescent population. Sexually transmitted infections (STIs) are prevalent among adolescents and young adults , 2. HoweTypically, screening events are implemented in collaboration with local public health departments, or screening is offered at school-based health centers, with the aim in either case to screen the entire school population within a short amount of time (e.g. 1 week) . School-In this study, we used mathematical modeling to evaluate the potential impact of school-based screening programs on chlamydia transmission dynamics, accounting for various epidemiological factors and programmatic features that may influence the program effects both within schools and in the surrounding communities. By modeling an array of different program scenarios in both high-burden and low-burden settings, we examined how specific features from existing programs, as well as possible implementation scenarios for hypothetical programs, could affect chlamydia transmission dynamics in different epidemiological contexts.The model in this study expanded on a previously developed chlamydia transmission model, described in detail elsewhere . A deterWe applied the model in three different settings, spanning a range of reported chlamydia diagnosis rates. Philadelphia and Chicago are large cities with relatively high rates of chlamydia diagnoses and with existing high-school-based screening programs. Rural Iowa has relatively low chlamydia diagnosis rates and does not have an existing school-based screening program. Among women aged 15-24y, rates of reported chlamydia diagnoses in 2015 were 7433 per 100,000 in Philadelphia , 6715 pePhiladelphia has the largest and longest-running high-school-based chlamydia screening program in the United States, established during the 2002–2003 school year. Between 2002 and 2015, there were 10,000–20,000 tests conducted annually, and the schools enrolled in the program covered approximately 55% of the 15–18-year-old population in the city. In Philadelphia’s intervention schools student participation declined over time, from approximately 30% of the student population during the 2002–2003 school year to 16% during the 2014–2015 school year. Chicago began a screening event program in 2010, following a pilot study in 2008. Between 2010 and 2013, the program was implemented in schools covering approximately 12.5% of the 15–18 year-old population in the city, and the number of tests conducted yearly increased from 2200 to 6900.Model calibration was undertaken using a Bayesian framework  operatioSex- and age-stratified chlamydia reported diagnosis rates were obtained for the three study settings –20, and Both Philadelphia and Chicago provided estimates of chlamydia positivity in intervention schools. For calibration, we used positivity estimates from the first year available for each program as a proxy for the baseline chlamydia prevalence among the population of sexually active 15–18 year-olds in each city. For Iowa, we used a chlamydia positivity estimate from rural family planning clinics . To caliThe model was initially run to equilibrium using time-invariant parameters. We introduced time-varying parameters governing rates of sexual initiation starting in 1991, to correspond to years of data availability from YRBS, and time-varying parameters relating to screening rates and completeness of reporting on diagnosed infections in the general population starting in 2000, to correspond to the first year of case report data included in the likelihood.We conducted two sets of analyses. In the first set of analyses, using only the models for Philadelphia and Chicago, we examined the potential influence of different observed programmatic features from the school-based screening programs in those cities on the estimated benefits from the programs. We used the calibrated models to estimate chlamydia prevalence in the 15–18 year-old population in the absence of school-based screening. We then estimated the potential impact of school-based screening using program data on the fraction of the 15–18 year-old population enrolled in intervention schools (“coverage”) and on student participation within intervention schools over time as input parameters. We used information on the highest and lowest yearly participation levels observed in Chicago and Philadelphia to determine ranges of participation levels. In Philadelphia, we modeled declining student participation over the 12 program years, from 30 to 16%, while holding population coverage constant at 55%. For Chicago, we modeled an increase in student participation over the 4 program years, from 13 to 40%, while holding coverage constant at 12.5%.In the second set of analyses, we used the calibrated models for all three settings to explore a set of hypothetical scenarios with varying coverage levels and participation trends, and each program scenario implemented over a 12-year period. Population coverage of school-based screening among ages 15–18 years was varied from 20 to 60% across cities. Three different time trends for participation were modeled: 1) stable participation at 50%; 2) declining participation, from 50 to 8%; and 3) increasing participation, from 50 to 90%.In both sets of analyses, parameter uncertainty was captured by running multiple model simulations, each based on sampling one set of parameter values from the joint posterior distribution of the parameters estimated through calibration. For each draw of model parameter values, we ran both a baseline (no-screening) scenario and each of the different program scenarios. Intervention effects are summarized in terms of prevalence in different scenarios among 15–18 year-olds as well as absolute differences in prevalence between intervention scenarios and the no-screening program baseline. Reduction in prevalence was calculated by taking the difference from each baseline draw and its respective counterfactual, and calculating an overall reduction across the reductions at the draw-level. We report the median and 95% credible interval for each outcome.Model calibration to the three settings produced baseline prevalence estimates in the absence of school-based screening. Figures SOur first set of analyses simulated observed programmatic features from the school-based screening programs in high-burden cities with existing programs (Philadelphia and Chicago). Figure At the end of the available data period for each program, the model estimated a modest population-level impact for the sexually active 15–18 year-old population in each city. In Philadelphia in 2014, chlamydia prevalence in this group was 4.61% (3.99–5.00%) compared to 5.03% (4.39–5.42%) in the absence of the program, representing a 0.38 percentage point (0.33–0.43) reduction. In Chicago in 2013, chlamydia prevalence was estimated as 6.85% (3.02–9.08%) compared to 7.00% (3.08–9.29%) in the absence of the program, representing a modest 0.16 (0.06–0.21) percentage point reduction.Our second set of analyses used models for all three settings to explore an array of hypothetical scenarios implemented over a 12-year period. Table For a school-based screening program covering 30% of the age group, and which maintained stable student participation, we estimated that the program would reduce prevalence among sexually active students in the intervention schools by 1.38 (1.22–1.49), 2.15 (0.93–2.85) and 0.40 (0.29–0.47) percentage points in Philadelphia, Chicago and Iowa, respectively. If student participation declined over time, benefits compared to baseline would shrink: reduction of 0.24 (0.20–0.27) percentage points in Philadelphia, 0.47 (0.19–0.68) percentage points in Chicago, and 0.09 (0.07–0.11) percentage points in Iowa. With increasing student participation, the reductions in prevalence in the three settings would be 2.09 (1.84–2.23), 3.17 (1.36–4.17) and 0.58 (0.42–0.69) percentage points, respectively.Holding participation constant but varying coverage from 30 to 60% of the 15–18 year-old population demonstrates the potential impact of doubling the size and reach of the program. Given the same level of participation within intervention schools, program effects expressed as reductions in prevalence among the students in intervention schools were relatively invariant to the overall population coverage of the program. On the other hand, considering the effects in the broader communities around the participating schools, with a 30% program coverage there was an estimated 0.45 (0.39–0.50) percentage point decrease in community prevalence among the sexually active population in Philadelphia, a 0.91 (0.33–1.31) percentage point decrease in Chicago, and a 0.16 (0.12–0.19) percentage point decrease in rural Iowa. With a 60% program coverage the corresponding decreases were 0.89 (0.78–0.99), 1.76 (0.64–2.54) and 0.31 (0.23–0.36) percentage points, respectively.In this study, we examined the impact of existing and hypothetical school-based chlamydia screening interventions on the prevalence in intervention schools and surrounding communities. Our results demonstrated that stable and sustained school-based screening programs are needed to maximize and maintain the impact of the interventions. In the Philadelphia intervention implemented during 2002–2014, we predicted an initial decline in chlamydia positivity. Although these initial declines were not entirely sustained, we estimated an overall reduction in prevalence compared to no school-based program baseline scenario after 12 years of program implementation. Reducing student participation in our model to the levels observed in some existing programs led to modelled prevalence estimates consistent with observed trends. Program coverage, measured in terms of the proportion of the 15–18 years old population enrolled in participating schools, was larger in Philadelphia than in Chicago, which resulted in larger population-level benefits in Philadelphia. In all hypothetical scenarios, school-based chlamydia screening had a larger impact in higher burden settings (modeled after Chicago and Philadelphia) than in a lower burden setting .Schools have been a venue for the delivery of complex sexual and reproductive health interventions such as sex education initiatives  and riskSustaining school-based screening requires considerable resources at the local level , 14. AllSchool-based screening programs often have limited information available to evaluate the impact of the intervention in the surrounding communities. As this mathematical modeling study demonstrated, school-based programs can theoretically reduce broader community STD rates. However, positivity trends are not directly comparable without understanding how the program was implemented and what coverage level was attained , 9. DiffOur model calibration was limited by the availability of data at the local level. We relied mostly on reported chlamydia diagnoses as a measure of chlamydia burden. We assumed that screening at schools would not influence the students’ screening outside of the program . If screening at schools made it more likely that students would not get screened in the community, this would lower the overall impact of the intervention compared to the results in our study. Conversely, if students were better informed due to the availability of school-based screening, they may be more likely to use community services in the future, which would increase the impact of the intervention. Once exposed to the opportunity to undergo STI screening, young people may have an increased awareness of their ability or right to access confidential health services, and they may seek out other sexual health services, including expanded HIV/STI testing or reproductive services, as has been suggested before . OverallThe highest burden of STIs remains in urban areas, where young people are a key population for STI control strategies. Interventions that can reach people outside of traditional clinic settings are an important component of comprehensive sexual health programs. Young people may not use sexual health services due to a lack of information regarding their rights to access these services on their own, concerns regarding privacy, cost, transportation, or some combination of these . By offeTo maximize the school-based screening programs’ impact on students and communities at large, a stable program with high participation among high school students is likely needed. Targeting the population with the highest burden of chlamydia will allow for the identification of the most infections, and will have a greater influence on chlamydia transmission dynamics.Additional file 1: Table S1. Parameter table describing the input parameters, and posterior parameter estimates. Figure S1. Calibration of the model to Philadelphia. Figure S2. Calibration of the model to Chicago. Figure S3. Calibration of the model to Rural-Iowa.


ARID1A) is one of the most frequently mutated genes in hepatocellular carcinoma (HCC), but its clinical significance is not clarified. We aimed to evaluate the clinical significance of low ARID1A expression in HCC. By analyzing the gene expression data of liver from Arid1a-knockout mice, hepatic Arid1a-specific gene expression signature was identified (p < 0.05 and 0.5-fold difference). From this signature, a prediction model was developed to identify tissues lacking Arid1a activity and was applied to gene expression data from three independent cohorts of HCC patients to stratify patients according to ARID1A activity. The molecular features associated with loss of ARID1A were analyzed using The Cancer Genome Atlas (TCGA) multi-platform data, and Ingenuity Pathway Analysis (IPA) was done to uncover potential signaling pathways associated with ARID1A loss. ARID1A inactivation was clinically associated with poor prognosis in all three independent cohorts and was consistently related to poor prognosis subtypes of previously reported gene signatures . Immune activity, indicated by significantly lower IFNG6 and cytolytic activity scores and enrichment of regulatory T-cell composition, was lower in the ARID1A-low subtype than ARID1A-high subtype. Ingenuity pathway analysis revealed that direct upstream transcription regulators of the ARID1A signature were genes associated with cell cycle, including E2F group, CCND1, and MYC, while tumor suppressors such as TP53, SMAD3, and CTNNB1 were significantly inhibited. ARID1A plays an important role in immune activity and regulating multiple genes involved in HCC development. Low-ARID1A subtype was associated with poor clinical outcome and suggests the possibility of ARID1A as a prognostic biomarker in HCC patients.AT-rich interactive domain 1A ( The prevalence of hepatocellular carcinoma (HCC) worldwide is 750,000 cases per year, making it the seventh most common cancer globally. With a mortality rate similar to the prevalence rate, HCC is the second most common cause of death from any type of cancer . DespiteFor HCC patients who are not candidates for locoregional therapies, the oral multi-kinase inhibitor sorafenib is currently the only drug approved in the U.S. that has been shown to prolong overall survival. However, most patients develop disease progression after an initial response to sorafenib; the average radiologic progression occurs at 5 months of treatment . Since tOne of the important roadblocks to treatment efficacy is the lack of understanding of the mechanism of HCC progression. Improving our understanding of this mechanism would allow the development of therapies directly targeting that mechanism and identification of patients who would respond to those therapies . While tARID1A) and BRCA1-associated protein 1 (BAP1) are highly mutated in HCC, along with other well-known genes such as TERT, TP53, and CTNNB1 [ARID1A as a key member of the switching defective/sucrose non-fermenting (SWI/SNF) complex, which acts as a tumor suppressor in a broad spectrum of human cancers [ARID1A has a context-specific role in liver cancer, whereby elevated ARID1A promotes tumor initiation, while reduced ARID1A in established tumors increases metastasis in mouse models [The role of epigenetics in cancer progression has been emphasized ever since the introduction of cancer genome-wide sequencing, which revealed significant alteration in genes responsible for modifying chromatin structure. Chromatin remodeling genes AT-rich interactive domain 1A  Arid1a [ARID1A mutation-related genes in predicting the outcome of HCC. Furthermore, we uncovered a potential connection between ARID1A and regulation of oncogenic signaling pathway and immune activity.Because the role of settings ,14, we a) Arid1a . Since iArid1a-WT and liver-specific Arid1a-knockout (KO) mice [Arid1a floxed mice, induced deletion between the two loxP sites produced cells lacking exon 8 of ARID1A, which created a frameshift mutation and induced nonsense-mediated decay in the resulting transcript. Genes whose expression differed significantly between Arid1a-WT and -KO mice (p < 0.05 and log2 ratio > 0.5) were considered part of the Arid1a gene signature, which was used for further analysis  with RNA-seq transcriptional profiling in liver tissues obtained after partial hepatectomy in KO) mice . The micanalysis .p-value < 0.05 derived using the Kolmogorov-Smirnov test was considered to have statistical significance.Biological significance of differentially expressed genes (DEGs) were explored by GO term enrichment analysis including biological process, cellular component, and molecular function based on Bioconductor packages “topGO.” A https://www.proteinatlas.org) and analyzed the correlation of expression between ARIDA1A and alpha-fetoprotein (AFP). The AFP level is positively correlated with HCC progression and higher expression is considered to have worse prognosis.The immunohistochemistry (IHC) data was derived from existing databased, The Human Protein Atlas  [n = 188, GSE16757 and GSE43619), and a U.S cohort  [To examine the clinical relevance of low SE14520) ,17, a Kohttp://linus.nci.nih.gov/BRB-ArrayTools.html) were used for gene expression data analysis and construction of a prediction model [http://www.r-project.org).BRB Array Tools software programs  algorithm [Arid1a gene signature, gene expression data from the human HCC cohort were normalized and centralized as described previously [ARID1A-WT (ARID1A-high) or ARID1A-mutant (ARID1A-low) gene signature, and tumors were dichotomized according to Bayesian probability (cutoff of 0.5). HCC patients from all three cohorts were classified as ARID1A-high or ARID1A-low based on the BCCP predictor algorithm [Expression data from lgorithm . Before eviously . The roblgorithm . After cARID1A-low HCC, further exploration was made through The Cancer Genome Atlas (TCGA) data portal. TCGA HCC patients were categorized as having ARID1A-high or ARID1A-low subtype HCC by the BCCP prediction method. Next, TCGA platforms were applied to these two groups to identify distinct molecular features associated with ARID1A mutation. The following platforms were used: (1) RNA sequencing, the Illumina HiSeq 2000 RNA Sequencing; (2) reverse-phase protein array (RPPA), the MD Anderson Cancer Center TCGA proteome characterization center RPPA core using 219 antibodies; (3) somatic mutations, the Illumina HiSeq and IlluminaGA system; (4) copy number alterations, the Affymetrix Genome-wide Human SNP Array 6.0 platform; and (5) DNA methylation profile, the Illumina Infinium HumanMethylation450 platform. To extend our understanding of the molecular features of ARID1A expression.These platforms were analyzed with the aim of gaining insight of correlation between RNA and protein data, to observe whether the copy number alteration and DNA hypermethylation could at least have partial effect on ARID1A-low type was analyzed.The National Cancer Institute Proliferation (NCIP) , hepaticCXCL9, CXCL10, IDO1, IFNG, HLA-DRA, and STAT1). This six-gene signature serve as predictive biomarker in the form of a composite score in pembrolizumab treated patients where a higher score is associated with better treatment response.The six-gene interferon-gamma (IFNG6) composite score, which is associated with clinical response to pembrolizumab therapy  is derivThe cytolytic score is based on the mean transcript levels of two key cytolytic effectors, granzyme and perforin  where hiARID1A-low subtype. The analysis used Ingenuity Pathway Analysis (IPA), and thus, was based on prior knowledge of expected effects of transcriptional factors on their target genes stored in the Ingenuity Knowledge Base. Genes whose expression differed significantly (p < 0.001 and log2 ratio > 0.5) between patients with ARID1A-low HCC and those with ARID1A-high HCC from the TCGA cohort were selected.Next, we used gene network analysis to identify potential upstream transcription factors that regulate gene expression patterns enriched in the ARID1A-low and -high groups from the MSigDB databases (www.broadinstitue.org/msigdb).Gene set enrichment analysis (GSEA) was performed to identify gene sets differentially expressed in ARID1A in the liver, we selected genes that had significant difference of expression between Arid1a-WT and Arid1a-KO mice. A total of 733 genes were identified  were placed in the ARID1A-low group; in cohort 3, 76 of the 188 patients (40.4%) were placed in the ARID1A-low group. After dichotomizing patients in these three cohorts into ARID1A-high and -low groups, generally, patients who were placed in the ARID1A-low group had higher baseline alpha-fetoprotein (AFP) levels  and advanced HCC stage  were categorized to the n of AFP .ARID1A gene signature in the three independent cohorts. This analysis identified a significant difference in survival between the patients in the ARID1A-high group and those in the ARID1A-low group. Overall, patients in the ARID1A-low group had a lower survival rate than those in the ARID1A-high group .We next validated the prognostic association of the gh group B–D. FurtARID1A-low and ARDI1A-high groups of cohort 1 were compared with NCIP subtypes (A or B). The NCIP gene signature was derived by using unsupervised analysis of genome-level expression data from human HCC tissues where subtype A was associated with poor prognosis. When this signature was applied, 67 of 94 (71.3%) ARID1A-low group patients and 21 of 153 (13.7%) of ARID1A-high group patients were categorized to the poor-prognosis NCIP A subtype .First, ARID1A-low HCC subtype was consistently associated with poor-prognosis and high-recurrence subtypes  the hepatic stem cell gene signature (vs. hepatocytes), derived from expression patterns resembling fetal hepatic stem cells, which was associated with poor prognosis; (2) the silence of Hippo gene signature (vs. activated Hippo), where the HIPPO pathway is a tumor suppressor in the liver and its inactivation is known to be associated with poor prognosis; and (3) the SNUR recurrence-high gene signature (vs. recurrence-low), which was derived using supervised approaches in selecting genes associated with early disease recurrence after curative-intent treatment. When each of these signatures was applied, the subtypes . Furthergnatures .ARID1A-low HCC subtype was significantly associated with the NCI high-proliferation subtype (NCIP A), the hepatic stem cell (HS) subtype, the silence of Hippo pathway (SOH) subtype, and the high-recurrence (SNUR high) subtype .The ARID1A-low subtype is significantly correlated with poor prognosis, we next investigated the molecular characteristics of HCCs by ARID1A subtype using the multi-platform data accessed through the TCGA data portal. When the ARID1A gene signature was applied to 371 patients in the TCGA cohort, 170 patients (46%) were classified to the ARID1A-high group and 201 patients (54%) to the ARID1A-low group. The pattern of survival in the TCGA cohort was similar to that in the three validation cohorts, with a lower survival rate and higher recurrence rate in the ARID1A-low group . The methylation level and chromosomal instability were significantly greater in the ARID1A-low group than in the ARID1A-high group  , while hypermethylation was negatively correlated with mRNA expression levels  (Since protein expression level would more accurately reflect the characteristics of < 0.001) . Further< 0.001) . Somatic mutations. To consolidate our findings indicating that our ARID1A gene signature truly identifies ARID1A-low subtypes with greater ARID1A mutation, further analysis was done for somatic mutations. Of the 371 patients, somatic mutation data was available for 367; 33 of those (9%) had ARID1A somatic mutation. The presence of ARID1A mutation in AIRD1A-low subtype was 71%, while it was 29% in ARID1A-high subtype (p = 0.054). Truncation was the most common type (72.7%) of mutation, followed by missense (24.3%) and in-frame (3%) mutations  treatment in cancer patients, was calculated on the basis of the genes reported by Seiwert et al. [ARID1A-low group than in the ARID1A-high group  were significantly suppressed in the ARID1A-low group, while inhibitory genes PD-L1 (CD276) and LAG3 were highly expressed in the ARID1A-high group , a total of 2503 genes, were selected for functional study based on the Ingenuity Knowledge Base repository [ARID1A signature were genes associated with cell cycle, including SMAD7, CCND1, MYC, and the E2F group comprising E2F1 (with activated z-scores > 2), while TP53, SMARCB1, SMARCA4, CDKN2A, SMAD3, and CTNNB1 were significantly inhibited (z-scores < -2) (To gain better insight into the molecular characteristics of the pository . Direct es < -2) .ARID1A, a subunit of SWI/SNF complexes that controls how much “read access” the cellular transcription machinery has to DNA sequences, can have profound consequences on gene expression, and genes encoding chromatin-remodeling proteins are some of the most frequently mutated genes in human cancer [ARID1A and hepatocarcinogenesis have been published, and a recent study reported controversial results [ARID1A as a predictor of outcome in HCC patients with the intent of identifying a mechanism underlying the suppression of ARID1A using a bioinformatic approach. Since ARID1A’s biological activity is best reflected in gene expression level, we have derived signature from Arid1a KO mouse dataset and applied to human dataset to stratify HCC tumors according to ARID1A activity. Since the best-known molecular activity of Arid1a is the regulation of gene expression through chromatin remodeling, the transcriptomic signature of Arid1a KO mice would best reflect its biological activity. Functional conservation of Arid1a’s role in liver as tumor suppressor is well-documented in a recent study from Zhu et al., demonstrating that Arid1a is one of frequently mutated genes in chronically diseased liver tissues and loss of Arid1a promote clonal expansion in damaged mouse liver [The SWI/SNF complex represents a novel link between chromatin remodeling and tumor suppression. Recurrent mutations in subunits of the complex have been identified in various cancers . ARID1A,n cancer . However results ,13,14. Tse liver . FurtherARID1A-related gene signature is effective in identifying HCC patients with poor prognosis. Patients in ARID1A-low subtype HCC had a significantly lower survival rate than ARID1A-high subtype in all four cohorts studied. Furthermore, when HCC subtypes derived from our ARID1A gene signature were systematically compared with previously validated gene signatures, we confirmed the correlation of our ARID1A-low subtype with the poor-prognosis subtypes NCIP A, which reflected high proliferation; hepatic stem cell, where the stem cell subtype is known to be associated with poor prognosis in all cancers; silence of the Hippo pathway, where the Hippo pathway is indispensable in restricting cell growth and proliferation and its inactivation leads to poor prognosis; and recurrence-high SNUR. Based on the analysis, first we confirmed that an ARID1A mRNA and protein expression is still not clear. Although the correlation between mRNA and protein level was not prominent with coefficient of 0.36, this result is in line with previous study by Chen et al., where positive correlation with coefficient averaging ρ = 0.3 was observed, while a median value of 0.45 was reported in another study [ARID1A was investigated using high-throughput platform data obtained from the TCGA portal. From the results, we speculated that the loss of ARID1A was due to hypermethylation, which is in accordance with a previous study proposing that promoter hypermethylation of the ARID1A gene is responsible for the low ARID1A mRNA expression in invasive breast cancers [ARID1A expression could be increased copy number alteration, leading to poor prognosis of HCC. Published evidence that gains in chromosomes 1q and 8q are linked to hepatocarcinogenesis [ARID1A-low subtype. The mutation rate (9%) of ARID1A in our present study is similar to study reported by Zucmann-Rossi et al., which showed 4–17% of ARID1A mutation and 10% according to study by Fujimoto et al. [The exact molecular mechanism underlying low er study ,33. In t cancers . Anotherogenesis ,35 is suo et al. .ARID1A-low and -high subtypes. One of these immune checkpoint–targeted agents, pembrolizumab, which targets PD-1, a negative co-stimulatory receptor and a strong inhibitor of T-cell response, is under clinical trial for treatment of HCC [ARID1A-low subtype had a lower IFNG6 score than the ARID1A-high subtype. Consistent with the lower IFNG6 score in the ARID1A-low subtype was the lower immune cytolytic activity, based on transcript levels of two key cytolytic effectors, granzyme A and perforin, which are dramatically up-regulated upon CD8+ T cell activation [ARID1A-low group. Likewise, IS score, derived from 105 genes based on response to anti-CTLA-4 treatment in melanoma patients, was lower in the ARID1A-low group. Low ARID1A expression had suppressed expression of immune-stimulatory genes, while it increased expression of inhibitory genes. Since immune-stimulatory genes are known to predict better prognosis and are found to be protective factor in cancer, while immune inhibitory genes lead to tumor promoting signals and have negative correlation with survival, our results are in accordance with the already known findings.Recent success for targeted therapies against immune checkpoints such as CTLA-4, PD-1 (PDCD1), and PD-L1 (CD274) ,39 led ut of HCC . When wetivation  during ptivation ,42, wereARID1A loss. Since ARID1A is a subunit of the SWI/SNF complex that coordinates activity of the proteins of the complex, it is responsible for altering the chromatin structure required to facilitate several cellular functions, including transcription, DNA synthesis, and DNA damage repair [TP53 and cyclin-dependent kinase genes such as CDKN2A are inactivated in the ARID1A-low subtype. TP53 down-regulation was observed in every analysis we did, including RPPA, somatic mutation analysis, and copy number alterations. The tumor suppressor function of ARID1A through collaboration with TP53 is disrupted by loss of ARID1A and this change could have led to disruption in TP53-regulated genes such as CDKN1A and SMAD3, which were all inhibited in IPA analysis. All these alterations in cell cycle–regulatory genes could have eventually caused hepatocarcinogenesis with poor prognosis.Gene network analysis revealed that cell proliferation and cell cycle dysregulation were activated as a consequence of e repair . TherefoARID1A promotes development of HCC at a very early stage while preventing HCC progression to metastatic tumor [ARID1A has tumor suppressor activity in the later stage of HCC. Another recent study, by Hu et al. [ARID1A was associated with increased angiogenesis and poor prognosis after HCC had established. ARID1A was expressed in the nucleus of all hepatocytes in normal liver and precancerous lesions, but ARID1A expression was heterogeneous in HCC. In accordance with their findings, our study showed an increased probability score for ARID1A loss in advanced stage HCC compared to early stage HCC, indicating that ARID1A deletion is likely to occur in late-stage disease. Sun et al. reported that ic tumor . Our obsu et al.  using moARID1A in translational application in the era where molecular profiling of HCC from the TCGA data is almost complete, while its translation into the clinic remains beyond the horizon. Future studies are required to establish the molecular relationships between ARID1A suppression and the identified pathway. Our present study was based on an in silico analysis and has a limitation in the functional validation of our results. Despite this limitation, our present findings set the stage to explore the potential of ARID1A subtype indicate that ARID1A may have tumor suppressive activity and suggests the possibility of ARID1A as a prognostic biomarker in HCC patients.The low immune activity along with activation of genes that are associated with HCC development in low-


Idiopathic granulomatous mastitis (IGM) is a chronic inflammatory disease of the breast, the etiology of which, has still not been elucidated. There have been several mechanisms proposed to explain the pathogenesis. Since the first description of the disease, it has proved itself to be a great diagnostic and therapeutic challenge. It is very often misdiagnosed as cancer, resulting in myriad workup by the physician and great distress to the patient. Clear guidelines as to the management have still not been described. Here, we describe two patients who presented with IGM and have been successfully treated. The first patient was treated with a combination of steroids and antibiotics. The second patient achieved remission of the disease with antibiotics alone. We also propose an algorithm for the management of IGM. Idiopathic granulomatous mastitis (IGM) is a chronic inflammatory disease of the breast of unclear etiology . Since tWe present a case series of two cases of IGM, which were successfully treated.Patient 1Corynebacterium kroppenstedtii. She was started on a course of doxycycline and trimethoprim-sulfamethoxazole. The steroids resulted in significant improvement of the disease, with the resolution of pain, erythema, and abscess formation. Her steroid dose has been slowly tapered but has been unable to come off steroids over the past two years due to disease recurrenceA 46-year-old woman presented with a left breast mass and associated breast pain. There was also a 1.4 cm lymph node within the left axilla. She underwent bilateral diagnostic mammography, which showed increased density in the left breast. She subsequently underwent a bilateral breast ultrasound, which revealed an ill-defined area of abnormal echogenicity in the left breast Figure . No evidPatient 2The second patient is a 30-year-old female, who initially presented with a right breast mass and erythema. She then underwent a mammogram, which revealed a possible mass in the right breast as well as left. Following this, she underwent an ultrasound, which showed no obvious lesion in the left breast but showed a hypoechoic region in the right breast Figure . She wasCorynebacterium in 34 cases of women with granulomatous mastitis [Corynebacterium is a gram-positive bacterium and is usually present as a normal commensal on the skin. Corynebacterium has frequently been implicated in IGM and is the most common bacterial strain found in cases of IGM [Corynebacterium kroppenstedtii, Corynebacterium amycolatum, and Corynebacterium tuberculostearicum have been isolated in decreasing frequency in cases of IGM [Corynebacterium kroppenstedtii was isolated. The hormonal etiology has not been proven either. Although IGM does occur more in women of childbearing age, with a peak incidence in pregnant and lactating women, there have been reports of IGM occurring in nulliparous women or even several years after the last pregnancy [ IGM is a rare, chronic inflammatory condition that can mimic malignancy and can often be difficult to diagnose and manage. Since the first report of the case in 1972, there have been several reports of the condition, but clear-cut management of the disease has still not emerged. As the name suggests, the etiology of IGM has been difficult to define. Three leading hypotheses for possible etiologies are: autoimmune, infectious, and hormonal . The hypmastitis . Corynebs of IGM . Corynebregnancy . Severalregnancy . High prregnancy . No causThe spectrum of presentation of IGM is extremely wide. The most common presentation is a unilateral mass that can occur in any quadrant of the breast ,9,17,18.The diagnosis of IGM requires a high index of suspicion after other causes of granulomatous mastitis have been excluded. These include causes like mycobacterial infections, sarcoidosis, foreign body reactions, and fat necrosis. Tuberculous mastitis must be ruled out because steroids are the mainstay of treatment of IGM, which can aggravate mycobacterial infections. All specimens must undergo Ziehl acid-fast staining . There are no specific radiological findings that could point towards a diagnosis of IGM. The workup for IGM can be done in the same way as a breast mass would be evaluated, i.e., with a mammogram in a woman above 30 years of age and with ultrasound in a woman below 30 years of age. There are no pathognomonic mammographic findings that would point towards a diagnosis. The most common mammographic findings include focal areas of asymmetry, which can be single or multiple. Skin thickening and axillary lymphadenopathy can also be seen . The mosA definitive diagnosis of IGM is based on histopathological examination of the tissue specimen. FNA has been historically used but is now losing favor in the diagnosis of IGM due to its lower sensitivity. Studies have shown that the diagnostic yield of FNA may be as low as 21%-39% . For a dAlthough there have been several reports of IGM, there has been no universally accepted algorithm for the treatment of IGM. The treatment options, which have been tried include observation, steroids, antibiotics, immunosuppressive agents like methotrexate and surgery. Observation can be sometimes used as an initial approach in patients who present with an initial episode of IGM. This observation is based on studies that reported successful management with conservative management. Lai et al. in their study reported spontaneous resolution of IGM in 50% of cases and stable disease in the other half . HoweverAntibiotics have also been used in treatment. Although, this is controversial as no direct causation has been established between bacterial infiltration and IGM. Antibiotic use can be guided by microbiological data available, with chosen agents being directed against gram-positive organisms . In our At this time, corticosteroids are the mainstay of treatment for IGM. Several studies have shown favorable results with steroid use. In a prospective study by Pandey et al., of the 44 patients treated with steroids, there was complete resolution of disease in 35 patients (79.5%) . The medBefore the introduction of steroids, IGM was managed primarily with surgery. In the retrospective study done by Oran et al., there were 46 patients being followed by the same surgical team. Wide local excision with negative margins was performed in 18 cases. Of these 18 cases, only three recurred who were then treated successfully with steroids and re-excision . The autHere, we propose an algorithm for the management of IGM Figure .IGM is a chronic and often debilitating disease. It requires a very astute clinician to diagnose the condition. With no accepted guidelines for the treatment of the disease, it becomes extremely challenging to adequately treat the condition. Further larger studies need to be undertaken to elucidate the most appropriate way of managing this condition.


Sorting nexins (SNX) are a family of PX domain-containing proteins with pivotal roles in trafficking and signaling. SNX-BARs, which also have a curvature-generating Bin/Amphiphysin/Rvs (BAR) domain, have membrane-remodeling functions, particularly at the endosome. The minimal PX-BAR module is a dimer mediated by BAR-BAR interactions. Many SNX-BAR proteins, however, additionally have low-complexity N-terminal regions of unknown function. Here, we present the cryo-EM structure of the full-length SNX-BAR Mvp1, which is an autoinhibited tetramer. The tetramer is a dimer of dimers, wherein the membrane-interacting BAR surfaces are sequestered and the PX lipid-binding sites are occluded. The N-terminal low-complexity region of Mvp1 is essential for tetramerization. Mvp1 lacking its N-terminus is dimeric and exhibits enhanced membrane association. Membrane binding and remodeling by Mvp1 therefore requires unmasking of the PX and BAR domain lipid-interacting surfaces. This work reveals a tetrameric configuration of a SNX-BAR protein that provides critical insight into SNX-BAR function and regulation. SNX-BAR proteins are a family of PX and BAR domain-containing proteins with pivotal roles in trafficking processes. Here authors present the cryo-EM structure of the full-length fungal SNX-BAR Mvp1, which is an autoinhibited tetramer and provides critical insight into SNX-BAR function and regulation. SNX–BAR proteins form a subfamily that is characterized by the presence of a membrane-remodeling or curvature-sensing Bin/Amphiphysin/Rvs (BAR) domain3 in addition to the PX domain which, typically, is a lipid-binding module4. Mammalian cells have at least 12 SNX–BAR proteins and yeast have 76. SNX–BAR proteins are involved in several cellular processes that depend upon membrane remodeling, including protein and lipid trafficking to and from the endosome, endocytosis, and autophagy7. Defects in SNX–BAR function are associated with tumorigenesis, neurodegenerative diseases, and cardiovascular defects9.Sorting nexins (SNX) are a large and varied family of phox-homology (PX) domain-containing proteins with functions in membrane trafficking and remodeling, signaling, and organelle movement10 have, in addition, a low-complexity N-terminal region, the function of which is unclear. In the case of the endocytic SNX–BAR SNX9, this low-complexity region is involved in allosteric regulation of SNX9 membrane binding and remodeling activites11.All SNX–BARs homo- or heterodimerize via extensive interactions between their BAR domains. Current models for SNX–BAR-mediated membrane remodeling propose that both the PX and BAR domains have to be engaged with the membrane to ensure specificity and efficient binding. While some SNX–BAR proteins consist only of their PX–BAR modules, others, including those involved in retromer-mediated retrograde trafficking and autophagic processes12 and is required for retrograde trafficking from the endosome15. Here, we use cryoelectron microscopy with single-particle averaging to determine the structure of the fungal SNX–BAR Mvp1. Full-length Mvp1 is a tetramer consisting of two Mvp1 dimers that self-interact in such a manner that the concave lipid-engaging surfaces of the BAR dimers are buried. Tetramerization depends on the presence of the Mvp1 N-terminal region, which is also required for Mvp1 sorting function in vivo. Mvp1 lacking the N-terminal region retains membrane-remodeling activity and exhibits enhanced membrane binding in vivo and in vitro. Together, this work reveals a mechanism of regulation of Mvp1 function by self-assembly.Mvp1 is a poorly characterized yeast SNX–BAR protein that shares conservation with the mammalian SNX–BAR SNX8. Mvp1 was initially identified as a genetic interaction partner for the fungal dynamin superfamily protein Vps114. Loss of Mvp1 results in several trafficking defects in the cell13. Previous findings and data mining from whole-genome synthetic genetic array screens17 provide strong evidence for Mvp1 function in retromer-dependent retrograde trafficking from the endosome to the TGN  cells did not  Fig. . Mvp1 re21. However, the constructs used for the structural analyses consisted of a minimal PX–BAR module, removing any low-complexity sequence that some SNX–BAR proteins have at their N-termini. We therefore generated an Mvp1 construct similar to those used for the structural analyses,  encompassing only the PX–BAR module and omitting the N-terminal low-complexity sequence (Mvp1 Δ2-78 and Mvp1 Δ2-100). Both Mvp1 Δ2-78  but it may reflect different modes, extents or regularity of assembly on the lipid surface.Existing structures of SNX–BAR proteins reported to date are invariably dimeric, with dimerization occurring via an extensive BAR–BAR interface that is conserved in all BAR domain-containing proteinsmvp1 cells as assessed by CPY secretion , similar to Mvp1, but also displayed an enhanced localization to the vacuolar membrane 20 and Chaetomium thermophilum Vps5 (PDB 6H7W)21 as a starting point  contact the distal end of BAR helix α2 from the trans Mvp1 dimer  . SNX1 heterodimerizes with either SNX5 or SNX6 to generate one of the membrane remodeling and cargo selecting modules that function with mammalian retromer26, which has been linked to neurodegeneration, diabetes, pathogen invasion, and various cancers27. Unlike the conservation with SNX7 and SNX8, which is restricted to the PX and BAR domains, Mvp1 is conserved with SNX1 throughout its sequence, including the low-complexity N-terminus. Interestingly, it has been reported that a portion of the SNX1 pool in the cytosol of HeLa cells elutes from a size-exclusion column at a volume consistent with tetramers29. In addition, the F-BAR protein PACSIN/syndapin has been reported to form tetrameric barrel-like structures, as assessed by size-exclusion chromatography, cross-linking and negative-stain electron microscopy30. These observations suggest that tetramerization may be a conserved mechanism of SNX–BAR and some other BAR protein regulation.Mvp1 shares homology with several mammalian SNX–BAR proteins. The highest conservation is with SNX8 and SNX7 (at ~22% identity across the PX–BAR module). SNX8 function is unclear but is involved in endosomal sorting31 and it was reported that a lysine within the N-terminus is ubiquitylated32. In the case of SNX1 and SNX2, both of which have similar low-complexity N-termini, multiple phosphorylation sites have been identified in different phosphoproteome analyses37. In the absence of the Mvp1 N-terminus, we observe increased lipid binding suggesting that the N-terminus may have an autoinhibitory function. Autoinhibition in BAR domain-containing proteins has been reported3. For example, SNX9 is autoinhibited in solution and this has been shown to be due to the linker preceding the PX–BAR module11. Other examples include the PACSIN/syndapin F-BAR, the I-BAR IRSp53, and the Drosophila F-BAR protein Nervous Wreck40. In all these cases, inhibition is mediated by auxiliary regions or domains adjacent to the BAR. Here, we propose the N-terminus promotes tetramerization as a mechanism of autoinhibition.Based on our data that the Mvp1 N-terminus is functionally important, we speculate that it will be a target for regulation, either by interaction with other proteins or by posttranslational modifications. Indeed, the NetPhos 3.1 server predicts several phosphorylation sites within the N-terminustrans BAR dimers, at least with the PX domains in the same positions as observed in the structure. Incubation of Mvp1 tetramers with the PI3P headgroup or DiC8 PI3P did not affect the tetramer, as assessed by SEC-MALS (data not shown). A membrane containing PI3P might therefore be required for productive disruption of the Mvp1 tetramer.Our sedimentation assays show that full-length Mvp1 interacts with liposomes, but only in the presence of PI3P. The structure of the tetramer suggests that the PX domains could not accommodate PI3P without steric clashes of the acyl chains with the trans BAR dimer has to change to accommodate the PI3P. This may lead to further opening of the tetramer. It has recently been reported that the SNX3 PX domain inserts its β1–β2 loop, as well as its membrane-inserting loop, into the bilayer when bound to membranes containing PI3P41. Mvp1 has a stretch of hydrophobic residues in its long β1–β2 loop. The equivalent of the membrane-inserting loop in Mvp1 is the top of the PPII loop, adjacent to Mut1, which we observe is critical for membrane binding. We therefore speculate that the initiating event in changing the conformation of the PX domain relative to the trans BAR dimer is insertion of the β1–β2 loop into the bilayer. This may be followed by PI3P binding and disruption of the tetramer. This would necessitate the presence of PI3P in a membrane context. The N-terminus may be displaced by sequential binding of the PX domain to PI3P followed by BAR interaction with the membrane’s negative charge. In the cell, tetramer opening may be further enhanced by cargo binding or posttranslational modifications of the N-terminus.How could tetrameric Mvp1 interact with membrane containing PI3P at all? The structure we describe was obtained in the absence of lipid or lipid headgroups. In the presence of membranes, we observe PI3P-dependent lipid binding so the conformation of the PX domain relative to the SNX–BAR tetramerization adds a layer of complexity to the regulation of SNX–BAR function in membrane remodeling and in cargo sorting. As SNX–BAR proteins are vital in well-characterized machineries such as retromer, and as deficiencies in SNX–BARs are associated with a range of health challenges, these findings provide a precedent and foundation for future studies on the functional regulation of SNX–BAR proteins via self-assembly.YPD  was used for routine growth. Synthetic complete  or SD  were used for growth prior to microscopy or to maintain plasmid selection. Diploids were sporulated by overnight incubation in YPA  followed by 1–2 days in SPO .Saccharomyces cerevisiae used in this work are listed in Supplementary Table Strains of S. cerevisiae MVP1, with flanking upstream and downstream regions of 250 and 150 nucleotides, was amplified from genomic DNA prepared from W303A/α diploids. All constructs were generated by splicing by overlap extension, if required, and Gibson assembly. Primers used in this work are listed in Supplementary Table 4Plasmids used in this work are listed in Supplementary Table 6 fusions followed by a thrombin cleavage site or from a homemade variant of pMW (a gift from Helen Kent), as a C-terminal MBP fusion preceded by a PreScission cleavage site. Constructs were expressed in Escherichiacoli strain BL21-Codon Plus(DE3)-RIPL (Agilent). Cells were grown in 2xTY at 37 °C until mid-log phase, after which expression was induced by addition of IPTG (42.5 μM). Proteins were expressed overnight at 21 °C. Cells were harvested and resuspended in TN150 buffer  before lysis by homogenization . Lysates were then clarified .Full-length Mvp1 (codons 1–511), its mutants and truncations were expressed from two vectors: pET-15b (Novagen), as N-terminal His6-Mvp1 fusions were applied, in batch, to Ni-IDA resin (Macherey-Nagel). The resins were washed with TN150, loaded into a column and fusion proteins were eluted using a 0–250 mM imidazole gradient in TN150. Peak fractions were pooled and dialyzed into TN150. The dialysates were then applied to an anion exchange column. Proteins were eluted with a 150–500 mM NaCl gradient. Peak fractions were pooled, concentrated, and passed over a Superdex 200 size-exclusion column, equilibrated with TN250 (TN with 250 mM NaCl). Peak fractions were concentrated and flash frozen in liquid nitrogen for storage until use.HisC-terminal MBP-tagged fusions were applied, in batch, to amylose resin (NEB). After washing in TN150, fusion proteins were eluted with 10 mM maltose in TN150. Peak fractions were pooled and dialyzed overnight into fresh TN150 at 4 °C, while being cleaved with PreScission protease . PreScission protease and cleaved MBP were removed by sequential incubations with glutathione sepharose (Macherey-Nagel) and amylose resin. Mvp1 proteins were then further purified by anion exchange and size-exclusion chromatography, as for the His6 fusions. A slight modification was used to purify Mvp1 Δ2-100. The uncleaved protein was purified in TN150 supplemented with 1 mM EDTA (TN150E) and 0.1 mM PMSF by amylose resin, elution, dialysis into TN150E, anion exchange, and size exclusion chromatography. Then the protein was cleaved in TN250 with PreScission, after which the PreScission was removed and the protein was passed over a Superdex 200 in TN250, before concentration and snap freezing.Yeast were grown at 30 °C overnight in YPD or appropriate SD to maintain plasmid selection. Yeast were then diluted in YPD and grown to mid-log phase. Vacuolar membranes were stained with FM 4–64  for 45 min, followed by washing and incubation in SC medium without dye for 1 h. Prior to imaging, cells were plated onto No. 1.5 glass-bottomed coverdishes (MatTek Corporation) previously treated with 15 μl 2 mg/ml concanavalin-A (Sigma-Aldrich).A Nikon (Melville) A1 confocal, equipped with a 100× Plan Apo 100× oil objective, was used to obtain confocal images and Z-stacks. NIS Elements Imaging software was used to control acquisition. Images were processed using Fiji.600/ml and fivefold serial dilutions were made in water. Two microliters of each dilution was spotted onto YPD or SD-Trp plates after which they were incubated at 30 °C for 24 h. The colonies were overlaid with nitrocellulose, followed by additional incubation at 30 °C for 16 h. The membranes were then extensively washed with TBST  and blocked for 1 h with TBST containing 5% bovine serum albumin. The membrane was probed for secreted CPY using an anti-CPY mouse monoclonal antibody  for 2 h at room temperature. The secondary antibody was IRDye 680RD-conjugated goat anti-mouse antibody . The signal was detected using a ChemiDoc MP Imaging System (Bio-Rad).After overnight growth to saturation in appropriate media at 30 °C, yeast were diluted and regrown to mid-logarithmic phase in YPD. Yeast were then diluted to 0.5 ODLiposomes were made from 100% PS  or 95% PS + 5% PI3P ). Lipid mixtures were dried using a stream of nitrogen gas and were desiccated for at least 3 h. The lipids were rehydrated in 20 mM TRIS/Cl pH 7.5, 250 mM NaCl to a concentration of 1 mg/ml. Liposomes were extruded through a 100 nm Nucleopore membrane (Whatman).g for 30 min at 4 °C in the S55-A2 rotor (Thermo Fisher Scientific). Pellets and supernatants were separated and equal proportions of each were analyzed by SDS–PAGE. Bands were quantified by integration using Fiji.Mvp1, its mutants or truncations were incubated in the absence or presence of 0.1 mg/ml 100 nm PS or PS/PI3P liposomes at 21 °C for 30 min in a final volume of 100 μl in TN250 and at a concentration of 1.2 μM. The samples were then centrifuged at 40,000 Mvp1, its mutants or truncations were subjected to size-exclusion chromatography using a Superdex 200 10/300 gl column  equilibrated 20 mM TRIS/Cl pH 7.4, 250 mM NaCl, 1.93 mM β-mercaptoethanol, unless explicitly stated. In general, 500 μl of 20–21.2 μM protein was injected, with the exception of Mvp1 Δ2-78, where sample limitations permitted injection of 500 μl of 14 μM protein. The Mvp1 with an engineered PreScission protease cleavage site after residue 99 was passed onto the column uncleaved or after cleavage with 10 U of PreScission protease in TN buffer for 90 min before removal of the PreScission protease by incubation with glutathione-sepharose beads. The column was coupled to a static 18-angle light-scattering detector (DAWN HELEOS-II) and a refractive index detector (Optilab T-rEX) (Wyatt Technology). Data were collected continuously at a flow rate of 0.3 ml/min. Data analysis was performed using the program Astra VII. Monomeric BSA (2.0 mg/ml) (Sigma) was used for data quality control.6 electron source, at the indicated magnification on a 4,000 × 4,000 Gatan Ultrascan charge-coupled device camera.For tubulation assays, 10 μg liposomes were incubated with 10 μM  Mvp1 or Mvp1 Mut1  for 30 min at room temperature. 3 μl sample aliquots  were adsorbed to glow-discharged 300-mesh carbon-coated copper grids and stained with 2% uranyl formate. Images were recorded on a Tecnai T12 Spirit electron microscope, operating at 120 kV with a LaB−/Å2/frame). SerialEM was used for automated data collection42. Defocus values used to collect the data set ranged from −0.5 to −3.5 μm. Further details are given in Supplementary Table Purified Mvp1 or Mvp1 Mut1 (~5 μM protein) was applied to glow-discharged holey carbon grids  and plunge-frozen in liquid ethane using an FEI Vitrobot Mark IV. Images were acquired at the University of Virginia School of Medicine Molecular Electron Microscopy Core on a Falcon 3EC detector in counting mode using an FEI Titan Krios at 300 kV with a nominal magnification of 75,000, corresponding to a final pixel size of 1.056 Å. For each image stack, a total dose of about 58 electrons per square angstrom was equally fractioned into 49 frames  0.143 criterion.Approximately 6500 particles of Mvp1 were manually picked from ~100 lowpass-filtered micrographs using e2boxer.py from the EMAN2 suiteThe 7 Å map was used as reference for further particle auto-picking from the whole data set. Approximately 1,230,000 particles were auto-picked from 1620 micrographs for further processing. The whole set of particles was cleaned to remove contaminants or junk particles by three rounds of 2d classification. Finally, ~200,000 particles were selected for further 3d reconstruction.We observed an apparent D2 symmetry existing in the 3d reconstruction from the side view and the 2d classification results. In order to reduce the impact of imposition of symmetry on the reconstruction, therefore, we performed the 3d reconstruction procedures without and with different symmetries .For C1 symmetry, after 3d classification into five classes, the most populated class, which accounted for 39.8% of the data set , was used for 3d-masked auto-refinement. This yielded a map of ~6.1 Å resolution (gold-standard FSC 0.143 criterion). A 5.3 Å map was generated after sharpening with a B-factor of −190 ÅC2 symmetry was imposed on three user-defined axes Fig. . For eac2),4.6 Å ,4.4 Å .4.7 Å , a final map of 4.2 Å resolution was obtained after sharpening with a B-factor of −152.8 Å47 or Coot48 and local map resolutions were calculated using RELION.Maps were visualized using ChimeraXThe same procedure was used for Mvp1 Mut1 data collection and processing. Due to the distribution of particle orientations  and Chaetomium thermophilum Vps5 (PDB 6HZW) were used as a starting point (Fig. 50, local distance restraints generated with ProSMART51, and existing SNX–BAR structures as guides. Some long loops could not be assigned well in the model including the β1–β2 loop in the PX domain, the loop connecting the PX domain to the BAR domain and the loop connecting BAR helices α2–α3. The model was refined against the 4.2 Å auto-sharpened map using phenix.real_space_refine. The final model statistics are listed in Supplementary Table The model for the Mvp1 tetramer was built with Cootint Fig. . The mapint Fig.  enabled 52. Sizes of tetramerization interfaces were calculated using the PISA web server53.The electrostatic potential of the surface of the Mvp1 tetramer was calculated, at a pH of 6.8, using APBS in the PDB2PQR serverFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary