RefSeq and the prokaryotic genome annotation pipeline in the age of metagenomes.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains over 315 000 bacterial and archaeal genomes and 236 million proteins with up-to-date and consistent annotation. In the past 3 years, we have expanded the diversity of the RefSeq collection by including the best quality metagenome-assembled genomes (MAGs) submitted to INSDC (DDBJ, ENA and GenBank), while maintaining its quality by adding validation checks. Assemblies are now more stringently evaluated for contamination and for completeness of annotation prior to acceptance into RefSeq. MAGs now account for over 17000 assemblies in RefSeq, split over 165 orders and 362 families. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP), which is used to annotate nearly all RefSeq assemblies include better detection of protein-coding genes. Nearly 83% of RefSeq proteins are now named by a curated Protein Family Model, a 4.7% increase in the past three years ago. In addition to literature citations, Enzyme Commission numbers, and gene symbols, Gene Ontology terms are now assigned to 48% of RefSeq proteins, allowing for easier multi-genome comparison. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/. PGAP is available as a stand-alone tool able to produce GenBank-ready files at https://github.com/ncbi/pgap.

Anatomic nomenclature and 3-dimensional regional model of the human ovary: call for a new paradigm.

The ovaries are the female gonads that are crucial for reproduction, steroid production, and overall health. Historically, the ovary was broadly divided into regions defined as the cortex, medulla, and hilum. This current nomenclature lacks specificity and fails to consider the significant anatomic variations in the ovary. Recent technological advances in imaging modalities and high-resolution omic analyses have brought about the need for revision of the existing definitions, which will facilitate the integration of generated data and enable the characterization of organ subanatomy and function at the cellular level. The creation of these high-resolution multimodal maps of the ovary will enhance collaboration and communication among disciplines and between clinicians and researchers. Beginning in March 2021, the Pediatric and Adolescent Gynecology Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development invited subject-matter experts to participate in a series of workshops and meetings to standardize ovarian nomenclature and define the organ's features. The goal was to develop a spatially defined and semantically consistent terminology of the ovary to support collaborative, team science-based endeavors aimed at generating reference atlases of the human ovary. The group recommended a standardized, 3-dimensional description of the ovary and an ontological approach to the subanatomy of the ovary and definition of follicles. This new greater precision in nomenclature and mapping will better reflect the ovary's heterogeneous composition and function, support the standardization of tissue collection, facilitate functional analyses, and enable clinical and research collaborations. The conceptualization process and outcomes of the effort, which spanned the better part of 2021 and early 2022, are introduced in this article. The institute and the workshop participants encourage researchers and clinicians to adopt the new systems in their everyday work to advance the overarching goal of improving human reproductive health.

RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) contains nearly 200 000 bacterial and archaeal genomes and 150 million proteins with up-to-date annotation. Changes in the Prokaryotic Genome Annotation Pipeline (PGAP) since 2018 have resulted in a substantial reduction in spurious annotation. The hierarchical collection of protein family models (PFMs) used by PGAP as evidence for structural and functional annotation was expanded to over 35 000 protein profile hidden Markov models (HMMs), 12 300 BlastRules and 36 000 curated CDD architectures. As a result, >122 million or 79% of RefSeq proteins are now named based on a match to a curated PFM. Gene symbols, Enzyme Commission numbers or supporting publication attributes are available on over 40% of the PFMs and are inherited by the proteins and features they name, facilitating multi-genome analyses and connections to the literature. In adherence with the principles of FAIR (findable, accessible, interoperable, reusable), the PFMs are available in the Protein Family Models Entrez database to any user. Finally, the reference and representative genome set, a taxonomically diverse subset of RefSeq prokaryotic genomes, is now recalculated regularly and available for download and homology searches with BLAST. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

Use of substances to cope during the COVID-19 pandemic among transgender and gender diverse adults.

Background: Studies have found changes in substance use during the COVID-19 pandemic in specific populations. Transgender and gender diverse (TGD) individuals have experienced greater distress compared to cisgender individuals during the pandemic; however, there is little research on substance use among TGD individuals during this sensitive time period.Objectives: The objective of this study is to examine distress from COVID-19 and coping via substance use including alcohol, cannabis, tobacco, and non-medical use of prescription drugs (NMUPD) among TGD adults.Method: An online survey assessing substance use, general psychiatric symptoms, and COVID-19 anxiety was completed by 342 TGD individuals (16.4% transfeminine, 19.6% transmasculine, 64.0% Gender Diverse) in June/July 2020. Chi-square and structural equation modeling (SEM) analyses examined the connections between distress, coping, and substance use.Results: Seventy-one percent of participants reported no changes in substance use since the start of the pandemic and 22% reported an increase in substance use. Increased substance use was associated with alcohol (p < .001), cannabis (p < .001), and combustible tobacco (p < .001) use in the prior three months. SEM showed significant direct effects between distress and substance use coping, substance use coping and recent drug use, and an indirect effect of distress on recent drug use through substance use coping (ß = .31, p = .001).Conclusion: Results highlight the risk of substance use to cope with COVID-19-related stress in a large sample of a minoritized population with mental health disparities. Transmasculine and gender diverse participants were especially likely to report using substances to cope.

A multi-institutional study of renal outcomes and renal-related pregnancy outcomes in uterus transplant recipients.

Uterus transplantation (UTx) is an effective treatment option for uterine factor infertility. However, the need for immunosuppression and congenital renal anomalies that coexist with uterine agenesis in about 30% of women with Mayer-Rokitansky-Kuster-Hauser syndrome create a risk for renal dysfunction. We therefore examined renal function trajectory and related pregnancy complications in an international cohort of 18 UTx recipients from September 2016-February 2020 who had at least one live birth. All UTx recipients had a diminution in their renal function that was apparent starting at 30 days posttransplant and in half the reduction in eGFR was at least 20%; the decrease in eGFR persisted into the early post-partum period. Half met criteria for Stage 1 acute kidney injury (AKI) as defined by the AKI Network criteria during their pregnancy. Overall, 28% of UTx recipients developed pre-eclampsia. eGFR was lower at embryo transfer and throughout pregnancy among those who developed pre-eclampsia, reaching statistical significance at week 16 of pregnancy. This effect was independent of tacrolimus levels. Mean eGFR remained significantly lower in the first 1-3 months after delivery. In the subgroup who reached 12 months of postpartum follow up and had a graft hysterectomy (n = 4), there was no longer a statistical difference in eGFR (pretransplant 106.7 ml/m ± 17.7 vs. 12 mos postpartum 92.6 ml/m ± 21.7, p = .13) but the number was small. Further study is required to delineate long term renal risks for UTx recipients, improve patient selection, and make decisions regarding a second pregnancy.

Evaluation of Firearm-Related Reinjury in Connecticut: An Opportunity for Gun Violence Prevention.

BACKGROUND: The regional extent of the risk of repeat firearm-related injury (FRI) and homicide mortality for victims of firearm injury in Connecticut is unknown. In this study, we evaluate the risk of repeat firearm injury in survivors of firearm violence in Connecticut. METHODS: Using medical record data from the Yale New Haven Health (YNHH) system and data from the Connecticut Office of the Chief Medical Examiner, we conducted a cohort study of patients with an FRI in 2014 to determine their risk of a repeat firearm injury or mortality from homicide in the ensuing 5 years compared with nonviolence-related trauma patient controls. RESULTS: We identified 94 patients with an FRI in the YNHH system from 2014 who survived to discharge. Of these patients, 8.5% (8 of 94) had a repeat FRI and 2% (2 of 94) died from homicide within the next 5 years. Compared with nonviolence-related trauma patients from 2014 (n = 2001), those with an FRI had 12 times the odds of a repeat firearm injury (odds ratio: 12.0, P = 0.047) in the next 5 years after adjustment for relevant covariates. CONCLUSIONS: Of the patients presenting with an initial FRI in the YNHH system, one in twelve will experience another firearm injury within the next 5 years. These data indicate that firearm-related reinjury is common in Connecticut and suggest the need for further violence prevention efforts.

Assessing the Race, Ethnicity, and Gender Inequities in Blood Alcohol Testing After Trauma.

INTRODUCTION: Alcohol use remains a significant contributing factor in traumatic injuries in the United States, resulting in substantial patient morbidity and societal cost. Because of this, the American College of Surgeons Verification, Review, and Consultation Program requires the screening of 80% of trauma admissions. Multiple studies suggest that patients who use alcohol are subject to stigma by health care providers and may ultimately face legal and financial ramifications of a positive alcohol screening test. There is also evidence that sociodemographic factors may dictate drug and alcohol screening patterns among patients. Because this screening target is often not uniformly achieved among all patients presenting with injury, we sought to investigate whether there are any discrepancies in screening across sociodemographic groups. METHODS: We investigated the Trauma Quality Program Participant User File for all trauma cases admitted during 2017 and compared the rates of the serum alcohol screening test across different demographic factors, including race and ethnicity. We then performed an adjusted multivariable logistic regression to determine the odds ratio (OR) for receiving a test based on these demographic factors adjusted for hospital and clinical factors. RESULTS: There were 729,174 traumas included in the study. Of this group, 345,315 (47.4%) were screened with a serum alcohol test. Screening rates varied by injury mechanism and were highest among motorcycle crashes (66.0% of patients screened) and lowest among falls (32.8% of patients screened). Overall, Asian and Pacific Islander (52.5% screened), Black (57.7% screened), and other race (58.4% screened) had higher rates of alcohol screening than White patients (43.7% screened, P < 0.001). Similarly, Hispanic patients were screened at higher rates than non-Hispanic patients (56.4% screening versus 46.2% screening, P < 0.001). These differences persisted across nearly all injury categories. In multivariable logistic regression, Asian and Pacific Islanders were associated with the highest odds of being screened (OR 1.34, P < 0.001) followed by other race (OR 1.25, P < 0.001) in comparison to White patients. CONCLUSIONS: There are consistent and significant differences in alcohol screening rates across race and ethnicity, despite accounting for injury mechanism and comorbidities.

Reproductive Technology Considerations in Uterus Transplant.

Uterus transplantation (UTx) provides a new pathway to parenthood for patients with absolute uterine factor infertility. The application of reproductive technologies, such as in vitro fertilization, embryo cryopreservation, and frozen embryo transfers, for this unique population, is particularly nuanced and continually evolving. There are important pretransplant and posttransplant reproductive considerations for physicians and patients anticipating UTx. As with any rapidly evolving medical innovation, efforts to consolidate experiences and knowledge by centers offering UTx is paramount.

Secreted indicators of androgen receptor activity in breast cancer pre-clinical models.

PURPOSE: Accumulating evidence has attracted attention to the androgen receptor (AR) as a biomarker and therapeutic target in breast cancer. We hypothesized that AR activity within the tumor has clinical implications and investigated whether androgen responsive serum factors might serve as a minimally invasive indicator of tumor AR activity. METHODS: Based on a comprehensive gene expression analysis of an AR-positive, triple negative breast cancer patient-derived xenograft (PDX) model, 163 dihydrotestosterone (DHT)-responsive genes were defined as an androgen responsive gene set. Among them, we focused on genes that were DHT-responsive that encode secreted proteins, namely KLK3, AZGP1 and PIP, that encode the secreted factors prostate specific antigen (PSA), zinc-alpha-2-glycoprotein (ZAG) and prolactin induced protein (PIP), respectively. Using AR-positive breast cancer cell lines representing all breast cancer subtypes, expression of candidate factors was assessed in response to agonist DHT and antagonist enzalutamide. Gene set enrichment analysis (GSEA) was performed on publically available gene expression datasets from breast cancer patients to analyze the relationship between genes encoding the secreted factors and other androgen responsive gene sets in each breast cancer subtype. RESULTS: Anti-androgen treatment decreased proliferation in all cell lines tested representing various tumor subtypes. Expression of the secreted factors was regulated by AR activation in the majority of breast cancer cell lines. In GSEA, the candidate genes were positively correlated with an androgen responsive gene set across breast cancer subtypes. CONCLUSION: KLK3, AZGP1 and PIP are AR regulated and reflect tumor AR activity. Further investigations are needed to examine the potential efficacy of these factors as serum biomarkers.

Life underneath the VCA umbrella: Perspectives from the US Uterus Transplant Consortium.

The parallel emergence of uterus transplantation (UTx) and other transplantation innovations including face and hand transplantation led to the categorization of the uterus as a vascular composite allograft (VCA). With >60 transplants and >20 births worldwide, UTx is transitioning rapidly from a research endeavor to an effective treatment option for women with uterine factor infertility. While it originally made sense to group the innovations under one umbrella, it is time to revisit the designation of UTx as a VCA. We describe how UTx needs unique policy, procedural codes, insurance contracts, and educational initiatives. We contend that separating UTx from VCAs may become necessary in the future to avoid hindering the growth and regulation of this field.

Guidelines for standardized nomenclature and reporting in uterus transplantation: An opinion from the United States Uterus Transplant Consortium.

Uterus transplantation is a nascent but growing field. To support this growth, the United States Uterus Transplant Consortium proposes guidelines for nomenclature related to operative technique, vascular anatomy, and donor, recipient, and offspring outcomes. In terms of anatomy, the group recommends reporting donor arterial inflow and recipient anastomotic site delivering inflow to the graft and offers standardization of the names for the 4 veins originating from the uterus because of current inconsistency in this particular nomenclature. Seven progressive stages with milestones of success are defined for reporting on uterus transplantation outcomes: (1) technical, (2) menstruation, (3) embryo implantation, (4) pregnancy, (5) delivery, (6) graft removal, and (7) long-term follow-up. The 3 primary metrics for success are recipient survival (as reported for other organ transplant recipients), graft survival, and uterus transplant live birth rate (defined as live birth per transplanted recipient). A number of secondary outcomes should also be reported, most of which capture stage-specific milestones, as well as data on graft failure. Outcome metrics for living donors include patient survival, survival free of operative intervention, and data on complications and hospitalizations. Finally, we make specific recommendations on follow-up for offspring born from uterine grafts, which includes specialty surveillance as well as collection and reporting of routine pediatric outcomes. The goal of standardization in reporting is to create consistency and improve the quality of evidence available on the efficacy and value of the procedure.

Financial Hardship After Traumatic Injury: Risk Factors and Drivers of Out-of-Pocket Health Expenses.

BACKGROUND: Trauma-related disorders rank among the top five most costly medical conditions to the health care system. However, the impact of out-of-pocket (OOP) health expenses for traumatic conditions is not known. In this cross-sectional study, we use nationally representative data to investigate whether patients with a traumatic injury experienced financial hardship from OOP health expenses. METHODS: Using data from the Medical Expenditure Panel Survey from 2010 to 2015, we analyzed the financial burden associated with a traumatic injury. Primary outcomes were excess financial burden (OOP>20% of annual income) and catastrophic medical expenses (OOP>40% of annual income). A multivariable logistic regression analysis evaluated whether these outcomes were associated with traumatic injury, adjusting for demographic, socioeconomic, and health care factors. We then completed a descriptive analysis to elucidate drivers of total OOP expenses. RESULTS: Of the 90,964 families in the cohort, 6434 families had a traumatic injury requiring a visit to the emergency room and 668 families had a traumatic injury requiring hospitalization. Overall 1 in 8 households with an injured family member requiring hospitalization experienced financial hardship. These families were more likely to experience excess financial burden (OR: 2.04, 95% CI: 1.13-3.64) and catastrophic medical expenses (OR: 3.08, 95% CI: 1.37-6.9). The largest burden of OOP expenses was due to prescription drug costs, with inpatient costs as a major driver of OOP expenses for those requiring hospitalization. CONCLUSIONS: Households with an injured family member requiring hospitalization are significantly more vulnerable to financial hardship from OOP health expenses than the noninjured population. Prescription drug and inpatient costs were the most significant drivers of OOP health expenses.

When to Admit to Observation: Predicting Length of Stay for Anticoagulated Elderly Fall Victims.

BACKGROUND: Geriatric patients who fall while taking an anticoagulant have a small but significant risk of delayed intracranial hemorrhage requiring observation for 24 h. However, the medical complexity associated with geriatric care may necessitate a longer stay in the hospital. Little is known about the factors associated with a successful observational status stay (<2 d) for this population. MATERIALS AND METHODS: Elderly patients who fell while taking an anticoagulant admitted from 2012 to 2017 at an ACS level II trauma center were included in a retrospective cohort study to determine what factors were associated with a stay consistent with observational status. INCLUSION CRITERIA: age> 65 y old, negative initial head CT, and one of the following: INR>3.5 if on warfarin, GCS<14, external signs of trauma, or focal neurological deficits. RESULTS: The cohort included 369 patients. Factors associated with decreased likelihood of successful observational status included the need for services after discharge such as an extended care facility (OR 0.06, 95% CI 0.02-0.19, P < 0.001) or visiting nurse agency services (OR 0.27, 95% CI 0.10-0.75, P < 0.001), a dementia diagnosis (OR 0.17, 95% CI 0.04-0.70, P = 0.014), increasing number of medications (OR 0.91, 95% CI 0.84-0.99, P = 0.031), and the use of coumadin (OR 0.28, 95% CI 0.12-0.70, P = 0.006). CONCLUSIONS: For trauma providers, knowing your patient's medication use and particularly type of anticoagulant, comorbidities including dementia, and likely need for services after discharge will help guide the decision to admit the patient for what may be a reasonably lengthy stay versus a brief observation in the hospital for elderly fall victims on anticoagulation.

RefSeq: an update on prokaryotic genome annotation and curation.

The Reference Sequence (RefSeq) project at the National Center for Biotechnology Information (NCBI) provides annotation for over 95 000 prokaryotic genomes that meet standards for sequence quality, completeness, and freedom from contamination. Genomes are annotated by a single Prokaryotic Genome Annotation Pipeline (PGAP) to provide users with a resource that is as consistent and accurate as possible. Notable recent changes include the development of a hierarchical evidence scheme, a new focus on curating annotation evidence sources, the addition and curation of protein profile hidden Markov models (HMMs), release of an updated pipeline (PGAP-4), and comprehensive re-annotation of RefSeq prokaryotic genomes. Antimicrobial resistance proteins have been reannotated comprehensively, improved structural annotation of insertion sequence transposases and selenoproteins is provided, curated complex domain architectures have given upgraded names to millions of multidomain proteins, and we introduce a new kind of annotation rule-BlastRules. Continual curation of supporting evidence, and propagation of improved names onto RefSeq proteins ensures that the functional annotation of genomes is kept current. An increasing share of our annotation now derives from HMMs and other sets of annotation rules that are portable by nature, and available for download and for reuse by other investigators. RefSeq is found at https://www.ncbi.nlm.nih.gov/refseq/.

Diet-induced obesity alters the maternal metabolome and early placenta transcriptome and decreases placenta vascularity in the mouse.

Maternal obesity is associated with an increased risk of obesity and metabolic disease in offspring. Increasing evidence suggests that the placenta plays an active role in fetal programming. In this study, we used a mouse model of diet-induced obesity to demonstrate that the abnormal metabolic milieu of maternal obesity sets the stage very early in pregnancy by altering the transcriptome of placenta progenitor cells in the preimplantation (trophectoderm [TE]) and early postimplantation (ectoplacental cone [EPC]) placenta precursors, which is associated with later changes in placenta development and function. Sphingolipid metabolism was markedly altered in the plasma of obese dams very early in pregnancy as was expression of genes related to sphingolipid processing in the early placenta. Upregulation of these pathways inhibits angiogenesis and causes endothelial dysfunction. The expression of many other genes related to angiogenesis and vascular development were disrupted in the TE and EPC. Other key changes in the maternal metabolome in obese dams that are likely to influence placenta and fetal development include a marked decrease in myo and chiro-inositol. These early metabolic and gene expression changes may contribute to phenotypic changes in the placenta, as we found that exposure to a high-fat diet decreased placenta microvessel density at both mid and late gestation. This is the first study to demonstrate that maternal obesity alters the transcriptome at the earliest stages of murine placenta development.

The influence of sociodemographic factors on operative decision-making in small bowel obstruction.

BACKGROUND: Current guidelines for small bowel obstruction (SBO) recommend a limited trial of nonoperative management of no more than 3-5 d. For patients requiring surgery, it is uncertain if sociodemographic factors are associated with disparities in the duration of the trial of nonoperative therapy. METHODS: The Healthcare Cost and Utilization Project National Inpatient Sample from 2012 to 2014 was queried for discharges with a primary diagnosis of SBO. Primary outcomes of interest were the effects of sociodemographic factors, including race, insurance status, and income on the rate of receiving any operative management for SBO, and subsequently, among patients managed surgically, the risk of operative delay, defined as operative management ≥ 5 d after admission. We did this by using logistic hierarchical generalized linear models, accounting for hospital clustering and adjusted for sex, age, comorbidity, and hospital factors. RESULTS: Of the 589,850 admissions for SBO between 2012 and 2014, 22.0% underwent operations. Overall, 26.2% were non-White, including 12.2% Black and 8.6% Hispanic patients, and the majority (56.0%) had Medicare insurance coverage. Income quartiles were evenly distributed across the overall study population. In adjusted logistic regression, operative delay was associated with increased odds of in-hospital mortality (odds ratio 1.30 95% confidence interval [1.10, 1.54]). Adjusted for patient and hospital factors, Black patients were significantly more likely to receive operations for SBO, whereas Medicaid and Medicare patients were significantly less likely. However, Black, Medicaid, and Medicare patients who were managed operatively were significantly more likely to have an operative delay of 5 or more d. There was no significant association between income and operative management in adjusted regression models. CONCLUSIONS: Significant disparities in the operative management were based on race and insurance status. Further research is warranted to understand the causes of, and solutions to, these sociodemographic disparities in care.

Reference sequence (RefSeq) database at NCBI: current status, taxonomic expansion, and functional annotation.

The RefSeq project at the National Center for Biotechnology Information (NCBI) maintains and curates a publicly available database of annotated genomic, transcript, and protein sequence records (http://www.ncbi.nlm.nih.gov/refseq/). The RefSeq project leverages the data submitted to the International Nucleotide Sequence Database Collaboration (INSDC) against a combination of computation, manual curation, and collaboration to produce a standard set of stable, non-redundant reference sequences. The RefSeq project augments these reference sequences with current knowledge including publications, functional features and informative nomenclature. The database currently represents sequences from more than 55,000 organisms (>4800 viruses, >40,000 prokaryotes and >10,000 eukaryotes; RefSeq release 71), ranging from a single record to complete genomes. This paper summarizes the current status of the viral, prokaryotic, and eukaryotic branches of the RefSeq project, reports on improvements to data access and details efforts to further expand the taxonomic representation of the collection. We also highlight diverse functional curation initiatives that support multiple uses of RefSeq data including taxonomic validation, genome annotation, comparative genomics, and clinical testing. We summarize our approach to utilizing available RNA-Seq and other data types in our manual curation process for vertebrate, plant, and other species, and describe a new direction for prokaryotic genomes and protein name management.

Gestational Diabetes Alters the Metabolomic Profile in 2nd Trimester Amniotic Fluid in a Sex-Specific Manner.

Maternal diabetes and obesity induce marked abnormalities in glucose homeostasis and insulin secretion in the fetus, and are linked to obesity, diabetes, and metabolic disease in the offspring, with specific metabolic characterization based on offspring sex. Gestational diabetes (GDM) has profound effects on the intrauterine milieu, which may reflect and/or modulate the function of the maternal⁻fetal unit. In order to characterize metabolic factors that affect offspring development, we profiled the metabolome of second trimester amniotic fluid (AF) from women who were subsequently diagnosed with gestational diabetes (GDM) using a targeted metabolomics approach, profiling 459 known biochemicals through gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) assays. Using a nested case-control study design, we identified 69 total biochemicals altered by GDM exposure, while sex-specific analysis identified 44 and 58 metabolites in male and female offspring, respectively. The most significant changes were in glucose, amino acid, glutathione, fatty acid, sphingolipid, and bile acid metabolism with specific changes identified based on the offspring sex. Targeted isotope dilution LC/MS confirmatory assays measured significant changes in docosahexaenoic acid and arachidonic acid. We conclude that the sex-specific alterations in GDM maternal⁻fetal metabolism may begin to explain the sex-specific metabolic outcomes seen in offspring exposed to GDM in utero.