With rapid advancements in single-cell RNA sequencing (scRNA-seq) technologies, exploration of the systemic coordination of critical physiological processes has entered a new era. Here, we generated a comprehensive Arabidopsis single-nucleus transcriptomic atlas using over 1 million nuclei from 20 tissues encompassing multiple developmental stages. Our analyses identified cell types that have not been characterized in previous single-protoplast studies and revealed cell-type conservation and specificity across different organs. Through time-resolved sampling, we revealed highly coordinated onset and progression of senescence among the major leaf cell types. We originally formulated two molecular indexes to quantify the aging state of leaf cells at single-cell resolution. Additionally, facilitated by weighted gene co-expression network analysis, we identified hundreds of promising hub genes that may integratively regulate leaf senescence. Inspired by the functional validation of identified hub genes, we built a systemic scenario of carbon and nitrogen allocation among different cell types from source leaves to sink organs.

Chimeric antigen receptor (CAR) T cell immunotherapy represents a breakthrough in the treatment of hematological malignancies, but poor specificity has limited its applicability to solid tumors. By contrast, natural T cells harboring T cell receptors (TCRs) can discriminate between neoantigen-expressing cancer cells and self-antigen-expressing healthy tissues but have limited potency against tumors. We used a high-throughput platform to systematically evaluate the impact of co-expressing a TCR and CAR on the same CAR T cell. While strong TCR-antigen interactions enhanced CAR activation, weak TCR-antigen interactions actively antagonized their activation. Mathematical modeling captured this TCR-CAR crosstalk in CAR T cells, allowing us to engineer dual TCR/CAR T cells targeting neoantigens (HHATL8F/p53R175H) and human epithelial growth factor receptor 2 (HER2) ligands, respectively. These T cells exhibited superior anti-cancer activity and minimal toxicity against healthy tissue compared with conventional CAR T cells in a humanized solid tumor mouse model. Harnessing pre-existing inhibitory crosstalk between receptors, therefore, paves the way for the design of more precise cancer immunotherapies.

Diverse microbes utilize redox shuttles to exchange electrons with their environment through mediated extracellular electron transfer (EET), supporting anaerobic survival. Although mediated EET has been leveraged for bioelectrocatalysis for decades, fundamental questions remain about how these redox shuttles are reduced within cells and their role in cellular bioenergetics. Here, we integrate genome editing, electrochemistry, and systems biology to investigate the mechanism and bioenergetics of mediated EET in Escherichia coli, elusive for over two decades. In the absence of alternative electron sinks, the redox cycling of 2-hydroxy-1,4-naphthoquinone (HNQ) via the cytoplasmic nitroreductases NfsB and NfsA enables E. coli respiration on an extracellular electrode. E. coli also exhibits rapid genetic adaptation in the outer membrane porin OmpC, enhancing HNQ-mediated EET levels coupled to growth. This work demonstrates that E. coli can grow independently of classic electron transport chains and fermentation, unveiling a potentially widespread new type of anaerobic energy metabolism.

N4-methylcytosine (4mC) is an important DNA modification in prokaryotes, but its relevance and even its presence in eukaryotes have been mysterious. Here we show that spermatogenesis in the liverwort Marchantia polymorpha involves two waves of extensive DNA methylation reprogramming. First, 5-methylcytosine (5mC) expands from transposons to the entire genome. Notably, the second wave installs 4mC throughout genic regions, covering over 50% of CG sites in sperm. 4mC requires a methyltransferase (MpDN4MT1a) that is specifically expressed during late spermiogenesis. Deletion of MpDN4MT1a alters the sperm transcriptome, causes sperm swimming and fertility defects, and impairs post-fertilization development. Our results reveal extensive 4mC in a eukaryote, identify a family of eukaryotic methyltransferases, and elucidate the biological functions of 4mC in reproductive development, thereby expanding the repertoire of functional eukaryotic DNA modifications.

Advanced gene editing methods have accelerated biomedical discovery and hold great therapeutic promise, but safe and efficient delivery of gene editors remains challenging. In this study, we present a virus-like particle (VLP) system featuring nucleocytosolic shuttling vehicles that retrieve pre-assembled Cas-effectors via aptamer-tagged guide RNAs. This approach ensures preferential loading of fully assembled editor ribonucleoproteins (RNPs) and enhances the efficacy of prime editing, base editing, trans-activators, and nuclease activity coupled to homology-directed repair in multiple immortalized, primary, stem cell, and stem-cell-derived cell types. We also achieve additional protection of inherently unstable prime editing guide RNAs (pegRNAs) by shielding the 3'-exposed end with Csy4/Cas6f, further enhancing editing performance. Furthermore, we identify a minimal set of packaging and budding modules that can serve as a platform for bottom-up engineering of enveloped delivery vehicles. Notably, our system demonstrates superior per-VLP editing efficiency in primary T lymphocytes and two mouse models of inherited retinal disease, highlighting its therapeutic potential.

Optical recording of intricate molecular dynamics is becoming an indispensable technique for biological studies, accelerated by the development of new or improved biosensors and microscopy technology. This creates major computational challenges to extract and quantify biologically meaningful spatiotemporal patterns embedded within complex and rich data sources, many of which cannot be captured with existing methods. Here, we introduce activity quantification and analysis (AQuA2), a fast, accurate, and versatile data analysis platform built upon advanced machine-learning techniques. It decomposes complex live-imaging-based datasets into elementary signaling events, allowing accurate and unbiased quantification of molecular activities and identification of consensus functional units. We demonstrate applications across a wide range of biosensors, cell types, organs, animal models, microscopy techniques, and imaging approaches. As exemplar findings, we show how AQuA2 identified drug-dependent interactions between neurons and astroglia, as well as distinct sensorimotor signal propagation patterns in the mouse spinal cord.

To maintain tissue homeostasis, many cells reside in a quiescent state until prompted to divide. The reactivation of quiescent cells is perturbed with aging and may underlie declining tissue homeostasis and resiliency. The unfolded protein response regulators IRE-1 and XBP-1 are required for the reactivation of quiescent cells in developmentally L1-arrested C. elegans. Utilizing a forward genetic screen in C. elegans, we discovered that macroautophagy targets protein aggregates to lysosomes in quiescent cells, leading to lysosome damage. Genetic inhibition of macroautophagy and stimulation of lysosomes via the overexpression of HLH-30 (TFEB/TFE3) synergistically reduces lysosome damage. Damaged lysosomes require IRE-1/XBP-1 for their repair following prolonged L1 arrest. Protein aggregates are also targeted to lysosomes by macroautophagy in quiescent cultured mammalian cells and are associated with lysosome damage. Thus, lysosome damage is a hallmark of quiescent cells, and limiting lysosome damage by restraining macroautophagy can stimulate their reactivation.

The plasma proteome is maintained by the influx and efflux of proteins from surrounding organs and cells. To quantify the extent to which different organs and cells impact the plasma proteome in healthy and diseased conditions, we developed a mass-spectrometry-based proteomics strategy to infer the tissue origin of proteins detected in human plasma. We first constructed an extensive human proteome atlas from 18 vascularized organs and the 8 most abundant cell types in blood. The atlas was interfaced with previous RNA and protein atlases to objectively define proteome-wide protein-organ associations to infer the origin and enable the reproducible quantification of organ-specific proteins in plasma. We demonstrate that the resource can determine disease-specific quantitative changes of organ-enriched protein panels in six separate patient cohorts, including sepsis, pancreatitis, and myocardial injury. The strategy can be extended to other diseases to advance our understanding of the processes contributing to plasma proteome dynamics.

Skeletal muscle contraction is triggered by acetylcholine (ACh) binding to its ionotropic receptors (AChRs) at neuromuscular junctions. In myasthenia gravis (MG), autoantibodies target AChRs, disrupting neurotransmission and causing muscle weakness. While treatments exist, variable patient responses suggest pathogenic heterogeneity. Progress in understanding the molecular basis of MG has been limited by the absence of structures of intact human muscle AChRs. Here, we present high-resolution cryoelectron microscopy (cryo-EM) structures of the human adult AChR in different functional states. Using six MG patient-derived monoclonal antibodies, we mapped distinct epitopes involved in diverse pathogenic mechanisms, including receptor blockade, internalization, and complement activation. Electrophysiological and binding assays revealed how these autoantibodies directly inhibit AChR channel activation. These findings provide critical insights into MG immunopathogenesis, uncovering unrecognized antibody epitope diversity and modes of receptor inhibition, and provide a framework for developing personalized therapies targeting antibody-mediated autoimmune disorders.

Cytokines interact with their receptor complexes to orchestrate diverse processes-from immune responses to behavioral modulation. Interleukin-17A (IL-17A) mediates protective immune responses by binding to IL-17 receptor A (IL-17RA) and IL-17RC subunits. IL-17A also modulates social interaction, yet the role of cytokine receptors in this process and their expression in the brain remains poorly characterized. Here, we mapped the brain-region-specific expression of all major IL-17R subunits and found that in addition to IL-17RA, IL-17RB-but not IL-17RC-plays a role in social behaviors through its expression in the cortex. We further showed that IL-17E, expressed in cortical neurons, enhances social interaction by acting on IL-17RA- and IL-17RB-expressing neurons. These findings highlight an IL-17 circuit within the cortex that modulates social behaviors. Thus, characterizing spatially restricted cytokine receptor expression can be leveraged to elucidate how cytokines function as critical messengers mediating neuroimmune interactions to shape animal behaviors.

Patients with autoimmune or infectious diseases can develop persistent mood alterations after inflammatory episodes. Peripheral immune molecules, like cytokines, can influence behavioral and internal states, yet their impact on the function of specific neural circuits in the brain remains unclear. Here, we show that cytokines act as neuromodulators to regulate anxiety by engaging receptor-expressing neurons in the basolateral amygdala (BLA). Heightened interleukin-17A (IL-17A) and IL-17C levels, paradoxically induced from treatment with anti-IL-17 receptor A (IL-17RA) antibodies, promote anxiogenic behaviors by increasing the excitability of IL-17RA/RE-expressing BLA neurons. Conversely, the anti-inflammatory IL-10, acting on the same population of BLA neurons via its receptor, exerts opposite effects on neuronal excitability and behavior. These findings reveal that inflammatory and anti-inflammatory cytokines bidirectionally modulate anxiety by engaging their respective receptors in the same BLA population. Our results highlight the role of cytokine signaling in shaping internal states through direct modulation of specific neural substrates.

Microglia, essential in the central nervous system (CNS), were historically considered absent from the peripheral nervous system (PNS). Here, we show a PNS-resident macrophage population that shares transcriptomic and epigenetic profiles as well as an ontogenetic trajectory with CNS microglia. This population (termed PNS microglia-like cells) enwraps the neuronal soma inside the satellite glial cell envelope, preferentially associates with larger neurons during PNS development, and is required for neuronal functions by regulating soma enlargement and axon growth. A phylogenetic survey of 24 vertebrates revealed an early origin of PNS microglia-like cells, whose presence is correlated with neuronal soma size (and body size) rather than evolutionary distance. Consistent with their requirement for soma enlargement, PNS microglia-like cells are maintained in vertebrates with large peripheral neuronal soma but absent when neurons evolve to have smaller soma. Our study thus reveals a PNS counterpart of CNS microglia that regulates neuronal soma size during both evolution and ontogeny.

Neural reflexes to chemicals in the throat protect the airway from aspiration and infection. Mechanistic understanding of these reflexes remains premature, exemplified by chronic cough-a sensitized cough reflex-being a prevalent unmet clinical need. Here, in mice, a whole-body search for channel synapses-featuring CALHM1/3 channel-mediated neurotransmitter release-and single-cell transcriptomics uncovered subclasses of the Pou2f3+ chemosensory cell family in the throat communicating with vagal neurons via this synapse. They express G protein-coupled receptors (GPCRs) for noxious chemicals, T2Rs, which upon stimulation trigger swallow and cough-like expulsive reflexes in the hypopharynx and larynx, respectively. These reflexes were abolished by Calhm3 and Pou2f3 knockout and could be triggered by targeted optogenetic stimulation. Furthermore, aeroallergen exposure augmented CALHM3-dependent expulsive reflex. This study identifies Pou2f3+ epithelial cells with channel synapses as chemosensory end organs of airway protective reflexes and sites of their hyperresponsiveness, advancing mechanistic understanding of airway defense programs with distinct therapeutic potential.

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.