The cryo-EM structure of the SF3b spliceosome complex bound to a splicing modulator reveals a pre-mRNA substrate competitive mechanism of action.

Somatic mutations in spliceosome proteins lead to dysregulated RNA splicing and are observed in a variety of cancers. These genetic aberrations may offer a potential intervention point for targeted therapeutics. SF3B1, part of the U2 small nuclear RNP (snRNP), is targeted by splicing modulators, including E7107, the first to enter clinical trials, and, more recently, H3B-8800. Modulating splicing represents a first-in-class opportunity in drug discovery, and elucidating the structural basis for the mode of action opens up new possibilities for structure-based drug design. Here, we present the cryogenic electron microscopy (cryo-EM) structure of the SF3b subcomplex (SF3B1, SF3B3, PHF5A, and SF3B5) bound to E7107 at 3.95 Å. This structure shows that E7107 binds in the branch point adenosine-binding pocket, forming close contacts with key residues that confer resistance upon mutation: SF3B1R1074H and PHF5AY36C The structure suggests a model in which splicing modulators interfere with branch point adenosine recognition and supports a substrate competitive mechanism of action (MOA). Using several related chemical probes, we validate the pose of the compound and support their substrate competitive MOA by comparing their activity against both strong and weak pre-mRNA substrates. Finally, we present functional data and structure-activity relationship (SAR) on the PHF5AR38C mutation that sensitizes cells to some chemical probes but not others. Developing small molecule splicing modulators represents a promising therapeutic approach for a variety of diseases, and this work provides a significant step in enabling structure-based drug design for these elaborate natural products. Importantly, this work also demonstrates that the utilization of cryo-EM in drug discovery is coming of age.

Historical Redlining and Present-Day Nonsuicide Firearm Fatalities.

BACKGROUND: Redlining began in the 1930s with the Home Owners' Loan Corporation (HOLC); this discriminatory practice limited mortgage availability and reinforced concentrated poverty that still exists today. It is important to understand the potential health implications of this federally sanctioned segregation. OBJECTIVE: To examine the relationship between historical redlining policies and present-day nonsuicide firearm fatalities. DESIGN: Maps from the HOLC were overlaid with incidence of nonsuicide firearm fatalities from 2014 to 2022. A multilevel negative binomial regression model tested the association between modern-day firearm fatalities and HOLC historical grading (A ["best"] to D ["hazardous"]), controlling for year, HOLC area-level demographics, and state-level factors as fixed effects and a random intercept for city. Incidence rates (IRs) per 100 000 persons, incidence rate ratios (IRRs), and adjusted IRRs (aIRRs) for each HOLC grade were estimated using A-rated areas as the reference. SETTING: 202 cities with areas graded by the HOLC in the 1930s. PARTICIPANTS: Population of the 8597 areas assessed by the HOLC. MEASUREMENTS: Nonsuicide firearm fatalities. RESULTS: From 2014 to 2022, a total of 41 428 nonsuicide firearm fatalities occurred in HOLC-graded areas. The firearm fatality rate increased as the HOLC grade progressed from A to D. In A-graded areas, the IR was 3.78 (95% CI, 3.52 to 4.05) per 100 000 persons per year. In B-graded areas, the IR, IRR, and aIRR relative to A areas were 7.43 (CI, 7.24 to 7.62) per 100 000 persons per year, 2.12 (CI, 1.94 to 2.32), and 1.42 (CI, 1.30 to 1.54), respectively. In C-graded areas, these values were 11.24 (CI, 11.08 to 11.40) per 100 000 persons per year, 3.78 (CI, 3.47 to 4.12), and 1.90 (CI, 1.75 to 2.07), respectively. In D-graded areas, these values were 16.26 (CI, 16.01 to 16.52) per 100 000 persons per year, 5.51 (CI, 5.05 to 6.02), and 2.07 (CI, 1.90 to 2.25), respectively. LIMITATION: The Gun Violence Archive relies on media coverage and police reports. CONCLUSION: Discriminatory redlining policies from 80 years ago are associated with nonsuicide firearm fatalities today. PRIMARY FUNDING SOURCE: Fred Lovejoy Housestaff Research and Education Fund.

Biochemical and structural basis for the pharmacological inhibition of nuclear hormone receptor PPARγ by inverse agonists.

Recent studies have reported that the peroxisome proliferator-activated receptor gamma (PPARγ) pathway is activated in approximately 40% of patients with muscle-invasive bladder cancer. This led us to investigate pharmacological repression of PPARγ as a possible intervention strategy. Here, we characterize PPARγ antagonists and inverse agonists and find that the former behave as silent ligands, whereas inverse agonists (T0070907 and SR10221) repress downstream PPARγ target genes leading to growth inhibition in bladder cancer cell lines. To understand the mechanism, we determined the ternary crystal structure of PPARγ bound to T0070907 and the corepressor (co-R) peptide NCOR1. The structure shows that the AF-2 helix 12 (H12) rearranges to bind inside the ligand-binding domain, where it forms stabilizing interactions with the compound. This dramatic movement in H12 unveils a large interface for co-R binding. In contrast, the crystal structure of PPARγ bound to a SR10221 analog shows more subtle structural differences, where the compound binds and pushes H12 away from the ligand-binding domain to allow co-R binding. Interestingly, we found that both classes of compound promote recruitment of co-R proteins in biochemical assays but with distinct conformational changes in H12. We validate our structural models using both site-directed mutagenesis and chemical probes. Our findings offer new mechanistic insights into pharmacological modulation of PPARγ signaling.

The Planetary Benefit of Suspending USMLE Step 2 CS: Estimating Carbon Emissions Associated with US Medical Students' Travel to Testing Centers.

Background: The discontinuation of the Step 2 Clinical Skills Exam (CS) by the United States Medical Licensing Examination (USMLE) eliminated the need for personal travel to testing centers. The carbon emissions associated with CS have not been previously quantified. Objective: To estimate the annual carbon emissions generated by travel to CS Testing Centers (CSTCs) and to explore differences across geographic regions. Methods: We conducted a cross-sectional, observational study by geocoding medical schools and CSTCs to calculate the distance between them. We obtained data from the 2017 matriculant databases of the Association of American Medical Colleges (AAMC) and the American Association of Colleges of Osteopathic Medicine (AACOM). The independent variable was the location as defined by USMLE geographic regions. The dependent variables were distance traveled to CSTCs and estimated carbon emissions in metric tons CO2 (mtCO2) calculated using three models. In model 1 all students used single occupancy vehicles; in model 2, all carpooled; and in model 3, half traveled by train and half by single occupancy vehicle. Results: Our analysis included 197 medical schools. The mean out-of-town travel distance was 280.67 miles (IQR: 97.49-383.42). The mtCO2 associated with travel was 2,807.46 for model 1; 3,135.55 for model 2; and 635.34 for model 3. The Western region traveled the farthest, while the Northeast traveled significantly less than other regions. Conclusion: The annual estimated carbon emissions from travel to CSTCs was approximately 3,000 mtCO2. Northeastern students traveled the shortest distances; the average US medical student expended 0.13 mtCO2. Medical leaders must consider the environmental impact of medical curricula and pursue accordant reforms.

An intestinal organoid-based platform that recreates susceptibility to T-cell-mediated tissue injury.

A goal in precision medicine is to use patient-derived material to predict disease course and intervention outcomes. Here, we use mechanistic observations in a preclinical animal model to design an ex vivo platform that recreates genetic susceptibility to T-cell-mediated damage. Intestinal graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation. We found that intestinal GVHD in mice deficient in Atg16L1, an autophagy gene that is polymorphic in humans, is reversed by inhibiting necroptosis. We further show that cocultured allogeneic T cells kill Atg16L1-mutant intestinal organoids from mice, which was associated with an aberrant epithelial interferon signature. Using this information, we demonstrate that pharmacologically inhibiting necroptosis or interferon signaling protects human organoids derived from individuals harboring a common ATG16L1 variant from allogeneic T-cell attack. Our study provides a roadmap for applying findings in animal models to individualized therapy that targets affected tissues.

Interleukin-22 protects intestinal stem cells from immune-mediated tissue damage and regulates sensitivity to graft versus host disease.

Little is known about the maintenance of intestinal stem cells (ISCs) and progenitors during immune-mediated tissue damage or about the susceptibility of transplant recipients to tissue damage mediated by the donor immune system during graft versus host disease (GVHD). We demonstrate here that deficiency of recipient-derived IL-22 increased acute GVHD tissue damage and mortality, that ISCs were eliminated during GVHD, and that ISCs as well as their downstream progenitors expressed the IL-22 receptor. Intestinal IL-22 was produced after bone marrow transplant by IL-23-responsive innate lymphoid cells (ILCs) from the transplant recipients, and intestinal IL-22 increased in response to pretransplant conditioning. However, ILC frequency and IL-22 amounts were decreased by GVHD. Recipient IL-22 deficiency led to increased crypt apoptosis, depletion of ISCs, and loss of epithelial integrity. Our findings reveal IL-22 as a critical regulator of tissue sensitivity to GVHD and a protective factor for ISCs during inflammatory intestinal damage.

A systematic review of cross-cultural adaptation of the National Institutes of Health Chronic Prostatitis Symptom Index.

BACKGROUND: The National Institutes of Health Chronic Prostatitis Symptom Index (NIH-CPSI) was developed to accurately assess the pain, urinary symptoms, and quality of life related to chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). This study aimed to evaluate the cross-cultural adaptations of the NIH-CPSI. METHOD: PubMed, Embase, CINAHL, and SciELO databases were searched from their established year to September 2020. Cross-cultural adaptations and the quality control of measurement properties of adaptations were conducted by two reviewers independently according to the Guidelines for the Process of Cross-Cultural Adaptation of Self-Report Measures and the Quality Criteria for Psychometric Properties of Health Status Questionnaire. RESULTS: Area total of 21 papers with 16 adaptations, and six studies of the original version of the NIH-CPSI were enrolled in the systematic review. Back translation was the weakest process for the quality assessment of the cross-cultural adaptations of the NIH-CPSI. Internal consistency was analyzed for most of the adaptations, but none of them met the standard. Only 11 adaptations reported test reliability, then only the Arabic-Egyptian, Chinese-Mainland, Danish, Italian, Persian, and Turkish adaptations met the criterion. Most adaptations reported the interpretability, but only the Danish adaptation reported the agreement. The other measurement properties, including responsiveness, and floor as well as ceiling effects were not reported in any of the adaptations. CONCLUSIONS: The overall quality of the NIH-CPSI cross-cultural adaptations was not organized as expected. Only the Portuguese-Brazilian, Italian, and Spanish adaptations reached over half the process for the cross-cultural adaptation. Only the Turkish adaptations finished half of the measurement properties of cross-cultural adaptations.

Nrf2 regulates CD4+ T cell-induced acute graft-versus-host disease in mice.

Nuclear factor erythroid-derived 2-like 2 (Nrf2) is a ubiquitously expressed transcription factor that is well known for its role in regulating the cellular redox pathway. Although there is mounting evidence suggesting a critical role for Nrf2 in hematopoietic stem cells and innate leukocytes, little is known about its involvement in T-cell biology. In this study, we identified a novel role for Nrf2 in regulating alloreactive T-cell function during allogeneic hematopoietic cell transplantation (allo-HCT). We observed increased expression and nuclear translocation of Nrf2 upon T-cell activation in vitro, especially in CD4+ donor T cells after allo-HCT. Allo-HCT recipients of Nrf2 -/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality, morbidity, and pathology. This reduction in GVHD was associated with the persistence of Helios+ donor regulatory T cells in the allograft, as well as defective upregulation of the gut-homing receptor LPAM-1 on alloreactive CD8+ T cells. Additionally, Nrf2 -/- donor CD8+ T cells demonstrated intact cytotoxicity against allogeneic target cells. Tumor-bearing allo-HCT recipients of Nrf2 -/- donor T cells had overall improved survival as a result of preserved graft-versus-tumor activity and reduced GVHD activity. Our findings characterized a previously unrecognized role for Nrf2 in T-cell function, as well as revealed a novel therapeutic target to improve the outcomes of allo-HCT.

Is Online Partner-Seeking Associated with Increased Risk of Condomless Sex and Sexually Transmitted Infections Among Individuals Who Engage in Heterosexual Sex? A Systematic Narrative Review.

Use of Internet websites and mobile applications to meet potential romantic and sexual partners is becoming increasingly popular. While the Internet might foster better communication and sexual negotiation between partners, it can also be a deceptive environment that instigates and accelerates sexual risk-taking. Given the complexities of the Internet, it is critical to examine the association between risky sexual behaviors (RSBs) and online partner-seeking. Five databases (i.e., Google Scholar, PubMed, PsycINFO, Web of Science, and Ovid Medline) were searched for articles published before September 10, 2017, that examined the association between online partner solicitation (either for romantic and/or sexual reasons) and RSBs. Studies were eligible for inclusion if they were empirical papers published in English-language peer-reviewed journals looking at samples of online partner seekers who practice heterosexual sex (with a comparison group) and reporting either condom use or sexually transmitted infections (STIs) status, which were the two primary outcomes. A total of 25 studies met the criteria to be included in our review. Results from this literature search do not indicate a clear association between online partner-seeking and condom use or STI status. Potential moderators (i.e., age, gender, reasons for online partner solicitation, duration of Internet correspondence, and Internet modalities) that should be included in future research were identified. Sexual health prevention efforts should address methods to negotiate safe practices between sexual partners and to encourage healthy non-virtual relationships, particularly among vulnerable populations.

Evaluation of a Digital Diabetes Prevention Program Adapted for Low-Income Patients, 2016-2018.

INTRODUCTION: We examined the effects of a digitally delivered, type 2 diabetes mellitus prevention program (DPP) for a low-income population. METHODS: We conducted a nonrandomized clinical trial with matched controls. The intervention group was offered a digital DPP, a web-based and mobile-based program including 52 weeks of participation in an educational curriculum, health coaching, and peer support. RESULTS: A total of 227 participants enrolled. At baseline, 34.6 was the mean body mass index, and 5.8 was the mean HbA1c. For the intervention group, mean weight loss was 4.4% at the 12-month follow-up. CONCLUSION: The modified DPP successfully engaged participants and resulted in weight loss. Low-income patients with prediabetes benefitted from a digitally delivered diabetes intervention. This prevention method should be accessible to a low-income population.

Mycobacterial Metabolic Syndrome: LprG and Rv1410 Regulate Triacylglyceride Levels, Growth Rate and Virulence in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) mutants lacking rv1411c, which encodes the lipoprotein LprG, and rv1410c, which encodes a putative efflux pump, are dramatically attenuated for growth in mice. Here we show that loss of LprG-Rv1410 in Mtb leads to intracellular triacylglyceride (TAG) accumulation, and overexpression of the locus increases the levels of TAG in the culture medium, demonstrating a role of this locus in TAG transport. LprG binds TAG within a large hydrophobic cleft and is sufficient to transfer TAG from donor to acceptor membranes. Further, LprG-Rv1410 is critical for broadly regulating bacterial growth and metabolism in vitro during carbon restriction and in vivo during infection of mice. The growth defect in mice is due to disrupted bacterial metabolism and occurs independently of key immune regulators. The in vivo essentiality of this locus suggests that this export system and other regulators of metabolism should be considered as targets for novel therapeutics.

An integrated cell purification and genomics strategy reveals multiple regulators of pancreas development.

The regulatory logic underlying global transcriptional programs controlling development of visceral organs like the pancreas remains undiscovered. Here, we profiled gene expression in 12 purified populations of fetal and adult pancreatic epithelial cells representing crucial progenitor cell subsets, and their endocrine or exocrine progeny. Using probabilistic models to decode the general programs organizing gene expression, we identified co-expressed gene sets in cell subsets that revealed patterns and processes governing progenitor cell development, lineage specification, and endocrine cell maturation. Purification of Neurog3 mutant cells and module network analysis linked established regulators such as Neurog3 to unrecognized gene targets and roles in pancreas development. Iterative module network analysis nominated and prioritized transcriptional regulators, including diabetes risk genes. Functional validation of a subset of candidate regulators with corresponding mutant mice revealed that the transcription factors Etv1, Prdm16, Runx1t1 and Bcl11a are essential for pancreas development. Our integrated approach provides a unique framework for identifying regulatory genes and functional gene sets underlying pancreas development and associated diseases such as diabetes mellitus.

Prevalence and Co-Occurrence of Addictive Behaviors Among Russian and Spanish Youth: A Replication Study.

Recently, an addiction matrix measure was assessed among U.S. former alternative high school youth. This presentation seeks to examine the generalizability of findings using this measure among Russian and Spanish high school adolescents. Latent class analysis was used to explore addiction subgroups among adolescents in Russia (average age = 16.27; n = 715) and Spain (average age = 14.9; n = 811). Last 30-day prevalence of one or more of 11 addictions reviewed in the previous work was the primary focus (i.e., cigarettes, alcohol, hard drugs, eating, gambling, Internet, love, sex, exercise, work, and shopping) among Russian youth, and last-30 prevalence of one or more of 8 addictions among Spanish youth (the three drug use items had not been included in the questionnaire for these youths). Results confirmed a two-class model (addicted class and non-addicted class) among both Russian and Spanish adolescents. The mean number of addictions reported was 1.39 (SD = 1.78) addictions among Russian youth and 1.56 (SD = 1.68) addictions among Spanish youth. The prevalence of the sample that constituted the "addicted group" in Russia and Spain was 32.2% and 28.6%, respectively. The most prevalent addictions (i.e., love, Internet, exercise) were similar. These results are similar to the findings previously reported for U.S. sample. Latent class structures for addictive behaviors are similar across international adolescent populations. Our results highlight the need to address multiple addictions in health education programming.

The central nervous system is a target of acute graft versus host disease in mice.

Despite significant advances in prevention and management, graft versus host disease (GVHD) is still a leading complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although skin, gut, liver, thymus, and lung are GVHD targets, neurological complications (NC) have also been reported following allo-HSCT. We demonstrate that the central nervous system (CNS) can be a direct target of alloreactive T cells following allo-HSCT in mice. We found significant infiltration of the CNS with donor T lymphocytes and cell death of neurons and neuroglia in allo-HSCT recipients with GVHD. We also found that allo-HSCT recipients with GVHD had deficits in spatial learning/memory and demonstrated increased anxious behavior. These findings highlight CNS sensitivity to damage caused by alloreactive donor T cells and represent the first characterization of target cell subsets and NC during GVHD. Therefore, these clinically relevant studies offer a novel and rational explanation for the well-described neurological symptoms observed after allo-HSCT.