The emergence of SARS-CoV in 2002 and SARS-CoV-2 in 2019 led to increased sampling of sarbecoviruses circulating in horseshoe bats. Employing phylogenetic inference while accounting for recombination of bat sarbecoviruses, we find that the closest-inferred bat virus ancestors of SARS-CoV and SARS-CoV-2 existed less than a decade prior to their emergence in humans. Phylogeographic analyses show bat sarbecoviruses traveled at rates approximating their horseshoe bat hosts and circulated in Asia for millennia. We find that the direct ancestors of SARS-CoV and SARS-CoV-2 are unlikely to have reached their respective sites of emergence via dispersal in the bat reservoir alone, supporting interactions with intermediate hosts through wildlife trade playing a role in zoonotic spillover. These results can guide future sampling efforts and demonstrate that viral genomic regions extremely closely related to SARS-CoV and SARS-CoV-2 were circulating in horseshoe bats, confirming their importance as the reservoir species for SARS viruses.

Chemically modified nucleotides in mRNA are critical regulators of gene expression, primarily through interactions with reader proteins that bind to these modifications. Here, we present a mechanism by which the epitranscriptomic mark N6-methyladenosine (m6A) is read by tRNAs during translation. Codons that are modified with m6A are decoded inefficiently by the ribosome, rendering them "non-optimal" and inducing ribosome collisions on cellular transcripts. This couples mRNA translation to decay. 5-Methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in the tRNA anticodon loop counteracts this effect. This unanticipated link between the mRNA and tRNA epitranscriptomes enables the coordinated decay of mRNA regulons, including those encoding oncogenic signaling pathways. In cancer, dysregulation of the m6A and mcm5s2U biogenesis pathways-marked by a shift toward more mcm5s2U-is associated with more aggressive tumors and poor prognosis. Overall, this pan-epitranscriptomic interaction represents a novel mechanism of post-transcriptional gene regulation with implications for human health.

The human gut harbors thousands of microbial species, each exhibiting significant inter-individual genetic variability. Although many studies have associated microbial relative abundances with human-health-related phenotypes, the substantial intraspecies genetic variability of gut microbes has not yet been comprehensively considered, limiting the potential of linking such genetic traits with host conditions. Here, we analyzed 32,152 metagenomes from 94 microbiome studies across the globe to investigate the human microbiome intraspecies genetic diversity. We reconstructed 583 species-specific phylogenies and linked them to geographic information and species' horizontal transmissibility. We identified 484 microbial-strain-level associations with 241 host phenotypes, encompassing human anthropometric factors, biochemical measurements, diseases, and lifestyle. We observed a higher prevalence of a Ruminococcus gnavus clade in nonagenarians correlated with distinct plasma bile acid profiles and a melanoma and prostate-cancer-associated Collinsella clade. Our large-scale intraspecies genetic analysis highlights the relevance of strain diversity as it relates to human health.

Vaccines generate long-lived plasma cells and memory B cells (Bmems) that may re-enter secondary germinal centers (GCs) to further mutate their B cell receptor upon boosting and re-exposure to antigen. We show in mouse models that lymph nodes draining the site of primary vaccination harbor a subset of Bmems that reside in the subcapsular niche, generate larger recall responses, and are more likely to re-enter GCs compared with circulating Bmems in non-draining lymph nodes. This location-dependent recall of Bmems into the GC in the draining lymph node was dependent on CD169+ subcapsular sinus macrophages (SSMs) in the subcapsular niche. In human participants, boosting of the BNT162b2 vaccine in the same arm generated more rapid secretion of broadly neutralizing antibodies, GC participation, and clonal expansion of SARS-CoV-2-specific B cells than boosting of the opposite arm. These data reveal an unappreciated role for primed draining lymph node SSMs in Bmem cell fate determination.

Tau accumulation is closely related to cognitive symptoms in Alzheimer's disease (AD). However, the cellular drivers of tau-dependent decline of memory-based cognition remain elusive. Here, we employed in vivo Neuropixels and patch-clamp recordings in mouse models and demonstrate that tau, independent of β-amyloid, selectively debilitates complex-spike burst firing of CA1 hippocampal neurons, a fundamental cellular mechanism underpinning learning and memory. Impaired bursting was associated with altered hippocampal network activities that are coupled to burst firing patterns (i.e., theta rhythms and high-frequency ripples) and was concurrent with reduced neuronal expression of CaV2.3 calcium channels, which are essential for burst firing in vivo. We subsequently identify soluble high molecular weight (HMW) tau, isolated from human AD brain, as the tau species responsible for suppression of burst firing. These data provide a cellular mechanism for tau-dependent cognitive decline in AD and implicate a rare species of intracellular HMW tau as a therapeutic target.

Lysosomes maintain an acidic pH of 4.5-5.0, optimal for macromolecular degradation. Whereas proton influx is produced by a V-type H+ ATPase, proton efflux is mediated by a fast H+ leak through TMEM175 channels, as well as an unidentified slow pathway. A candidate screen on an orphan lysosome membrane protein (OLMP) library enabled us to discover that SLC7A11, the protein target of the ferroptosis-inducing compound erastin, mediates a slow lysosomal H+ leak through downward flux of cystine and glutamate, two H+ equivalents with uniquely large but opposite concentration gradients across lysosomal membranes. SLC7A11 deficiency or inhibition caused lysosomal over-acidification, reduced degradation, accumulation of storage materials, and ferroptosis, as well as facilitated α-synuclein aggregation in neurons. Correction of abnormal lysosomal acidity restored lysosome homeostasis and prevented ferroptosis. These studies have revealed an unconventional H+ transport conduit that is integral to lysosomal flux of protonatable metabolites to regulate lysosome function, ferroptosis, and Parkinson's disease (PD) pathology.

In humans, selective and promiscuous interactions between 46 secreted chemokine ligands and 23 cell surface chemokine receptors of the G-protein-coupled receptor (GPCR) family form a complex network to coordinate cell migration. While chemokines and their GPCRs each share common structural scaffolds, the molecular principles driving selectivity and promiscuity remain elusive. Here, we identify conserved, semi-conserved, and variable determinants (i.e., recognition elements) that are encoded and decoded by chemokines and their receptors to mediate interactions. Selectivity and promiscuity emerge from an ensemble of generalized ("public/conserved") and specific ("private/variable") determinants distributed among structured and unstructured protein regions, with ligands and receptors recognizing these determinants combinatorially. We employ these principles to engineer a viral chemokine with altered GPCR coupling preferences and provide a web resource to facilitate sequence-structure-function studies and protein design efforts for developing immuno-therapeutics and cell therapies.

Mutations in RNA splicing factors are prevalent across cancers and generate recurrently mis-spliced mRNA isoforms. Here, we identified a series of bona fide neoantigens translated from highly stereotyped splicing alterations promoted by neomorphic, leukemia-associated somatic splicing machinery mutations. We utilized feature-barcoded peptide-major histocompatibility complex (MHC) dextramers to isolate neoantigen-reactive T cell receptors (TCRs) from healthy donors, patients with active myeloid malignancy, and following curative allogeneic stem cell transplant. Neoantigen-reactive CD8+ T cells were present in the blood of patients with active cancer and had a distinct phenotype from virus-reactive T cells with evidence of impaired cytotoxic function. T cells engineered with TCRs recognizing SRSF2 mutant-induced neoantigens arising from mis-splicing events in CLK3 and RHOT2 resulted in specific recognition and cytotoxicity of SRSF2-mutant leukemia. These data identify recurrent RNA mis-splicing events as sources of actionable public neoantigens in myeloid leukemias and provide proof of concept for genetically redirecting T cells to recognize these targets.

Leptin acts in the brain to suppress appetite, yet the responsible neurocircuitries underlying leptin's anorectic effect are incompletely defined. Prepronociceptin (PNOC)-expressing neurons mediate diet-induced hyperphagia and weight gain in mice. Here, we show that leptin regulates appetite and body weight via PNOC neurons, and that loss of leptin receptor (Lepr) expression in PNOC-expressing neurons in the arcuate nucleus of the hypothalamus (ARC) causes hyperphagia and obesity. Restoring Lepr expression in PNOC neurons on a Lepr-null obese background substantially reduces body weight. Lepr inactivation in PNOC neurons increases neuropeptide Y (Npy) expression in a subset of hypothalamic PNOC neurons that do not express agouti-related peptide (Agrp). Selective chemogenetic activation of PNOC/NPY neurons promotes feeding to the same extent as activating all PNOCARC neurons, and overexpression of Npy in PNOCARC neurons promotes hyperphagia and obesity. Thus, we introduce PNOC/NPYARC neurons as an additional critical mediator of leptin action and as a promising target for obesity therapeutics.

Virtually all individuals aged 65 or older develop at least early pathology of Alzheimer's disease (AD), yet most lack disease-causing mutations in APP, PSEN, or MAPT, and many do not carry the APOE4 risk allele. This raises questions about AD development in the general population. Although transcriptional dysregulation has not traditionally been a hallmark of AD, recent studies reveal significant epigenomic changes in late-onset AD (LOAD) patients. We show that altered expression of the LOAD biomarker phosphoglycerate dehydrogenase (PHGDH) modulates AD pathology in mice and human brain organoids independent of its enzymatic activity. PHGDH has an uncharacterized role in transcriptional regulation, promoting the transcription of inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKa) and high-mobility group box 1 (HMGB1) in astrocytes, which suppress autophagy and accelerate amyloid pathology. A blood-brain-barrier-permeable small-molecule inhibitor targeting PHGDH's transcriptional function reduces amyloid pathology and improves AD-related behavioral deficits. These findings highlight transcriptional regulation in LOAD and suggest therapeutic strategies beyond targeting familial mutations.

The folding of the genome in the 3D nuclear space is fundamental for regulating all DNA-related processes. The association of the genome with the nuclear lamina into lamina-associated domains (LADs) represents the earliest feature of nuclear organization during development. Here, we performed a gain-of-function screen in mouse embryos to obtain mechanistic insights. We find that perturbations impacting histone H3 modifications, heterochromatin, and histone content are crucial for the establishment of nuclear architecture in zygotes and/or 2-cell-stage embryos. Notably, some perturbations exerted differential effects on zygotes versus 2-cell-stage embryos. Moreover, embryos with disrupted LADs can rebuild nuclear architecture at the 2-cell stage, indicating that the initial establishment of LADs in zygotes might be dispensable for early development. Our findings provide valuable insights into the functional interplay between chromatin and structural components of the nucleus that guide genome-lamina interactions during the earliest developmental stages.

Rice tillering is an important agronomic trait regulated by plant genetic and environmental factors. However, the role and mechanism of the root microbiota in modulating rice tillering have not been explored. Here, we examined the root microbiota composition and tiller numbers of 182 genome-sequenced rice varieties grown under field conditions and uncovered a significant correlation between root microbiota composition and rice tiller number. Using cultivated bacterial isolates, we demonstrated that various members of the root microbiota can regulate rice tillering in both laboratory and field conditions. Genetic, biochemical, and structural analyses revealed that cyclo(Leu-Pro), produced by the tiller-inhibiting bacterium Exiguobacterium R2567, activates the rice strigolactone (SL) signaling pathway by binding to the SL receptor OsD14, thus regulating tillering. The present work provides insight into how the root microbiota regulates key agronomic traits and offers a promising strategy for optimizing crop growth by harnessing the root microbiota in sustainable agriculture.

The prefrontal cortex (PFC) is crucial for economic decision-making. However, how PFC value representations facilitate flexible decisions remains unknown. We reframe economic decision-making as a navigation process through a cognitive map of choice values. We found rhesus macaques represented choices as navigation trajectories in a value space using a grid-like code. This occurred in ventromedial PFC (vmPFC) local field potential theta frequency across two datasets. vmPFC neurons deployed the same grid-like code and encoded chosen value. However, both signals depended on theta phase: occurring on theta troughs but on separate theta cycles. Finally, we found sharp-wave ripples-a key signature of planning and flexible behavior-in vmPFC. Thus, vmPFC utilizes cognitive map-based computations to organize and compare values, suggesting an alternative architecture for economic choice in PFC.

Passive diffusion does not explain why many drugs are too large and/or too polar for rule-breaking membrane penetration, such as proteolysis-targeting chimeras (PROTACs, generally of a molecular weight > 800 Da). Here, using biotinylated chemical-probe-based target fishing and genetic knockdown/knockin approaches, we discovered that the membrane cluster of differentiation 36 (CD36) binds to and facilitates the uptake and efficacy of diverse PROTACs (e.g., SIM1-Me, MZ1, and clinical ARV-110) and large and/or polar small-molecule drugs (e.g., rapalink-1, rapamycin, navitoclax, birinapant, tubacin, and doxorubicin) via the CD36-mediated early endosome antigen 1 (EEA1)/Ras-related protein 5A (Rab5) endosomal cascade in vitro and/or in vivo. We then devised a novel chemical endocytic medicinal chemistry strategy to improve binding of PROTACs to CD36 using structural modifications via the prodrug approach, markedly enhancing PROTAC anti-tumor efficacy through spontaneously augmenting permeability and solubility.

Human blood vessel organoids (hBVOs) have emerged as a system to model human vascular development and disease. Here, we use single-cell multi-omics together with genetic and signaling pathway perturbations to reconstruct hBVO development. Mesodermal progenitors bifurcate into endothelial and mural fates in vitro, and xenografted BVOs acquire definitive arteriovenous endothelial cell specification. We infer a gene regulatory network and use single-cell genetic perturbations to identify transcription factors (TFs) and receptors involved in cell fate specification, including a role for MECOM in endothelial and mural specification. We assess the potential of BVOs to generate organotypic states, identify TFs lacking expression in hBVOs, and find that induced LEF1 overexpression increases brain vasculature specificity. Finally, we map vascular disease-associated genes to hBVO cell states and analyze an hBVO model of diabetes. Altogether, we provide a comprehensive cell state atlas of hBVO development and illuminate the power and limitation of hBVOs for translational research.

Mycobacteriophage Bxb1 is a well-characterized virus of Mycobacterium smegmatis with double-stranded DNA and a long, flexible tail. Mycobacteriophages show considerable potential as therapies for Mycobacterium infections, but little is known about the structural details of these phages or how they bind to and traverse the complex Mycobacterium cell wall. Here, we report the complete structure and atomic model of phage Bxb1, including the arrangement of immunodominant domains of both the capsid and tail tube subunits, as well as the assembly of the protein subunits in the tail-tip complex. The structure contains protein assemblies with 3-, 5-, 6-, and 12-fold symmetries, which interact to satisfy several symmetry mismatches. Cryoelectron tomography of phage particles bound to M. smegmatis reveals the structural transitions that occur for free phage particles to bind to the cell surface and navigate through the cell wall to enable DNA transfer into the cytoplasm.

Polyglutamine (polyQ) expansion is associated with pathogenic protein aggregation in neurodegenerative disorders. However, long polyQ tracts are also found in many transcription factors (TFs), such as FOXP2, a TF implicated in human speech. Here, we explore how FOXP2 and other glutamine-rich TFs avoid unscheduled assembly. Throughout interphase, DNA binding, irrespective of sequence specificity, has a solubilizing effect. During mitosis, multiple phosphorylation events promote FOXP2's eviction from chromatin and supplant the solubilizing function of DNA. Further, human-specific amino acid substitutions linked to the evolution of speech map to a mitotic phospho-patch, the "EVO patch," and reduce the propensity of the human FOXP2 to assemble. Fusing the pathogenic form of Huntingtin to either a DNA-binding domain, a phosphomimetic variant of this EVO patch, or a negatively charged peptide is sufficient to diminish assembly formation, suggesting that hijacking mechanisms governing solubility of glutamine-rich TFs may offer new strategies for treatment of polyQ expansion diseases.