Functional mapping of epigenetic regulators uncovers coordinated tumor suppression by the HBO1 and MLL1 complexes.

Epigenetic dysregulation is widespread in cancer. However, the specific epigenetic regulators and the processes they control to drive cancer phenotypes are poorly understood. Here, we employed a novel, scalable and high-throughput in vivo method to perform iterative functional screens of over 250 epigenetic regulatory genes within autochthonous oncogenic KRAS-driven lung tumors. We identified multiple novel epigenetic tumor suppressor and tumor dependency genes. We show that a specific HBO1 complex and the MLL1 complex are among the most impactful tumor suppressive epigenetic regulators in lung. The histone modifications generated by the HBO1 complex are frequently absent or reduced in human lung adenocarcinomas. The HBO1 and MLL1 complexes regulate chromatin accessibility of shared genomic regions, lineage fidelity and the expression of canonical tumor suppressor genes. The HBO1 and MLL1 complexes are epistatic during lung tumorigenesis, and their functional correlation is conserved in human cancer cell lines. Together, these results demonstrate the value of quantitative methods to generate a phenotypic roadmap of epigenetic regulatory genes in tumorigenesis in vivo .

CD4 + T cells recruit, then engage macrophages in cognate interactions to clear Mycobacterium tuberculosis from the lungs.

IFN-γ-producing CD4 + T cells are required for protection against lethal Mycobacterium tuberculosis ( Mtb ) infections. However, the ability of CD4 + T cells to suppress Mtb growth cannot be fully explained by IFN-γ or other known T cell products. In this study, we show that CD4 + T cell-derived IFN-γ promoted the recruitment of monocyte-derived macrophages (MDMs) to the lungs of Mtb -infected mice. Although the recruited MDMs became quickly and preferentially infected with Mtb , CD4 + T cells rapidly disinfected the MDMs. Clearance of Mtb from MDMs was not explained by IFN-γ, but rather by MHCII-mediated cognate interactions with CD4 + T cells. These interactions promoted MDM expression of glycolysis genes essential for Mtb control. Thus, by recruiting MDMs, CD4 + T cells initiate a cycle of bacterial phagocytosis, Mtb antigen presentation and disinfection in an attempt to clear the bacteria from the lungs.

"They're not my daughter, and yet they're also not my son": Parents negotiating their adult child's nonbinary gender identity.

Scholars are increasingly recognizing that substantial gender heterogeneity exists among transgender populations; that is, gender identities that defy the ubiquitous binary categories of male and female. However, the developing research base on the families of transgender adults focuses almost exclusively on the family members of transgender persons with binary gender identities, a noteworthy shortcoming considering the prevalence of nonbinary gender identities among transgender populations and the pervasive assumption that only two genders exist. To address this gap, the current study sought to uncover how the parents of transgender adults with nonbinary gender identities come to understand, make sense of, and negotiate nonbinary gender identities in their families. Fourteen parents-12 mothers and 2 fathers-completed in-depth, semi-structured interviews, and the collected data were analyzed using reflexive thematic analysis. Analyses generated three broad themes that best-described these parents' experience with their child's gender, which was heavily shaped by the pervasiveness of cisnormativity: (a) varied attempts to understand nonbinary gender; (b) a nonbinary "double-edged sword"; and (c) familial resilience. Directions for future research, clinical practice, and policy change are discussed, including the therapeutic benefit of dialectical thinking and the need for legislation that legally affirms and protects nonbinary persons.

Tensions on the actin cytoskeleton and apical cell junctions in the C. elegans spermatheca are influenced by spermathecal anatomy, ovulation state and activation of myosin.

Cells generate mechanical forces mainly through myosin motor activity on the actin cytoskeleton. In C. elegans, actomyosin stress fibers drive contractility of the smooth muscle-like cells of the spermatheca, a distensible, tube-shaped tissue in the hermaphrodite reproductive system and the site of oocyte fertilization. Stretching of the spermathecal cells by oocyte entry triggers activation of the small GTPase Rho. In this study, we asked how forces are distributed in vivo using the spermatheca, and explored how this tissue responds to alterations in myosin activity. Using laser ablation, we show that the basal actomyosin fibers are under tension in the occupied spermatheca. Reducing actomyosin contractility by depletion of the phospholipase C-ε/PLC-1 or non-muscle myosin II/NMY-1, leads to distended spermathecae occupied by one or more embryos, but does not alter tension on the basal actomyosin fibers. This suggests that much of the tension on the basal actin fibers in the occupied spermatheca is due to the presence of the embryo. However, activating myosin through depletion of the Rho GAP SPV-1 increases tension on the actomyosin fibers, consistent with earlier studies showing Rho drives spermathecal contractility. On the inner surface of the spermathecal tube, tension on the apical junctions is decreased by depletion of PLC-1 and NMY-1. Surprisingly, when basal contractility is increased through SPV-1 depletion, the tension on apical junctions also decreases, with the most significant effect on the junctions aligned in perpendicular to the axis of the spermatheca. This suggests tension on the outer basal surface may compress the apical side, and suggests the three-dimensional shape of the spermatheca plays a role in force distribution and contractility during ovulation.

Regulation of immune responses to food by commensal microbes.

The increasing prevalence of immune-mediated non-communicable chronic diseases, such as food allergies, has prompted a deeper investigation into the role of the gut microbiome in modulating immune responses. Here, we explore the complex interactions between commensal microbes and the host immune system, highlighting the critical role of gut bacteria in maintaining immune homeostasis. We examine how modern lifestyle practices and environmental factors have disrupted co-evolved host-microbe interactions and discuss how changes in microbiome composition impact epithelial barrier function, responses to food allergens, and susceptibility to allergic diseases. Finally, we examine the potential of bioengineered microbiome-based therapies, and live biotherapeutic products, for reestablishing immune homeostasis to prevent or treat food allergies.

A New Mechanism for Formation of Glycine from Glyoxylic acid: the Aza-Cannizzaro Reaction.

Glyoxylic acid and glycine are widely considered to have been important prebiotic building blocks. Several mechanistic routes have been previously examined for conversion of glyoxylic acid to glycine. Here we provide evidence for a new mechanistic path. Glycine is spontaneously formed from glyoxylic acid in ammonium-rich aqueous solutions at neutral pH; oxamic acid is generated as well. Hydride transfer from the glyoxylate-derived hemiaminal to the corresponding iminium ion appears to underlie this transformation. This proposed mechanism parallels the well-known Cannizzaro reaction mechanism, which leads us to suggest the designation "aza-Cannizzaro reaction." This discovery offers a new perspective on prebiotic nitrogen incorporation because glycine can be a source of nitrogen for more complex molecules, including other α-amino acids.

Streamlining LC-MS Characterization of Pharmaceutical Polymers by Fourier-Transform-Based Deconvolution and Macromolecular Mass Defect Analysis.

Polymer conjugation has risen in importance over the past three decades as a means of increasing the in vivo half-life of biotherapeutics, with benefits including better stability, greater drug efficacy, and lower toxicity. However, the intrinsic variability of polymer synthesis results in products with broad distributions in chain length and branching structure, complicating quality control for successful functionalization and downstream conjugation. Frequently, a combination of several analytical techniques is required for comprehensive characterization. While liquid chromatography-mass spectrometry (LC-MS) is a powerful platform that can provide detailed molecular features of polymers, the mass spectra are inherently challenging to interpret due to high mass polydispersity and overlapping charge distributions. Here, by leveraging Fourier transform-based deconvolution and macromolecular mass defect analysis, we demonstrate a new way to streamline pharmaceutical polymer analysis, shedding light on polymer size, composition, branching, and end-group functionalization with the capability for reaction monitoring.

An international study presenting a federated learning AI platform for pediatric brain tumors.

While multiple factors impact disease, artificial intelligence (AI) studies in medicine often use small, non-diverse patient cohorts due to data sharing and privacy issues. Federated learning (FL) has emerged as a solution, enabling training across hospitals without direct data sharing. Here, we present FL-PedBrain, an FL platform for pediatric posterior fossa brain tumors, and evaluate its performance on a diverse, realistic, multi-center cohort. Pediatric brain tumors were targeted due to the scarcity of such datasets, even in tertiary care hospitals. Our platform orchestrates federated training for joint tumor classification and segmentation across 19 international sites. FL-PedBrain exhibits less than a 1.5% decrease in classification and a 3% reduction in segmentation performance compared to centralized data training. FL boosts segmentation performance by 20 to 30% on three external, out-of-network sites. Finally, we explore the sources of data heterogeneity and examine FL robustness in real-world scenarios with data imbalances.

Structure-guided engineering of type I-F CASTs for targeted gene insertion in human cells.

Conventional genome editing tools rely on DNA double-strand breaks (DSBs) and host recombination proteins to achieve large insertions, resulting in a heterogeneous mixture of undesirable editing outcomes. We recently leveraged a type I-F CRISPR-associated transposase (CAST) from the Pseudoalteromonas Tn 7016 transposon ( Pse CAST) for DSB-free, RNA-guided DNA integration in human cells, taking advantage of its programmability and large payload capacity. Pse CAST is the only characterized CAST system that has achieved human genomic DNA insertions, but multiple lines of evidence suggest that DNA binding may be a critical bottleneck that limits high-efficiency activity. Here we report structural determinants of target DNA recognition by the Pse CAST QCascade complex using single-particle cryogenic electron microscopy (cryoEM), which revealed novel subtype-specific interactions and RNA-DNA heteroduplex features. By combining our structural data with target DNA library screens and rationally engineered protein mutations, we uncovered CAST variants that exhibit increased integration efficiency and modified PAM stringency. Structure predictions of key interfaces in the transpososome holoenzyme also revealed opportunities for the design of hybrid CASTs, which we leveraged to build chimeric systems that combine high-activity DNA binding and DNA integration modules. Collectively, our work provides unique structural insights into type I-F CAST systems while showcasing multiple diverse strategies to investigate and engineer new RNA-guided transposase architectures for human genome editing applications.

Association of Cardiopulmonary Hemodynamics and Outcomes in Pulmonary Hypertension Following Kidney Transplant: A Multicenter Retrospective Cohort Study.

BACKGROUND: Pulmonary Hypertension (PH) frequently complicates the evaluation of kidney transplant (KT) candidates, and is associated with increased adverse outcomes (mortality, delayed graft function (DGF), and major adverse cardiovascular events (MACE)) following KT. RESEARCH QUESTION: What is the relationship between cardiopulmonary hemodynamics and post-KT outcomes? STUDY DESIGN AND METHODS: We conducted a multicenter retrospective cohort study of adults undergoing KT between 10/1/11 and 10/1/21, who underwent right heart catheterization (RHC) to assess cardiopulmonary hemodynamics within a year of transplantation. Frailty models and logistic regression models were used to evaluate the association between cardiopulmonary hemodynamics and outcomes (mortality, DGF, MACE) following KT. RESULTS: A total of 117 patients were included in the final analysis, predominantly male (72%), with a median age of 57 years. PH, defined as mean pulmonary artery pressure (mPAP) > 20mmHg, was present in the majority of the cohort (N=93, 79%). The cohort was followed for a median of 29.9 months post-KT, during which about one-fourth experienced mortality (23%) or DGF (25%) events, and approximately one-third (34%) experienced MACE. Though echocardiographic measures of pulmonary artery pressure failed to identify post-KT outcomes, a mPAP of ≥ 30mmHg on RHC was associated with post-KT MACE (Hazard Ratio 2.60, 95% Confidence Interval [1.10, 6.10]) and more prevalent in those experiencing post-KT mortality (63% vs 32%, p=0.001). Pre-capillary pulmonary hypertension was also associated with post-KT mortality (Hazard Ratio 3.71, 95% Confidence Interval [1.07, 12.90]). INTERPRETATION: Pre-capillary pulmonary hypertension and a mPAP of ≥ 30mmHg on RHC, but not echocardiographic evidence of PH, was associated with mortality and MACE following KT. These data suggest that RHC hemodynamics are superior to echocardiographic measures of PH in associating with outcomes following KT, and RHC-derived mPAP in particular may have value in predicting MACE and mortality post-KT.

De novo gene synthesis by an antiviral reverse transcriptase.

Defense-associated reverse transcriptase (DRT) systems perform DNA synthesis to protect bacteria against viral infection, but the identities and functions of their DNA products remain largely unknown. Here we show that DRT2 systems encode an unprecedented immune pathway that involves de novo gene synthesis via rolling circle reverse transcription of a non-coding RNA (ncRNA). Programmed template jumping on the ncRNA generates a concatemeric cDNA, which becomes double-stranded upon viral infection. Remarkably, this DNA product constitutes a protein-coding, nearly endless ORF (neo) gene whose expression leads to potent cell growth arrest, thereby restricting the viral infection. Our work highlights an elegant expansion of genome coding potential through RNA-templated gene creation, and challenges conventional paradigms of genetic information encoded along the one-dimensional axis of genomic DNA.

Strategies for optimal management of pediatric acute agitation in emergency settings.

Acute agitation in youth is a challenging presentation to the emergency department. In many cases, however, youth can be behaviorally de-escalated using a combination of environmental modification and verbal de-escalation. In cases where additional strategies such as pharmacologic de-escalation or physical restraint are needed, using the least restrictive means possible, including the youth in the decision-making process, and providing options are important. This paper reviews specific considerations on the approach to a youth with acute agitation and strategies and techniques to successfully de-escalate agitated youth who pose a danger to themselves and/or others.

Regulation of bacterial stringent response by an evolutionarily conserved ribosomal protein L11 methylation.

Lysine and arginine methylation is an important regulator of enzyme activity and transcription in eukaryotes. However, little is known about this covalent modification in bacteria. In this work, we investigated the role of methylation in bacteria. By reanalyzing a large phyloproteomics data set from 48 bacterial strains representing six phyla, we found that almost a quarter of the bacterial proteome is methylated. Many of these methylated proteins are conserved across diverse bacterial lineages, including those involved in central carbon metabolism and translation. Among the proteins with the most conserved methylation sites is ribosomal protein L11 (bL11). bL11 methylation has been a mystery for five decades, as the deletion of its methyltransferase PrmA causes no cell growth defects. Comparative proteomics analysis combined with inorganic polyphosphate and guanosine tetra/pentaphosphate assays of the ΔprmA mutant in Escherichia coli revealed that bL11 methylation is important for stringent response signaling. In the stationary phase, we found that the ΔprmA mutant has impaired guanosine tetra/pentaphosphate production. This leads to a reduction in inorganic polyphosphate levels, accumulation of RNA and ribosomal proteins, and an abnormal polysome profile. Overall, our investigation demonstrates that the evolutionarily conserved bL11 methylation is important for stringent response signaling and ribosomal activity regulation and turnover. IMPORTANCE: Protein methylation in bacteria was first identified over 60 years ago. Since then, its functional role has been identified for only a few proteins. To better understand the functional role of methylation in bacteria, we analyzed a large phyloproteomics data set encompassing 48 diverse bacteria. Our analysis revealed that ribosomal proteins are often methylated at conserved residues, suggesting that methylation of these sites may have a functional role in translation. Further analysis revealed that methylation of ribosomal protein L11 is important for stringent response signaling and ribosomal homeostasis.

The potential role of finerenone in patients with type 1 diabetes and chronic kidney disease.

Chronic kidney disease (CKD) represents a global health concern, associated with an increased risk of cardiovascular morbidity and mortality and decreased quality of life. Many patients with type 1 diabetes (T1D) will develop CKD over their lifetime. Uncontrolled glucose levels, which occur in patients with T1D as well as type 2 diabetes (T2D), are associated with substantial mortality and cardiovascular disease burden. T2D and T1D share common pathological features of CKD, which is thought to be driven by haemodynamic dysfunction, metabolic disturbances, and subsequently an influx of inflammatory and profibrotic mediators, both of which are major interrelated contributors to CKD progression. The mineralocorticoid receptor is also involved, and, under conditions of oxidative stress, salt loading and hyperglycaemia, it switches from homeostatic regulator to pathophysiological mediator by promoting oxidative stress, inflammation and fibrosis. Progressive glomerular and tubular injury leads to macroalbuminuria a progressive reduction in the glomerular filtration rate and eventually end-stage renal disease. Finerenone, a non-steroidal, selective mineralocorticoid receptor antagonist, is approved for treatment of patients with CKD associated with T2D; however, the benefit of finerenone in patients with T1D has yet to be determined. This narrative review will discuss treatment of CKD in T1D and the potential future role of finerenone in this setting.

Antigenic cartography using variant-specific hamster sera reveals substantial antigenic variation among Omicron subvariants.

Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2) has developed substantial antigenic variability. As the majority of the population now has pre-existing immunity due to infection or vaccination, the use of experimentally generated animal immune sera can be valuable for measuring antigenic differences between virus variants. Here, we immunized Syrian hamsters by two successive infections with one of nine SARS-CoV-2 variants. Their sera were titrated against 16 SARS-CoV-2 variants, and the resulting titers were visualized using antigenic cartography. The antigenic map shows a condensed cluster containing all pre-Omicron variants (D614G, Alpha, Delta, Beta, Mu, and an engineered B.1+E484K variant) and considerably more diversity among a selected panel of Omicron subvariants (BA.1, BA.2, BA.4/BA.5, the BA.5 descendants BF.7 and BQ.1.18, the BA.2.75 descendant BN.1.3.1, the BA.2-derived recombinants XBB.2 and EG.5.1, and the BA.2.86 descendant JN.1). Some Omicron subvariants were as antigenically distinct from each other as the wildtype is from the Omicron BA.1 variant. Compared to titers measured in human sera, titers in hamster sera are of higher magnitude, show less fold change, and result in a more compact antigenic map topology. The results highlight the potential of sera from hamsters for the continued antigenic characterization of SARS-CoV-2.

Auditory associative word learning in adults: The effects of musical experience and stimulus ordering.

Evidence for sequential associative word learning in the auditory domain has been identified in infants, while adults have shown difficulties. To better understand which factors may facilitate adult auditory associative word learning, we assessed the role of auditory expertise as a learner-related property and stimulus order as a stimulus-related manipulation in the association of auditory objects and novel labels. We tested in the first experiment auditorily-trained musicians versus athletes (high-level control group) and in the second experiment stimulus ordering, contrasting object-label versus label-object presentation. Learning was evaluated from Event-Related Potentials (ERPs) during training and subsequent testing phases using a cluster-based permutation approach, as well as accuracy-judgement responses during test. Results revealed for musicians a late positive component in the ERP during testing, but neither an N400 (400-800 ms) nor behavioral effects were found at test, while athletes did not show any effect of learning. Moreover, the object-label-ordering group only exhibited emerging association effects during training, while the label-object-ordering group showed a trend-level late ERP effect (800-1200 ms) during test as well as above chance accuracy-judgement scores. Thus, our results suggest the learner-related property of auditory expertise and stimulus-related manipulation of stimulus ordering modulate auditory associative word learning in adults.

Versatile roles of protein flavinylation in bacterial extracyotosolic electron transfer.

Bacteria perform diverse redox chemistries in the periplasm, cell wall, and extracellular space. Electron transfer for these extracytosolic activities is frequently mediated by proteins with covalently bound flavins, which are attached through post-translational flavinylation by the enzyme ApbE. Despite the significance of protein flavinylation to bacterial physiology, the basis and function of this modification remain unresolved. Here we apply genomic context analyses, computational structural biology, and biochemical studies to address the role of ApbE flavinylation throughout bacterial life. We identify ApbE flavinylation sites within structurally diverse protein domains and show that multi-flavinylated proteins, which may mediate longer distance electron transfer via multiple flavinylation sites, exhibit substantial structural heterogeneity. We identify two novel classes of flavinylation substrates that are related to characterized proteins with non-covalently bound flavins, providing evidence that protein flavinylation can evolve from a non-covalent flavoprotein precursor. We further find a group of structurally related flavinylation-associated cytochromes, including those with the domain of unknown function DUF4405, that presumably mediate electron transfer in the cytoplasmic membrane. DUF4405 homologs are widespread in bacteria and related to ferrosome iron storage organelle proteins that may facilitate iron redox cycling within ferrosomes. These studies reveal a complex basis for flavinylated electron transfer and highlight the discovery power of coupling comparative genomic analyses with high-quality structural models. IMPORTANCE: This study explores the mechanisms bacteria use to transfer electrons outside the cytosol, a fundamental process involved in energy metabolism and environmental interactions. Central to this process is a phenomenon known as flavinylation, where a flavin molecule-a compound related to vitamin B2-is covalently attached to proteins, to enable electron transfer. We employed advanced genomic analysis and computational modeling to explore how this modification occurs across different bacterial species. Our findings uncover new types of proteins that undergo this modification and highlight the diversity and complexity of bacterial electron transfer mechanisms. This research broadens our understanding of bacterial physiology and informs potential biotechnological applications that rely on microbial electron transfer, including bioenergy production and bioremediation.

Simultaneous time-resolved inorganic haloamine measurements enable analysis of disinfectant degradation kinetics and by-product formation.

We demonstrate the application of proton transfer time-of-flight mass spectrometry (PTR-TOF-MS) in monitoring the kinetics of disinfectant decay in water with a sensitivity one to three orders of magnitude greater than other analytical methods. Chemical disinfection inactivates pathogens during water treatment and prevents regrowth as water is conveyed in distribution system pipes, but it also causes formation of toxic disinfection by-products. Analytical limits have hindered kinetic models, which aid in ensuring water quality and protecting public health by predicting disinfection by-products formation. PTR-TOF-MS, designed for measuring gas phase concentrations of organic compounds, was able to simultaneously monitor aqueous concentrations of five inorganic haloamines relevant to chloramine disinfection under drinking water relevant concentrations. This novel application to aqueous analytes opens a new range of applications for PTR-TOF-MS.