Despite the fundamental importance of gut microbes, the genetic basis of their colonization remains largely unexplored. Here, by applying cross-species genotype-habitat association at the tree-of-life scale, we identify conserved microbial gene modules associated with gut colonization. Across thousands of species, we discovered 79 taxonomically diverse putative colonization factors organized into operonic and non-operonic modules. They include previously characterized colonization pathways such as autoinducer-2 biosynthesis and novel processes including tRNA modification and translation. In vivo functional validation revealed YigZ (IMPACT family) and tRNA hydroxylation protein-P (TrhP) are required for E. coli intestinal colonization. Overexpressing YigZ alone is sufficient to enhance colonization of the poorly colonizing MG1655 E. coli by >100-fold. Moreover, natural allelic variations in YigZ impact inter-strain colonization efficiency. Our findings highlight the power of large-scale comparative genomics in revealing the genetic basis of microbial adaptations. These broadly conserved colonization factors may prove critical for understanding gastrointestinal (GI) dysbiosis and developing therapeutics.

Western equine encephalitis virus (WEEV) is an arbovirus that historically caused large outbreaks of encephalitis throughout the Americas. WEEV binds protocadherin 10 (PCDH10) as a receptor, and highly virulent ancestral WEEV strains also bind low-density lipoprotein receptor (LDLR)-related proteins. As WEEV declined as a human pathogen in North America over the past century, isolates have lost the ability to bind mammalian receptors while still recognizing avian receptors. To explain shifts in receptor dependencies and assess the risk of WEEV re-emergence, we determined cryoelectron microscopy structures of WEEV bound to human PCDH10, avian PCDH10, and human very-low-density lipoprotein receptor (VLDLR). We show that one to three E2 glycoprotein substitutions are sufficient for a nonpathogenic strain to regain the ability to bind mammalian receptors. A soluble VLDLR fragment protects mice from lethal challenge by a virulent ancestral WEEV strain. Because WEEV recently re-emerged in South America after decades of inactivity, our findings have important implications for outbreak preparedness.

The structural basis for shifts in receptor usage remains poorly understood despite the implications for virus adaptation and emergence. Western equine encephalitis virus (WEEV) strains exhibit different patterns of engagement for two of their entry receptors: very-low-density lipoprotein receptor (VLDLR) and protocadherin 10 (PCDH10). Using structural and functional studies, we show that while all WEEV strains have a lipoprotein class A (LA) domain binding site near the E1 fusion loop, VLDLR engagement requires a second binding site in E2 that can vary with single nucleotide substitutions. We also resolve a structure of PCDH10 bound to WEEV, which reveals interactions near the E1 fusion loop with residues that also mediate LA domain binding. Evolutionary analysis enabled the generation of a PCDH10 decoy that protects in vivo against all WEEV strains tested. Our experiments demonstrate how viruses can engage multiple receptors using shared determinants, which likely impacts cellular tropism and virulence.

Claustrum orchestrates brain functions via its connections with numerous brain regions, but its molecular and cellular organization remains unresolved. Single-nucleus RNA sequencing of 227,750 macaque claustral cells identified 48 transcriptome-defined cell types, with most glutamatergic neurons similar to deep-layer insular neurons. Comparison of macaque, marmoset, and mouse transcriptomes revealed macaque-specific cell types. Retrograde tracer injections at 67 cortical and 7 subcortical regions defined four distinct distribution zones of retrogradely labeled claustral neurons. Joint analysis of whole-brain connectivity and single-cell spatial transcriptome showed that these four zones containing distinct compositions of glutamatergic (but not GABAergic) cell types preferentially connected to specific brain regions with a strong ipsilateral bias. Several macaque-specific glutamatergic cell types in ventral vs. dorsal claustral zones selectively co-projected to two functionally related areas-entorhinal cortex and hippocampus vs. motor cortex and putamen, respectively. These data provide the basis for elucidating the neuronal organization underlying diverse claustral functions.

The intestinal immune system maintains tolerance to harmless food proteins and gut microbiota through peripherally derived RORγt+ Tregs (pTregs), which prevent food intolerance and inflammatory bowel disease. Recent studies suggested that RORγt+ antigen-presenting cells (APCs), which encompass rare dendritic cell (DC) subsets and type 3 innate lymphoid cells (ILC3s), are key to pTreg induction. Here, we developed a mouse with reduced RORγt+ APCs by deleting a specific cis-regulatory element of Rorc encoding RORγt. Single-cell RNA sequencing and flow cytometry analyses confirmed the depletion of a RORγt+ DC subset and ILC3s. These mice showed a secondary reduction in pTregs, impaired tolerance to oral antigens, and an increase in T helper (Th)2 cells. Conversely, ILC3-deficient mice showed no pTregs or Th2 cell abnormalities. Lineage tracing revealed that RORγt+ DCs share a lymphoid origin with ILC3s, consistent with their similar phenotypic traits. These findings highlight the role of lymphoid RORγt+ DCs in maintaining intestinal immune balance and preventing conditions like food allergies.

Alveolar rhabdomyosarcoma (ARMS) patients harboring paired-box fusion proteins (PAX3/7-FOXO1) exhibit a greater incidence of tumor relapse, metastasis, and poor survival outcome, thereby underscoring the urgent need to develop effective therapies to treat this subtype of childhood cancer. To uncover mechanisms that contribute to tumor initiation, we develop a muscle progenitor model and use epigenomic approaches to unravel genome rewiring events mediated by PAX3/7 fusion proteins. Among the key targets of PAX3/7 fusion proteins, we identify a cohort of oncogenes, fibroblast growth factor (FGF) receptors, tRNA-modifying enzymes, and genes essential for mitochondrial metabolism and protein translation, which we successfully targeted in preclinical trials. We identify leucine usage as a key factor driving the growth of aggressive PAX-fusion tumors, as limiting its bioavailability impaired oxidative phosphorylation and mitochondrial metabolism, delaying tumor progression and improving survival in vivo. Our data provide a compelling list of actionable targets and suggest promising new strategies to treat this tumor.

Twenty years ago, histone lysine demethylases (KDMs) were discovered. Since their discovery, they have been increasingly studied and shown to be important across species, development, and diseases. Considerable advances have been made toward understanding their (1) enzymology, (2) role as critical components of biological complexes, (3) role in normal cellular processes and functions, (4) implications in pathological conditions, and (5) therapeutic potential. This Review covers these key relationships related to the KDM field with the awareness that numerous laboratories have contributed to this field. The current knowledge coupled with future insights will shape our understanding about cell function, development, and disease onset and progression, which will allow for novel biomarkers to be identified and for optimal therapeutic options to be developed for KDM-related diseases in the years ahead.

Targeting ubiquitin E3 ligases is therapeutically attractive; however, the absence of an active-site pocket impedes computational approaches for identifying inhibitors. In a large, unbiased biochemical screen, we discover inhibitors that bind a cryptic cavity distant from the catalytic cysteine of the homologous to E6-associated protein C terminus domain (HECT) E3 ligase, SMAD ubiquitin regulatory factor 1 (SMURF1). Structural and biochemical analyses and engineered escape mutants revealed that these inhibitors restrict an essential catalytic motion by extending an α helix over a conserved glycine hinge. SMURF1 levels are increased in pulmonary arterial hypertension (PAH), a disease caused by mutation of bone morphogenetic protein receptor-2 (BMPR2). We demonstrated that SMURF1 inhibition prevented BMPR2 ubiquitylation, normalized bone morphogenetic protein (BMP) signaling, restored pulmonary vascular cell homeostasis, and reversed pathology in established experimental PAH. Leveraging this deep mechanistic understanding, we undertook an in silico machine-learning-based screen to identify inhibitors of the prototypic HECT E6AP and confirmed glycine-hinge-dependent allosteric activity in vitro. Inhibiting HECTs and other glycine-hinge proteins opens a new druggable space.

The cytoskeletal protein actin is crucial for cell shape and integrity throughout eukaryotes. Actin filaments perform essential biological functions, including muscle contraction, cell division, and tissue morphogenesis. These diverse activities are achieved through the ability of actin filaments to be arranged into precise architectures. Much progress has been made in defining the proteome of the actin cytoskeleton, but a detailed appreciation of the dynamic organizational state of the actin filaments themselves has been hindered by available tools. Fluorescence polarization microscopy is uniquely placed for measuring actin filament organization by exploiting the sensitivity of polarized light excitation to the orientation of fluorophores attached to actin filaments. By engineering fusions of five widely used actin localization reporters to fluorescent proteins with constrained mobility, we have succeeded in developing genetically encoded, green- and red-fluorescent-protein-based reporters for non-invasive, quantitative measurements of actin filament organization in living cells and tissues by fluorescence polarization microscopy.

Amoebal predation exerts a strong evolutionary selection pressure on bacteria, thus driving the development of effective predator-defense strategies. However, little is known about the molecular interplay between bacteria and predators, particularly how bacteria can sense and kill their microbial predators. We show how the ubiquitous bacterium Pseudomonas syringae detects and kills the social amoeba Polysphondylium pallidum. Combining comparative genomics, molecular biology, and chemical analyses, we identified a chemical radar system. The system relies on P. syringae secreting the lipopeptide syringafactin, which is deacylated by the amoeba. The resulting peptides are sensed via the bacterial sensor protein chemical radar regulator (CraR) that activates genes for converting the predator-derived signal into the amoebicide pyrofactin. This system is widespread in P. syringae and enables bacteria to infect A. thaliana in the presence of amoebae. Our study advances the understanding of microbial sensing and opens new avenues for the discovery of natural products.

Membranes are molecular interfaces that compartmentalize cells to control the flow of nutrients and information. These functions are facilitated by diverse collections of lipids, nearly all of which are distributed asymmetrically between the two bilayer leaflets. Most models of biomembrane structure and function include the implicit assumption that these leaflets have similar abundances of phospholipids. Here, we show that this assumption is generally invalid and investigate the consequences of lipid abundance imbalances in mammalian plasma membranes (PMs). Using lipidomics, we report that cytoplasmic leaflets of human erythrocyte membranes have >50% overabundance of phospholipids compared with exoplasmic leaflets. This imbalance is enabled by an asymmetric interleaflet distribution of cholesterol, which regulates cellular cholesterol homeostasis. These features produce unique functional characteristics, including low PM permeability and resting tension in the cytoplasmic leaflet that regulates protein localization.

Chimeric antigen receptor (CAR) T cell therapy for solid tumors encounters challenges such as on-target off-tumor toxicity, exhaustion, and limited T cell persistence. Here, we engineer sonogenetic EchoBack-CAR T cells using an ultrasensitive heat-shock promoter screened from a library and integrated with a positive feedback loop from CAR signaling, enabling long-lasting CAR expression upon focused-ultrasound (FUS) stimulation. EchoBack-hGD2CAR T cells, targeting disialoganglioside GD2, exhibited potent cytotoxicity and persistence in 3D glioblastoma (GBM) models. In mice, EchoBack-hGD2CAR T cells suppressed GBM without off-tumor toxicity and outperformed their constitutive counterparts. Single-cell RNA sequencing revealed enhanced cytotoxicity and reduced exhaustion in EchoBack-CAR T cells compared with the standard CAR T cells. This EchoBack design was further adapted to target prostate-specific membrane antigen (EchoBack-PSMACAR) for prostate cancer treatment, demonstrating long-lasting tumor suppression with minimal off-tumor toxicity. Thus, the sonogenetic EchoBack-CAR T cells can serve as a versatile, efficient, and safe strategy for solid tumor treatment.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a cornerstone treatment for a broad spectrum of malignant and nonmalignant hematological disorders. However, the success of allo-HSCT is often overshadowed by acute graft-versus-host disease (aGVHD), a life-threatening complication. Here, we show in patients and murine models that the circadian timing of stem cell infusion dictates the development of aGVHD. Early-infused patients exhibit a significantly lower incidence and severity of aGVHD, as well as improved survival. We observed time-of-day variations in the levels of cytokines, especially IL-1α, which controls donor T cell responses after transplantation. The levels of IL-1α in patients were strongly associated with the development of aGVHD. Furthermore, preclinical results showed that the administration of IL-1α neutralizing antibodies markedly alleviated aGVHD and increased survival. Our study suggests that scheduling stem cell infusions early in the day could be a simple yet transformative intervention for preventing aGVHD.

Single-cell proteomics (SCPs) has advanced significantly, yet it remains largely unidimensional, focusing primarily on protein abundances. In this study, we employed a pulsed stable isotope labeling by amino acids in cell culture (pSILAC) approach to simultaneously analyze protein abundance and turnover in single cells (SC-pSILAC). Using a state-of-the-art SCP workflow, we demonstrated that two SILAC labels are detectable from ∼4,000 proteins in single HeLa cells recapitulating known biology. We performed a large-scale time-series SC-pSILAC analysis of undirected differentiation of human induced pluripotent stem cells (iPSCs) encompassing 6 sampling times over 2 months and analyzed >1,000 cells. Protein turnover dynamics highlighted differentiation-specific co-regulation of protein complexes with core histone turnover, discriminating dividing and non-dividing cells. Lastly, correlating cell diameter with the abundance of individual proteins showed that histones and some cell-cycle proteins do not scale with cell size. The SC-pSILAC method provides a multidimensional view of protein dynamics in single-cell biology.

Ring-like structural maintenance of chromosome (SMC) complexes are crucial for genome organization and operate through mechanisms of DNA entrapment and loop extrusion. Here, we explore the DNA loading process of the bacterial SMC complex MukBEF. Using cryoelectron microscopy (cryo-EM), we demonstrate that ATP binding opens one of MukBEF's three potential DNA entry gates, exposing a DNA capture site that positions DNA at the open neck gate. We discover that the gp5.9 protein of bacteriophage T7 blocks this capture site by DNA mimicry, thereby preventing DNA loading and inactivating MukBEF. We propose a comprehensive and unidirectional loading mechanism in which DNA is first captured at the complex's periphery and then ingested through the DNA entry gate, powered by a single cycle of ATP hydrolysis. These findings illuminate a fundamental aspect of how ubiquitous DNA organizers are primed for genome maintenance and demonstrate how this process can be disrupted by viruses.

Anti-PD-(L)1 treatment is standard for non-small cell lung cancer (NSCLC), but patients show variable responses to the same regimen. The tumor immune microenvironment (TIME) is associated with immunotherapy response, yet the heterogeneous underlying therapeutic outcomes remain underexplored. We applied single-cell RNA and TCR sequencing (scRNA/TCR-seq) to analyze surgical tumor samples from 234 NSCLC patients post-neoadjuvant chemo-immunotherapy. Analyses revealed five distinct TIME subtypes with varying major pathological response (MPR) rates. MPR patients had elevated levels of FGFBP2+ NK/NK-like T cells, memory B cells, or effector T cells, while non-MPR patients showed higher CCR8+ Tregs. T cell clonal expansion analyses unveiled heterogeneity in non-MPR patients, marked by varying expansions of Tex-relevant cells and CCR8+ Tregs. Precursor exhausted T cells (Texp cells) correlated with recurrence-free survival, identifying a patient subgroup with reduced recurrence risk despite lack of MPR. Our study dissects TIME heterogeneity in response to chemoimmunotherapy, offering insights for NSCLC management.

Efforts to use genome-wide assays or brain scans to diagnose autism have seen diminishing returns. Yet the clinical intuition of healthcare professionals, based on longstanding first-hand experience, remains the gold standard for diagnosis of autism. We leveraged deep learning to deconstruct and interrogate the logic of expert clinician intuition from clinical reports to inform our understanding of autism. After pre-training on hundreds of millions of general sentences, we finessed large language models (LLMs) on >4,000 free-form health records from healthcare professionals to distinguish confirmed versus suspected autism cases. By introducing an explainability strategy, our extended language model architecture could pin down the most salient single sentences in what drives clinical thinking toward correct diagnoses. Our framework flagged the most autism-critical DSM-5 criteria to be stereotyped repetitive behaviors, special interests, and perception-based behaviors, which challenges today's focus on deficits in social interplay, suggesting necessary revision of long-trusted diagnostic criteria in gold-standard instruments.

Nipah virus recurrently spills over to humans, causing fatal infections. The viral receptor-binding protein (RBP or G) attaches to host receptors and is a major target of neutralizing antibodies. Here, we use deep mutational scanning to measure how all amino-acid mutations to the RBP affect cell entry, receptor binding, and escape from neutralizing antibodies. We identify functionally constrained regions of the RBP, including sites involved in oligomerization, along with mutations that differentially modulate RBP binding to its two ephrin receptors. We map escape mutations for six anti-RBP antibodies and find that few antigenic mutations are present in natural Nipah strains. Our findings offer insights into the potential for functional and antigenic evolution of the RBP that can inform the development of antibody therapies and vaccines.

The gastrointestinal (GI) tract experiences a myriad of mechanical forces while orchestrating digestion and barrier immunity. A central conductor of these processes, the enteric nervous system (ENS), detects luminal pressure to regulate peristalsis independently of extrinsic input from the central and peripheral nervous systems. However, how the ∼500 million enteric neurons that reside in the GI tract sense and respond to force remains unknown. Herein, we establish that the mechanosensor Piezo1 is functionally expressed in cholinergic enteric neurons. Optogenetic stimulation of Piezo1+ cholinergic enteric neurons drives colonic motility, while Piezo1 deficiency reduces cholinergic neuronal activity and slows peristalsis. Additionally, Piezo1 deficiency in cholinergic enteric neurons abolishes exercise-induced acceleration of GI motility. Finally, we uncover that enteric neuronal Piezo1 function is required for motility alterations in colitis and acts to prevent aberrant inflammation and tissue damage. This work uncovers how the ENS senses and responds to mechanical force.

How genomic DNA is folded during cell division to form the characteristic rod-shaped mitotic chromosomes essential for faithful genome inheritance is a long-standing open question in biology. Here, we use nanoscale DNA tracing in single dividing cells to directly visualize how the 3D fold of genomic DNA changes during mitosis at scales from single loops to entire chromosomes. Our structural analysis reveals a characteristic genome scaling minimum of 6-8 megabases in mitosis. Combined with data-driven modeling and molecular perturbations, we can show that very large and strongly overlapping loops formed by condensins are the fundamental structuring principle of mitotic chromosomes. These loops compact chromosomes locally and globally to the limit set by chromatin self-repulsion. The characteristic length, density, and increasingly overlapping structure of mitotic loops we observe in 3D fully explain how the rod-shaped mitotic chromosome structure emerges by self-organization during cell division.