An atlas of protein-protein interactions across mouse tissues.

Cellular processes arise from the dynamic organization of proteins in networks of physical interactions. Mapping the interactome has therefore been a central objective of high-throughput biology. However, the dynamics of protein interactions across physiological contexts remain poorly understood. Here, we develop a quantitative proteomic approach combining protein correlation profiling with stable isotope labeling of mammals (PCP-SILAM) to map the interactomes of seven mouse tissues. The resulting maps provide a proteome-scale survey of interactome rewiring across mammalian tissues, revealing more than 125,000 unique interactions at a quality comparable to the highest-quality human screens. We identify systematic suppression of cross-talk between the evolutionarily ancient housekeeping interactome and younger, tissue-specific modules. Rewired proteins are tightly regulated by multiple cellular mechanisms and are implicated in disease. Our study opens up new avenues to uncover regulatory mechanisms that shape in vivo interactome responses to physiological and pathophysiological stimuli in mammalian systems.

The RNA editor ADAR2 promotes immune cell trafficking by enhancing endothelial responses to interleukin-6 during sterile inflammation.

Immune cell trafficking constitutes a fundamental component of immunological response to tissue injury, but the contribution of intrinsic RNA nucleotide modifications to this response remains elusive. We report that RNA editor ADAR2 exerts a tissue- and stress-specific regulation of endothelial responses to interleukin-6 (IL-6), which tightly controls leukocyte trafficking in IL-6-inflamed and ischemic tissues. Genetic ablation of ADAR2 from vascular endothelial cells diminished myeloid cell rolling and adhesion on vascular walls and reduced immune cell infiltration within ischemic tissues. ADAR2 was required in the endothelium for the expression of the IL-6 receptor subunit, IL-6 signal transducer (IL6ST; gp130), and subsequently, for IL-6 trans-signaling responses. ADAR2-induced adenosine-to-inosine RNA editing suppressed the Drosha-dependent primary microRNA processing, thereby overwriting the default endothelial transcriptional program to safeguard gp130 expression. This work demonstrates a role for ADAR2 epitranscriptional activity as a checkpoint in IL-6 trans-signaling and immune cell trafficking to sites of tissue injury.

The Mettl3 epitranscriptomic writer amplifies p53 stress responses.

The p53 transcription factor drives anti-proliferative gene expression programs in response to diverse stressors, including DNA damage and oncogenic signaling. Here, we seek to uncover new mechanisms through which p53 regulates gene expression using tandem affinity purification/mass spectrometry to identify p53-interacting proteins. This approach identified METTL3, an m6A RNA-methyltransferase complex (MTC) constituent, as a p53 interactor. We find that METTL3 promotes p53 protein stabilization and target gene expression in response to DNA damage and oncogenic signals, by both catalytic activity-dependent and independent mechanisms. METTL3 also enhances p53 tumor suppressor activity in in vivo mouse cancer models and human cancer cells. Notably, METTL3 only promotes tumor suppression in the context of intact p53. Analysis of human cancer genome data further supports the notion that the MTC reinforces p53 function in human cancer. Together, these studies reveal a fundamental role for METTL3 in amplifying p53 signaling in response to cellular stress.

ATM-dependent Phosphorylation of Nemo SQ Motifs Is Dispensable for Nemo-mediated Gene Expression Changes in Response to DNA Double-Strand Breaks.

In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes.

The Cyclin D3 Protein Enforces Monogenic TCRß Expression by Mediating TCRß Protein-Signaled Feedback Inhibition of Vß Recombination.

In jawed vertebrates, adaptive immunity depends on the process of V(D)J recombination creating vast numbers of T and B lymphocytes that each expresses unique Ag receptors of uniform specificity. The asynchronous initiation of V-to-(D)J rearrangement between alleles and the resulting protein from one allele signaling feedback inhibition of V recombination on the other allele ensures homogeneous receptor specificity of individual cells. Upon productive Vß-to-DßJß rearrangements in noncycling double-negative thymocytes, TCRß protein signals induction of the cyclin D3 protein to accelerate cell cycle entry, thereby driving proliferative expansion of developing αß T cells. Through undetermined mechanisms, the inactivation of cyclin D3 in mice causes an increased frequency of αß T cells that express TCRß proteins from both alleles, producing lymphocytes of heterogeneous specificities. To determine how cyclin D3 enforces monogenic TCRß expression, we used our mouse lines with enhanced rearrangement of specific Vß segments due to replacement of their poor-quality recombination signal sequence (RSS) DNA elements with a better RSS. We show that cyclin D3 inactivation in these mice elevates the frequencies of αß T cells that display proteins from RSS-augmented Vß segments on both alleles. By assaying mature αß T cells, we find that cyclin D3 deficiency increases the levels of Vß rearrangements that occur within developing thymocytes. Our data demonstrate that a component of the cell cycle machinery mediates TCRß protein-signaled feedback inhibition in thymocytes to achieve monogenic TCRß expression and resulting uniform specificity of individual αß T cells.

Plastid terminal oxidase is required for chloroplast biogenesis in barley.

Chloroplast biogenesis is critical for crop biomass and economic yield. However, chloroplast development is a very complicated process coordinated by cross-communication between the nucleus and plastids, and the underlying mechanisms have not been fully revealed. To explore the regulatory machinery for chloroplast biogenesis, we conducted map-based cloning of the Grandpa 1 (Gpa1) gene regulating chloroplast development in barley. The spontaneous mutation gpa1.a caused a variegation phenotype of the leaf, dwarfed growth, reduced grain yield, and increased tiller number. Genetic mapping anchored the Gpa1 gene onto 2H within a gene cluster functionally related to photosynthesis or chloroplast differentiation. One gene (HORVU.MOREX.r3.2HG0213170) in the delimited region encodes a putative plastid terminal oxidase (PTOX) in thylakoid membranes, which is homologous to IMMUTANS (IM) of Arabidopsis. The IM gene is required for chloroplast biogenesis and maintenance of functional thylakoids in Arabidopsis. Using CRISPR technology and gene transformation, we functionally validated that the PTOX-encoding gene, HORVU.MOREX.r3.2HG0213170, is the causal gene of Gpa1. Gene expression and chemical analysis revealed that the carotenoid biosynthesis pathway is suppressed by the gpa1 mutation, rendering mutants vulnerable to photobleaching. Our results showed that the overtillering associated with the gpa1 mutation was caused by the lower accumulation of carotenoid-derived strigolactones (SLs) in the mutant. The cloning of Gpa1 not only improves our understanding of the molecular mechanisms underlying chloroplast biosynthesis but also indicates that the PTOX activity is conserved between monocots and dicots for the establishment of the photosynthesis factory.

Five-year follow-up of KEYNOTE-087: pembrolizumab monotherapy for relapsed/refractory classical Hodgkin lymphoma.

Previous analyses of the phase 2 KEYNOTE-087 (NCT02453594) trial of pembrolizumab monotherapy demonstrated effective antitumor activity with acceptable safety in patients with relapsed or refractory (R/R) classical Hodgkin lymphoma (cHL). However, long-term response durability and outcome of patients who receive a second course after treatment discontinuation after complete response (CR) remain of clinical interest. We present KEYNOTE-087 data after >5 years of median follow-up. Patients with R/R cHL and progressive disease (PD) after autologous stem cell transplantation (ASCT) and brentuximab vedotin (BV; cohort 1), salvage chemotherapy and BV without ASCT (cohort 2), or ASCT without subsequent BV (cohort 3), received pembrolizumab for ≤2 years. Patients in CR who discontinued treatment and subsequently experienced PD were eligible for second-course pembrolizumab. Primary end points were the objective response rate (ORR) using blinded central review and safety. The median follow-up was 63.7 months. ORR was 71.4% (95% confidence interval [CI], 64.8-77.4; CR, 27.6%; partial response, 43.8%). Median duration of response (DOR) was 16.6 months; median progression-free survival was 13.7 months. A quarter of responders, including half of complete responders, maintained a response for ≥4 years. Median overall survival was not achieved. Among 20 patients receiving second-course pembrolizumab, ORR for 19 evaluable patients was 73.7% (95% CI, 48.8-90.8); median DOR was 15.2 months. Any-grade treatment-related adverse events occurred in 72.9% of patients and grade 3 or 4 adverse events occurred in 12.9% of patients; no treatment-related deaths occurred. Single-agent pembrolizumab can induce durable responses, particularly in patients achieving CR. Second-course pembrolizumab frequently reinduced sustained responses after relapse from initial CR.

Genetic Mapping, Identification, and Characterization of a Candidate Susceptibility Gene for Powdery Mildew in Cannabis sativa.

Powdery mildew (PM) in Cannabis sativa is most frequently caused by the biotrophic fungus Golovinomyces ambrosiae. Based on previously characterized variation in susceptibility to PM, biparental populations were developed by crossing the most resistant cultivar evaluated, 'FL 58', with a susceptible cultivar, 'TJ's CBD'. F1 progeny were evaluated and displayed a range of susceptibility, and two were self-pollinated to generate two F2 populations. In 2021, the F2 populations (n = 706) were inoculated with PM and surveyed for disease severity. In both F2 populations, 25% of the progeny were resistant, while the remaining 75% showed a range of susceptibility. The F2 populations, as well as selected F1 progeny and the parents, were genotyped with a single-nucleotide polymorphism array, and a consensus genetic map was produced. A major effect quantitative trait locus on C. sativa chromosome 1 (Chr01) and other smaller-effect quantitative trait loci (QTL) on four other chromosomes were identified. The most associated marker on Chr01 was located near CsMLO1, a candidate susceptibility gene. Genomic DNA and cDNA sequencing of CsMLO1 revealed a 6.8-kb insertion in FL 58, relative to TJ's CBD, of which 846 bp are typically spliced into the mRNA transcript encoding a premature stop codon. Molecular marker assays were developed using CsMLO1 sequences to distinguish PM-resistant and PM-susceptible genotypes. These data support the hypothesis that a mutated MLO susceptibility gene confers resistance to PM in C. sativa and provides new genetic resources to develop resistant cultivars. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Familial Associations of Prevalence and Cause-Specific Mortality for Thoracic Aortic Disease and Bicuspid Aortic Valve in a Large-Population Database.

BACKGROUND: Thoracic aortic disease and bicuspid aortic valve (BAV) likely have a heritable component, but large population-based studies are lacking. This study characterizes familial associations of thoracic aortic disease and BAV, as well as cardiovascular and aortic-specific mortality, among relatives of these individuals in a large-population database. METHODS: In this observational case-control study of the Utah Population Database, we identified probands with a diagnosis of BAV, thoracic aortic aneurysm, or thoracic aortic dissection. Age- and sex-matched controls (10:1 ratio) were identified for each proband. First-degree relatives, second-degree relatives, and first cousins of probands and controls were identified through linked genealogical information. Cox proportional hazard models were used to quantify the familial associations for each diagnosis. We used a competing-risk model to determine the risk of cardiovascular-specific and aortic-specific mortality for relatives of probands. RESULTS: The study population included 3 812 588 unique individuals. Familial hazard risk of a concordant diagnosis was elevated in the following populations compared with controls: first-degree relatives of patients with BAV (hazard ratio [HR], 6.88 [95% CI, 5.62-8.43]); first-degree relatives of patients with thoracic aortic aneurysm (HR, 5.09 [95% CI, 3.80-6.82]); and first-degree relatives of patients with thoracic aortic dissection (HR, 4.15 [95% CI, 3.25-5.31]). In addition, the risk of aortic dissection was higher in first-degree relatives of patients with BAV (HR, 3.63 [95% CI, 2.68-4.91]) and in first-degree relatives of patients with thoracic aneurysm (HR, 3.89 [95% CI, 2.93-5.18]) compared with controls. Dissection risk was highest in first-degree relatives of patients who carried a diagnosis of both BAV and aneurysm (HR, 6.13 [95% CI, 2.82-13.33]). First-degree relatives of patients with BAV, thoracic aneurysm, or aortic dissection had a higher risk of aortic-specific mortality (HR, 2.83 [95% CI, 2.44-3.29]) compared with controls. CONCLUSIONS: Our results indicate that BAV and thoracic aortic disease carry a significant familial association for concordant disease and aortic dissection. The pattern of familiality is consistent with a genetic cause of disease. Furthermore, we observed higher risk of aortic-specific mortality in relatives of individuals with these diagnoses. This study provides supportive evidence for screening in relatives of patients with BAV, thoracic aneurysm, or dissection.

Distinct Transcriptomic and Proteomic Profile Specifies Patients Who Have Heart Failure With Potential of Myocardial Recovery on Mechanical Unloading and Circulatory Support.

BACKGROUND: Extensive evidence from single-center studies indicates that a subset of patients with chronic advanced heart failure (HF) undergoing left ventricular assist device (LVAD) support show significantly improved heart function and reverse structural remodeling (ie, termed "responders"). Furthermore, we recently published a multicenter prospective study, RESTAGE-HF (Remission from Stage D Heart Failure), demonstrating that LVAD support combined with standard HF medications induced remarkable cardiac structural and functional improvement, leading to high rates of LVAD weaning and excellent long-term outcomes. This intriguing phenomenon provides great translational and clinical promise, although the underlying molecular mechanisms driving this recovery are largely unknown. METHODS: To identify changes in signaling pathways operative in the normal and failing human heart and to molecularly characterize patients who respond favorably to LVAD unloading, we performed global RNA sequencing and phosphopeptide profiling of left ventricular tissue from 93 patients with HF undergoing LVAD implantation (25 responders and 68 nonresponders) and 12 nonfailing donor hearts. Patients were prospectively monitored through echocardiography to characterize their myocardial structure and function and identify responders and nonresponders. RESULTS: These analyses identified 1341 transcripts and 288 phosphopeptides that are differentially regulated in cardiac tissue from nonfailing control samples and patients with HF. In addition, these unbiased molecular profiles identified a unique signature of 29 transcripts and 93 phosphopeptides in patients with HF that distinguished responders after LVAD unloading. Further analyses of these macromolecules highlighted differential regulation in 2 key pathways: cell cycle regulation and extracellular matrix/focal adhesions. CONCLUSIONS: This is the first study to characterize changes in the nonfailing and failing human heart by integrating multiple -omics platforms to identify molecular indices defining patients capable of myocardial recovery. These findings may guide patient selection for advanced HF therapies and identify new HF therapeutic targets.

Factors that determine patients considering medication for the disease of obesity: an IMI2 SOPHIA study.

OBJECTIVE: Obesity-related problems can now be managed with effective nutritional therapy, pharmacotherapy, and surgeries that achieve >10% weight loss. Assessing patient preferences, treatment choices, and factors affecting patients can improve treatment compliance and efficacy. Our aim was to identify factors affecting patient preference and subsequent choice of pharmacotherapy among those seeking treatment for obesity-related disorders. METHODS: A participatory action study using purposeful sampling recruited 33 patients with obesity complications. They were referred to specialist clinics in non-alcoholic fatty liver disease, diabetes mellitus, hypertension, and chronic kidney disease. Sixteen males and seventeen females aged 18-70 years, with BMI > 35 kg/m2 were recruited. Before the interview, participants watched a 60-minute video explaining nutritional therapy, pharmacotherapy, and surgery in equipoise. Data were collected in semi-structured interviews; Reflective thematic analysis was used. This sub study focuses only on patients who expressed specific attitudes (positive or negative) towards pharmacotherapy. RESULTS: Ten (30%) patients expressed a view on pharmacotherapy. Eight (24%) patients chose pharmacotherapy alone, whereas two (6%) patients chose pharmacotherapy combined with nutritional therapy. In this sub study focusing on pharmacotherapy, five themes were identified related to choosing whether or not to take medication: (1) attitudes towards pharmacotherapy, (2) attitudes toward size of obesity and its complications, (3) weighing the benefits and risks of treatment, (4) knowledge and reassurance of health professionals, and (5) costs associated with drug therapy. CONCLUSION: The primary concerns regarding pharmacotherapy for intentional weight loss were efficacy, side effects, lifelong dosing, pharmacokinetics, and cost. Providing access to information about all the pharmacotherapies and the benefits is likely to result in greater penetrance of treatment.

Immunochemotherapy plus lenalidomide for high-risk mantle cell lymphoma with measurable residual disease evaluation.

Chemoimmunotherapy followed by consolidative high-dose therapy with autologous stem cell rescue was a standard upfront treatment for fit patients with mantle cell lymphoma (MCL) in first remission; however, treatment paradigms are evolving in the era of novel therapies. Lenalidomide is an immunomodulatory agent with known efficacy in treating MCL. We conducted a single-center, investigator-initiated, phase II study of immunochemotherapy incorporating lenalidomide, without autologous stem cell transplant consolidation, enriching for patients with high-risk MCL (clinicaltrials gov. Identifier: NCT02633137). Patients received four cycles of lenalidomide-R-CHOP, two cycles of R-HiDAC, and six cycles of R-lenalidomide. The primary endpoint was rate of 3-year progression-free survival. We measured measurable residual disease (MRD) using a next-generation sequencing-based assay after each phase of treatment and at 6 months following end-oftreatment. We enrolled 49 patients of which 47 were response evaluable. By intent-to-treat, rates of overall and complete response were equivalent at 88% (43/49), one patient with stable disease, and two patients had disease progression during study; 3-year progression-free survival was 63% (primary endpoint not met) and differed by TP53 status (78% wild-type vs. 38% ALT; P=0.043). MRD status was prognostic and predicted long-term outcomes following R-HiDAC and at 6 months following end-of-treatment. In a high-dose therapy-sparing, intensive approach, we achieved favorable outcomes in TP53- wild-type MCL, including high-risk cases. We confirmed that sequential MRD assessment is a powerful prognostic tool in patients with MCL.

Core Descriptor Sets for Rectal Prolapse Outcomes Research Using a Modified Delphi Consensus.

BACKGROUND: There is wide variation in prolapse care. OBJECTIVE: To determine core descriptor sets for rectal prolapse to enhance outcomes research. DESIGN: Descriptors for patients undergoing rectal prolapse surgery were generated through a systematic review and expert opinion. Stakeholders were recruited internationally via listserv and social media. Experts were encouraged to consider the minimum descriptors that could be considered during clinical care, and descriptors were grouped into core descriptor sets. Consensus was defined as greater than 70% agreement. SETTING: A 3-round Delphi process using a 9-point Likert scale based on expert results was distributed via survey. The final interactive meeting used a polling platform. PARTICIPANTS: The Pelvic Floor Disorders Consortium interdisciplinary group convened to advance the clinical care of pelvic floor disorders. MAIN OUTCOME MEASURES: To achieve expert consensus for core descriptor sets for rectal prolapse using a modified Delphi method. RESULTS: A total of 206 providers participated, with survey response rates of 82% and 88%, respectively. Responders were from North America (56%), Europe (29%), and Latin America, Asia, Australia, New Zealand, and Africa (15%). Ninety-one percent of participants identified as colorectal surgeons and 80% reported >5 years of experience (35% reported >15 years). Fifty-seven attendees participated in the final meeting and voted on core descriptor sets. Ninety-three percent of participants agreed that descriptors such as age, BMI, frailty, nutrition, and the American Society of Anesthesiology score correlated to physiologic status. One hundred percent of participants agreed to include baseline bowel function. One hundred percent of participants reported willingness to complete a synoptic operative report. Follow-up intervals 1, 3, and 5 years after surgery (76%) with a collection of recurrence and functional outcomes at those time periods reached an agreement. LIMITATIONS: Individual bias, self-identification of experts, and paucity of knowledge related to rectal prolapse. CONCLUSIONS: This represents the first steps toward international consensus to unify language and data collection processes for rectal prolapse. See Video Abstract . CONJUNTOS DE DESCRIPTORES BSICOS PARA LA INVESTIGACIN DE RESULTADOS DE PROLAPSO RECTAL MEDIANTE UN CONSENSO DELPHI MODIFICADO: ANTECEDENTES:Existe una amplia variación en la atención del prolapso.OBJETIVO:Determinar conjuntos de descriptores básicos para el prolapso rectal para mejorar los resultados de la investigación.DISEÑO:Los descriptores para pacientes sometidos a cirugía de prolapso rectal se generaron a través de una revisión sistemática y la opinión de expertos. Las partes interesadas fueron reclutadas internacionalmente a través de listas de servicio y redes sociales. Se animó a los expertos a considerar los descriptores mínimos que podrían considerarse durante la atención clínica, y los descriptores se agruparon en conjuntos de descriptores básicos. El consenso se definió como > 70% de acuerdo.AJUSTE:Se distribuyó mediante encuesta un proceso Delphi de tres rondas que utiliza una escala Likert de 9 puntos basada en resultados de expertos. La reunión interactiva final utilizó una plataforma de votación.PARTICIPANTES:El grupo interdisciplinario del Consorcio de Trastornos del Suelo Pélvico se reunió para avanzar en la atención clínica de los trastornos del suelo pélvico.MEDIDAS PRINCIPALES DE RESULTADOS:Lograr el consenso de expertos para los conjuntos de descriptores básicos para el prolapso rectal utilizando un método Delphi modificado.RESULTADOS:Participaron 206 proveedores con tasas de respuesta a la encuesta del 82% y 88% respectivamente. Los encuestados procedían de América del Norte (56%), Europa (29%) y América Latina, Asia, Australia, Nueva Zelanda y África (15%). El noventa y uno por ciento se identificó como cirujanos colorrectales y el 80% reportó más de 5 años de experiencia (35% > 15 años). Cincuenta y siete asistentes participaron en la reunión final y votaron sobre conjuntos de descriptores básicos. El noventa y tres por ciento estuvo de acuerdo en que descriptores como edad, índice de masa corporal, fragilidad, nutrición y puntuación de la Sociedad Estadounidense de Anestesiología se correlacionaban con el estado fisiológico. El cien por ciento estuvo de acuerdo en incluir la función intestinal inicial. El 100% refirió disposición para realizar un informe operativo sinóptico. Los intervalos de seguimiento 1,3,5 años después de la cirugía (76%) con un conjunto de recurrencias y los resultados funcionales en esos períodos de tiempo coincidieron.LIMITACIONES:Sesgo individual, autoidentificación de los expertos y escasez de conocimientos relacionados con el prolapso rectal.CONCLUSIONES:Esto representa los primeros pasos hacia un consenso internacional para unificar el lenguaje y los procesos de recolección de datos para el prolapso rectal. (Traducción-Yesenia Rojas-Khalil ).

Development of a Consensus Derived Synoptic Operative Report for Rectal Prolapse: A Report From the Pelvic Floor Disorders Consortium.

BACKGROUND: Narrative operative reports may frequently omit or obscure data from an operation. OBJECTIVE: We aim to develop a synoptic operative report for rectal prolapse that includes core descriptors as developed by an international consensus of expert pelvic floor surgeons. DESIGN: Descriptors for patients undergoing rectal prolapse surgery were generated through review. Members of the Pelvic Floor Disorders Consortium were recruited to participate in a 3 round Delphi process using a 9-point Likert scale. Descriptors that achieved 70% agreement were kept from the first round, descriptors scoring 40-70% agreement were recirculated in subsequent rounds. A final list of operative descriptors was determined at a consensus meeting, with a final consensus meeting more than 70% agreement. SETTINGS: This was a survey administered to members of the Pelvic Floor Disorders Consortium. MAIN OUTCOME MEASURES: Descriptors meeting greater than 70% agreement were selected. RESULTS: One-hundred seventy six surgeons representing colorectal surgeons, urogynecologists, and urologists distributed throughout North America (56%), Latin America (4%), Western Europe (29%), Asia (4%), and Africa (1%) participated in the first round of Delphi voting. After two additional rounds and a final consensus meeting, 16 of 30 descriptors met 70% consensus. Descriptors that met consensus were: surgery type, posterior dissection, ventral dissection, mesh used, type of mesh used, mesh location, sutures used, suture type, pouch of Douglas and peritoneum reclosed, length of rectum imbricated, length of bowel resected, levatoroplasty, simultaneous vaginal procedure, simultaneous gynecologic procedure, simultaneous enterocele repair, and simultaneous urinary incontinence procedure. LIMITATIONS: Survey represents views of members of the Delphi panel, and may not represent viewpoints of all surgeons. CONCLUSIONS/DISCUSSION: This Delphi survey establishes international consensus descriptors for intraoperative variables that have been used to produce a synoptic operative report. This will help establish defined operative reporting to improve clinical communication, quality measures, and clinical research. See Video Abstract.

Canine Model of Ischemia-Induced Ventricular Tachycardia.

INTRODUCTION: Despite advances in antiarrhythmia therapies, ventricular tachycardia (VT) is a leading cause of sudden cardiac death. Investigation into the characteristics and new treatments for this arrhythmia is required to improve outcomes and a reproducible model of VT would be useful in these endeavors. We therefore created a canine model of ischemia-induced VT. MATERIALS AND METHODS: A pacing lead was implanted in the right ventricle in canines (n = 13) and the left anterior descending artery was occluded in two locations for 2 h and subsequently released to create an ischemia-reperfusion injury. In the 10 dogs that survived the first 48 h following the initial study, a terminal study was conducted 4-7 d later and VT was induced using premature stimulation or burst pacing through the right ventricle lead. The arrhythmia was terminated using either antitachycardia pacing or a defibrillatory shock. Multiple inductions into sustained VT were attempted. RESULTS: Sustained VT was induced in eight of 10 dogs with an average cycle length of 335 ± 70 bpm. Multiple episodes of VT were induced. Episodes of VT exhibited different electrocardiogram morphologies and cycle lengths in individual animals. CONCLUSIONS: This canine model provides a consistent technique for inducing multiple episodes of sustained VT. It may be useful for investigating VT mechanisms and testing novel therapeutics and treatments for patients with VT.

Bridge-to-transplant temporary mechanical circulatory support and risk of allosensitization.

INTRODUCTION: Since the 2018 change in the US adult heart allocation policy, more patients are bridged-to-transplant on temporary mechanical circulatory support (tMCS). Previous studies indicate that durable left ventricular assist devices (LVAD) may lead to allosensitization. The goal of this study was to assess whether tMCS implantation is associated with changes in sensitization. METHODS: We included patients evaluated for heart transplants between 2015 and 2022 who had alloantibody measured before and after MCS implantation. Allosensitization was defined as development of new alloantibodies after tMCS implant. RESULTS: A total of 41 patients received tMCS before transplant. Nine (22.0%) patients developed alloantibodies following tMCS implantation: 3 (12.0%) in the intra-aortic balloon pump group (n = 25), 2 (28.6%) in the microaxial percutaneous LVAD group (n = 7), and 4 (44.4%) in the veno-arterial extra-corporeal membrane oxygenation group (n = 9)-p = .039. Sensitized patients were younger (44.7 ± 11.6 years vs. 54.3 ± 12.5 years, p = .044), were more likely to be sensitized at baseline - 3 of 9 (33.3%) compared to 2 out of 32 (6.3%) (p = .028) and received more transfusions with red blood cells (6 (66.6%) vs. 8 (25%), p = .02) and platelets (6 (66.6%) vs. 5 (15.6%), p = .002). There was no significant difference in tMCS median duration of support (4 [3,15] days vs. 8.5 [5,14.5] days, p = .57). Importantly, out of the 11 patients who received a durable LVAD after tMCS, 5 (45.5%) became sensitized, compared to 4 out of 30 patients (13.3%) who only had tMCS-p = .028. CONCLUSIONS: Our findings suggest that patients bridged-to-transplant with tMCS, without significant blood product transfusions and a subsequent durable LVAD implant, have a low risk of allosensitization. Further studies are needed to confirm our findings and determine whether risk of sensitization varies by type of tMCS and duration of support.

Monogenic TCRß Assembly and Expression Are Paramount for Uniform Antigen Receptor Specificity of Individual αß T Lymphocytes.

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αß T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRß and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRß protein. However, whether this mechanism operates in the physiological context where each dual-TCRß cell expresses one set of a vast number of different TCRß proteins remains an open question, largely because there is a low, but significant, portion of cells carrying two in-frame TCRß genes. To resolve this issue, we inactivated one allelic copy of the TCRα locus in a new mouse strain that assembles two in-frame TCRß genes in an elevated fraction of cells. This genetic manipulation has no effect on the frequency of cells that display multiple types of αß TCR, yet increases the representation of cells displaying TCRß proteins that generate more highly expressed TCRs. Our data demonstrate that some TCRß proteins exhibit differential functional pairing with TCRα proteins, but these restrictions have negligible contribution for ensuring uniform specificity of cells that express two types of TCRß protein. Therefore, we conclude that mechanisms governing monogenic assembly and expression of TCRß genes in individual cells are paramount for uniform specificity of αß T lymphocytes.

Nemo-Dependent, ATM-Mediated Signals from RAG DNA Breaks at Igk Feedback Inhibit V κ Recombination to Enforce Igκ Allelic Exclusion.

Monoallelic AgR gene expression underlies specific adaptive immune responses. AgR allelic exclusion is achieved by sequential initiation of V(D)J recombination between alleles and resultant protein from one allele signaling to prevent recombination of the other. The ATM kinase, a regulator of the DNA double-strand break (DSB) response, helps enforce allelic exclusion through undetermined mechanisms. ATM promotes repair of RAG1/RAG2 (RAG) endonuclease-induced DSBs and transduces signals from RAG DSBs during Igk gene rearrangement on one allele to transiently inhibit RAG1 protein expression, Igk accessibility, and RAG cleavage of the other allele. Yet, the relative contributions of ATM functions in DSB repair versus signaling to enforce AgR allelic exclusion remain undetermined. In this study, we demonstrate that inactivation in mouse pre-B cells of the NF-κB essential modulator (Nemo) protein, an effector of ATM signaling, diminishes RAG DSB-triggered repression of Rag1/Rag2 transcription and Igk accessibility but does not result in aberrant repair of RAG DSBs like ATM inactivation. We show that Nemo deficiency increases simultaneous biallelic Igk cleavage in pre-B cells and raises the frequency of B cells expressing Igκ proteins from both alleles. In contrast, the incidence of biallelic Igκ expression is not elevated by inactivation of the SpiC transcriptional repressor, which is induced by RAG DSBs in an ATM-dependent manner and suppresses Igk accessibility. Thus, we conclude that Nemo-dependent, ATM-mediated DNA damage signals enforce Igκ allelic exclusion by orchestrating transient repression of RAG expression and feedback inhibition of additional Igk rearrangements in response to RAG cleavage on one Igk allele.

Poor-Quality Vß Recombination Signal Sequences and the DNA Damage Response ATM Kinase Collaborate to Establish TCRß Gene Repertoire and Allelic Exclusion.

The monoallelic expression (allelic exclusion) of diverse lymphocyte Ag receptor genes enables specific immune responses. Allelic exclusion is achieved by asynchronous initiation of V(D)J recombination between alleles and protein encoded by successful rearrangement on the first allele signaling permanent inhibition of V rearrangement on the other allele. The ATM kinase that guides DNA repair and transiently suppresses V(D)J recombination also helps impose allelic exclusion through undetermined mechanisms. At the TCRß locus, one Vß gene segment (V31) rearranges only by inversion, whereas all other Vß segments rearrange by deletion except for rare cases in which they rearrange through inversion following V31 rearrangement. The poor-quality recombination signal sequences (RSSs) of V31 and V2 help establish TCRß gene repertoire and allelic exclusion by stochastically limiting initiation of Vß rearrangements before TCRß protein-signaled permanent silencing of Vß recombination. We show in this study in mice that ATM functions with these RSSs and the weak V1 RSS to shape TCRß gene repertoire by restricting their Vß segments from initiating recombination and hindering aberrant nonfunctional Vß recombination products, especially during inversional V31 rearrangements. We find that ATM collaborates with the V1 and V2 RSSs to help enforce allelic exclusion by facilitating competition between alleles for initiation and functional completion of rearrangements of these Vß segments. Our data demonstrate that the fundamental genetic DNA elements that underlie inefficient Vß recombination cooperate with ATM-mediated rapid DNA damage responses to help establish diversity and allelic exclusion of TCRß genes.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.