A host-microbiota interactome reveals extensive transkingdom connectivity.

The myriad microorganisms that live in close association with humans have diverse effects on physiology, yet the molecular bases for these impacts remain mostly unknown1-3. Classical pathogens often invade host tissues and modulate immune responses through interactions with human extracellular and secreted proteins (the 'exoproteome'). Commensal microorganisms may also facilitate niche colonization and shape host biology by engaging host exoproteins; however, direct exoproteome-microbiota interactions remain largely unexplored. Here we developed and validated a novel technology, BASEHIT, that enables proteome-scale assessment of human exoproteome-microbiome interactions. Using BASEHIT, we interrogated more than 1.7 million potential interactions between 519 human-associated bacterial strains from diverse phylogenies and tissues of origin and 3,324 human exoproteins. The resulting interactome revealed an extensive network of transkingdom connectivity consisting of thousands of previously undescribed host-microorganism interactions involving 383 strains and 651 host proteins. Specific binding patterns within this network implied underlying biological logic; for example, conspecific strains exhibited shared exoprotein-binding patterns, and individual tissue isolates uniquely bound tissue-specific exoproteins. Furthermore, we observed dozens of unique and often strain-specific interactions with potential roles in niche colonization, tissue remodelling and immunomodulation, and found that strains with differing host interaction profiles had divergent interactions with host cells in vitro and effects on the host immune system in vivo. Overall, these studies expose a previously unexplored landscape of molecular-level host-microbiota interactions that may underlie causal effects of indigenous microorganisms on human health and disease.

Within-host evolution of a gut pathobiont facilitates liver translocation.

Gut commensal bacteria with the ability to translocate across the intestinal barrier can drive the development of diverse immune-mediated diseases1-4. However, the key factors that dictate bacterial translocation remain unclear. Recent studies have revealed that gut microbiota strains can adapt and evolve throughout the lifetime of the host5-9, raising the possibility that changes in individual commensal bacteria themselves over time may affect their propensity to elicit inflammatory disease. Here we show that within-host evolution of the model gut pathobiont Enterococcus gallinarum facilitates bacterial translocation and initiation of inflammation. Using a combination of in vivo experimental evolution and comparative genomics, we found that E. gallinarum diverges into independent lineages adapted to colonize either luminal or mucosal niches in the gut. Compared with ancestral and luminal E. gallinarum, mucosally adapted strains evade detection and clearance by the immune system, exhibit increased translocation to and survival within the mesenteric lymph nodes and liver, and induce increased intestinal and hepatic inflammation. Mechanistically, these changes in bacterial behaviour are associated with non-synonymous mutations or insertion-deletions in defined regulatory genes in E. gallinarum, altered microbial gene expression programs and remodelled cell wall structures. Lactobacillus reuteri also exhibited broadly similar patterns of divergent evolution and enhanced immune evasion in a monocolonization-based model of within-host evolution. Overall, these studies define within-host evolution as a critical regulator of commensal pathogenicity that provides a unique source of stochasticity in the development and progression of microbiota-driven disease.

Prominent role of gut dysbiosis in the pathogenesis of cystic fibrosis-related liver disease in mice.

BACKGROUND & AIMS: Cystic fibrosis-related liver disease (CFLD) is a chronic cholangiopathy that increases morbidity and mortality in patients with CF. Current treatments are unsatisfactory, and incomplete understanding of CFLD pathogenesis hampers therapeutic development. We have previously shown that mouse CF cholangiocytes respond to lipopolysaccharide with excessive inflammation. Thus, we investigated the role of the gut-liver axis in the pathogenesis of CFLD. METHODS: Wild-type (WT), whole-body Cftr knockout (CFTR-KO) and gut-corrected (CFTR-KO-GC) mice were studied. Liver changes were assessed by immunohistochemistry and single-cell transcriptomics (single-cell RNA sequencing), inflammatory mediators were analysed by proteome array, faecal microbiota by 16S ribosomal RNA sequencing and gut permeability by FITC-dextran assay. RESULTS: The livers of CFTR-KO mice showed ductular proliferation and periportal inflammation, whereas livers of CFTR-KO-GC mice had no evident pathology. Single-cell RNA sequencing analysis of periportal cells showed increased presence of neutrophils, macrophages and T cells, and activation of pro-inflammatory and pathogen-mediated immune pathways in CFTR-KO livers, consistent with a response to gut-derived stimuli. CFTR-KO mice exhibited gut dysbiosis with enrichment of Enterobacteriaceae and Enterococcus spp., which was associated with increased intestinal permeability and mucosal inflammation, whereas gut dysbiosis and inflammation were absent in CFTR-KO-GC mice. Treatment with nonabsorbable antibiotics ameliorated intestinal permeability and liver inflammation in CFTR-KO mice. Faecal microbiota transfer from CFTR-KO to germ-free WT mice did not result in dysbiosis nor liver pathology, indicating that defective intestinal CFTR is required to maintain dysbiosis. CONCLUSION: Defective CFTR in the gut sustains a pathogenic microbiota, creates an inflammatory milieu, and alters intestinal permeability. These changes are necessary for the development of cholangiopathy. Restoring CFTR in the intestine or modulating the microbiota could be a promising strategy to prevent or attenuate liver disease. IMPACT AND IMPLICATIONS: Severe cystic fibrosis-related liver disease (CFLD) affects 10% of patients with cystic fibrosis (CF) and contributes to increased morbidity and mortality. Treatment options remain limited due to a lack of understanding of disease pathophysiology. The cystic fibrosis transmembrane conductance regulator (CFTR) mediates Cl- and HCO3- secretion in the biliary epithelium and its defective function is thought to cause cholestasis and excessive inflammatory responses in CF. However, our study in Cftr-knockout mice demonstrates that microbial dysbiosis, combined with increased intestinal permeability caused by defective CFTR in the intestinal mucosa, acts as a necessary co-factor for the development of CFLD-like liver pathology in mice. These findings uncover a major role for the gut microbiota in CFLD pathogenesis and call for further investigation and clinical validation to develop targeted therapeutic strategies acting on the gut-liver axis in CF.

Association of Socioeconomic Status, Sex, Racial, and Ethnic Identity With Sustained and Cultivated Careers in Surgery.

OBJECTIVE: Examine the association between sex, race, ethnicity, and family income, and the intersectionality between these identities, and sustained or cultivated paths in surgery in medical school. METHODS: This retrospective cohort study examines US medical students who matriculated in academic years 2014-2015 and 2015-2016. Data were provided by the Association of American Medical Colleges, including self-reported sex, race, ethnicity, family income, interest in surgery at matriculation, and successful placement into a surgical residency at graduation. This study examined 2 outcomes: (1) sustained path in surgery between matriculation and graduation for students who entered medical school with an interest in surgery and (2) cultivated path in surgery for students who entered medical school not initially interested in surgery and who applied to and were successfully placed into a surgical residency at graduation. RESULTS: Among the 5074 students who reported interest in surgery at matriculation, 2108 (41.5%) had sustained path in surgery. Compared to male students, female students were significantly less likely to have sustained path in surgery [adjusted relative risk (aRR): 0.92 (0.85-0.98)], while Asian (aRR: 0.82, 95% CI: 0.74-0.91), Hispanic (aRR: 0.70, 95% CI: 0.59-0.83), and low-income (aRR: 0.85, 95% CI: 0.78-0.92) students were less likely to have a sustained path in surgery compared to their peers. Among the 17,586 students who reported an initial interest in a nonsurgical specialty, 1869 (10.6%) were placed into a surgical residency at graduation. Female students, regardless of race/ethnic identity and income, were significantly less likely to have cultivated paths in surgery compared to male students, with underrepresented in medicine female students reporting the lowest rates. CONCLUSIONS AND RELEVANCE: This study demonstrates the significant disparity in sustained and cultivated paths in surgery during undergraduate medical education. Innovative transformation of the surgical learning environment to promote surgical identity development and belonging for females, underrepresented in medicine, and low-income students is essential to diversify the surgical workforce.

Internal Medicine Program Directors' Perceptions About Accommodating Residents with Disabilities: A Qualitative Study.

BACKGROUND: While 26% of US adults are disabled, only 3.1 to 9.3% of practicing physicians report having a disability. Ableism within medical training and practice diminishes physician diversity and wellbeing and contributes to healthcare disparities. OBJECTIVE: Explore physician barriers to disability equity and inclusion by examining internal medicine (IM) program directors' (PD) perspectives about recruiting and accommodating residents with disabilities (RWD). DESIGN: Qualitative study involving semi-structured virtual interviews (conducted December 2022-September 2023; analyzed through December 2023). PARTICIPANTS: PDs were recruited via email. Purposive sampling captured program diversity in size, location, and affiliations. Convenience sampling ensured PD diversity by gender, race/ethnicity, and age. APPROACH: Coders analyzed thematic and discursive content of interview transcripts to characterize PD perspectives about RWDs and accommodations. KEY RESULTS: Of the 15 programs represented, 4 had ≤ 49 and 8 had ≥ 100 total residents. Three were community-based; the rest had academic affiliations. On average, PDs had 17 (SD 8.2) years in practice. Most (11/15) identified as White race; 8/15 as female; and none as disabled. PDs characterized disability as a source of grit and empathy but also as an intrinsic deficit. They worried RWDs could have unpredictable absences and clinical incompetencies. Perceived accommodation challenges included inexperience, workload distribution, information asymmetry about accommodation needs or options, barriers to disclosure (e.g., discrimination concerns), and insufficient accommodation advertising. Perceived facilitators included advanced planning; clear, publicized processes; and access to expertise (e.g., occupational health, ombudsmen). CONCLUSIONS: PDs held contradictory views of RWDs. PD insights revealed opportunities to alleviate PD-RWD information asymmetry in recruitment/accommodation processes, which could help align needs and improve representation and inclusion.

National Institutes of Health Diversity Supplement Awards by Medical School.

BACKGROUND: Increasing medical school faculty diversity is an urgent priority. National Institutes of Health (NIH) diversity supplements, which provide funding and career development opportunities to individuals underrepresented in research, are an important mechanism to increase faculty diversity. OBJECTIVE: Analyze diversity supplement utilization by medical schools. DESIGN: Retrospective cohort study. PARTICIPANTS: All R01 grant-associated diversity supplements awarded to medical schools from 2005 to 2020. Diversity supplements were identified using the publicly available NIH RePORTER database. MAIN MEASURES: Main measures were the number of R01-associated diversity supplements awarded to medical schools each year by medical school NIH funding status and the number of R01-associated diversity supplements awarded to individual medical schools in the NIH top 40 by funding status. We also examined the percentage of R01 grants with an associated diversity supplement by NIH funding status and individual medical school in the NIH top 40. KEY RESULTS: From 2005 to 2020, US medical school faculty received 1389 R01-associated diversity supplements. The number of diversity supplements awarded grew from 2012 to 2020, from ten to 187 for top 40 schools, and from seven to 83 for non-top 40 schools. The annual growth rate for diversity supplement awards at NIH top 40 schools (44.2%) was not significantly different than the annual growth rate among non-top 40 schools (36.2%; p = 0.68). From 2005 to 2020, the highest number of diversity supplements that an individual medical school received was 56 and the lowest number was four (mean = 24.6, SD = 11.7). The highest percentage of R01 grants with an associated diversity supplement received by a school was 4.5% and the lowest percentage was 0.79% (mean = 2.3%, SD = 0.98). CONCLUSION: Medical schools may be missing an opportunity to address the continuing shortage of individuals historically underrepresented in biomedical science and should consider additional mechanisms to enhance diversity supplement utilization.

Revolutionizing Health Professions Admissions to Achieve an Inclusive Workforce.

This article describes the "The Admissions Revolution: Bold Strategies for Diversifying the Healthcare Workforce" conference, which preceded the 2022 Beyond Flexner Alliance Conference and called for health professions institutions to boldly reimagine the admission process to diversify the health care workforce. Proposed strategies encompassed 4 key themes: admission metrics, aligning admission practices with institutional mission, community partnerships to fulfill social mission, and student support and retention. Transformation of the health professions admission process requires broad institutional and individual effort. Careful consideration and implementation of these practices will help institutions achieve greater workforce diversity and catalyze progress toward health equity.

Medical student intentions to practice internal medicine in underserved areas associated with debt, identity and extracurricular participation.

BACKGROUND: Currently, Internal Medicine (IM) physicians do not reflect the ethno-racial diversity of the US population. Moreover, there is a shortage of IM physicians in Medically Underserved Areas (MUAs) in the US. The purpose of this study was to determine factors that influence medical students' intent to practice IM in MUAs. We hypothesized students with intentions to pursue a career in IM and work in MUAs were more likely than their peers to identify as underrepresented in medicine (URiM), report greater student debt loads, and report medical school experiences in cultural competencies. METHODS: We analyzed de-identified data of 67,050 graduating allopathic medical students who completed the Association of American Medical Colleges' (AAMC) Medical School annual Graduation Questionnaire (GQ) between 2012-2017 by multivariate logistic regression models, examining intent to practice IM in MUAs based on respondent characteristics. RESULTS: Of 8,363 students indicating an intent to pursue IM, 1,969 (23.54%) students also expressed an intent to practice in MUAs. Students awarded scholarships, (aOR: 1.23, [1.03-1.46]), with debt greater than $300,000 (aOR: 1.54, [1.21-1.95], and self-identified non-Hispanic Black/African American (aOR: 3.79 [2.95-4.87]) or Hispanic (aOR: 2.53, [2.05-3.11]) students were more likely than non-Hispanic White students to indicate intent to practice in MUAs. This pattern also existed for students who participated in a community-based research project (aOR: 1.55, [1.19-2.01]), had experiences related to health disparities (aOR: 2.13, [1.44-3.15]), or had experiences related to global health (aOR: 1.75, [1.34-2.28]). CONCLUSIONS: We identified experiences and characteristics that associate with intention to practice IM in MUAs, which can aid future curricular redesign by medical schools to expand and deepen comprehension of health disparities, access to community-based research, and global health experiences. Loan forgiveness programs and other initiatives to increase recruitment and retention of future physicians should also be developed.

Outcomes of the Main Residency Match for Applicants With Disability.

This cohort study assesses match rates of US applicants with and without disability into specialty residence programs.

Leave of Absence and Medical Student Placement Into Graduate Medical Education by Race and Ethnicity.

This study examines the association between taking a leave of absence from medical school and placement into graduate medical education (GME) by race and ethnicity.

Transition From Mentored to Independent NIH Funding by Gender and Department.

This study uses National Institutes of Health RePORTER data for mentored K awards and R01-equivalent grants to all departments in US schools of medicine to characterize K-award distribution and K-to-R transition by gender and department between 1997 and 2021.

Temporal Trends in Childhood Household Income Among Applicants and Matriculants to Medical School and the Likelihood of Acceptance by Income, 2014-2019.

This study examines trends in childhood household income among applicants and matriculants to medical school and the likelihood of acceptance by income.

Development of a Tool to Measure Student Perceptions of Equity and Inclusion in Medical Schools.

Importance: Creating an inclusive and equitable learning environment is a national priority. Nevertheless, data reflecting medical students' perception of the climate of equity and inclusion are limited. Objective: To develop and validate an instrument to measure students' perceptions of the climate of equity and inclusion in medical school using data collected annually by the Association of American Medical Colleges (AAMC). Design, Setting, and Participants: The Promoting Diversity, Group Inclusion, and Equity tool was developed in 3 stages. A Delphi panel of 9 members identified survey items from preexisting AAMC data sources. Exploratory and confirmatory factor analysis was performed on student responses to AAMC surveys to construct the tool, which underwent rigorous psychometric validation. Participants were undergraduate medical students at Liaison Committee on Medical Education-accredited medical schools in the US who completed the 2015 to 2019 AAMC Year 2 Questionnaire (Y2Q), the administrations of 2016 to 2020 AAMC Graduation Questionnaire (GQ), or both. Data were analyzed from August 2020 to November 2023. Exposures: Student race and ethnicity, sex, sexual orientation, and socioeconomic status. Main Outcomes and Measures: Development and psychometric validation of the tool, including construct validity, internal consistency, and criterion validity. Results: Delphi panel members identified 146 survey items from the Y2Q and GQ reflecting students' perception of the climate of equity and inclusion, and responses to these survey items were obtained from 54 906 students for the Y2Q cohort (median [IQR] age, 24 [23-26] years; 29 208 [52.75%] were female, 11 389 [20.57%] were Asian, 4089 [7.39%] were multiracial, and 33 373 [60.28%] were White) and 61 998 for the GQ cohort (median [IQR] age, 27 [26-28] years; 30 793 [49.67%] were female, 13 049 [21.05%] were Asian, 4136 [6.67%] were multiracial, and 38 215 [61.64%] were White). Exploratory and confirmatory factor analyses of student responses identified 8 factors for the Y2Q model (faculty role modeling; student empowerment; student fellowship; cultural humility; faculty support for students; fostering a collaborative and safe environment; discrimination: race, ethnicity, and gender; and discrimination: sexual orientation) and 5 factors for the GQ model (faculty role modeling; student empowerment; faculty support for students; discrimination: race, ethnicity, and gender; and discrimination: sexual orientation). Confirmatory factor analysis indicated acceptable model fit (root mean square error of approximation of 0.05 [Y2Q] and 0.06 [GQ] and comparative fit indices of 0.95 [Y2Q] and 0.94 [GQ]). Cronbach α for individual factors demonstrated internal consistency ranging from 0.69 to 0.92 (Y2Q) and 0.76 to 0.95 (GQ). Conclusions and Relevance: This study found that the new tool is a reliable and psychometrically valid measure of medical students' perceptions of equity and inclusion in the learning environment.

Sequential Chromogenic IHC: Spatial Analysis of Lymph Nodes Identifies Contact Interactions between Plasmacytoid Dendritic Cells and Plasmablasts.

Recent clinical observations have emphasized the critical role that the spatial organization of immune cells in lymphoid structures plays in the success of cancer immunotherapy and patient survival. However, implementing sequential chromogenic IHC (scIHC) to analyze multiple biomarkers on a single tissue section has been limited because of a lack of a standardized, rigorous guide to the development of customized biomarker panels and a need for user-friendly analysis pipelines that can extract meaningful data. In this context, we provide a comprehensive guide for the development of novel biomarker panels for scIHC, using practical examples and illustrations to highlight the most common complications that can arise during the setup of a new biomarker panel, and provide detailed instructions on how to prevent and detect cross-reactivity between secondary reagents and carryover between detection antibodies. We also developed a novel analysis pipeline based on non-rigid tissue deformation correction, Cellpose-inspired automated cell segmentation, and computational network masking of low-quality data. We applied this biomarker panel and pipeline to study regional lymph nodes from patients with head and neck cancer, identifying novel contact interactions between plasmablasts and plasmacytoid dendritic cells in vivo. Given that Toll-like receptors, which are highly expressed in plasmacytoid dendritic cells, play a key role in vaccine efficacy, the significance of this cell-cell interaction decisively warrants further studies. In summary, this work provides a streamlined approach to the development of customized biomarker panels for scIHC that will ultimately improve our understanding of immune responses in cancer. Significance: We present a comprehensive guide for developing customized biomarker panels to investigate cell-cell interactions in the context of immune responses in cancer. This approach revealed novel contact interactions between plasmablasts and plasmacytoid dendritic cells in lymph nodes from patients with head and neck cancer.