Plant growth relies on the activity of key transcription factors. Here, we uncover a mechanism for organ-specific growth driven by opposing electrochemical signals that propagate in a cell-type-specific manner. Using a genetically encoded pH sensor and a pH-sensitive dye, we show that apoplastic pH in epidermal cells oscillates antiphasically relative to phloem pH. The clock component CCA1 lowers apoplastic pH in hypocotyl epidermal cells while increasing it in companion cells. This opposing regulation promotes hypocotyl growth but inhibits root elongation. Mechanistically, CCA1 activates auxin signaling in shoots while repressing sucrose transporter 2 and the electrogenic (H+)-pump ATPase AHA3 by directly binding their promoters. The repression decreases sucrose loading into the phloem and slows transport velocity. Expressing CCA1 in the phloem is sufficient to inhibit root elongation, whereas AHA3 overexpression in CCA1 overexpressing seedlings rescues root growth. Thus, a circadian rheostat orchestrates electrochemical signals to optimize source capacity with sink demand.

Bifidobacterium longum and B. infantis are pioneer colonizers of the neonatal gut and are widely used as probiotics to support infant growth, development, and disease resistance. However, commercial strains derived largely from high-income countries (HICs) may be suboptimal for infants in low- and middle-income countries (LMICs). We assembled a global genomic atlas of more than 4,000 genomes from 48 countries, increasing representation from LMICs by 12- to 17-fold. High-resolution phylogenomic and functional analyses support delineating B. longum and B. infantis as distinct species with divergent functions and epidemiological patterns. B. infantis dominates early-life microbiota in LMICs but is rarely detected in HICs. Natural B. infantis strains show extreme biogeographic stratification and predicted adaptations to local plant-glycan-rich diets and breast-milk-derived substrates, including urea and B vitamins. This genomic resource enables genome-guided selection of geographically matched strains to inform more effective probiotics and precision microbiome therapeutics for diverse infant populations.

We present PocketXMol, an atom-level model that unifies generative tasks related to protein pocket interactions. Using atomic prompts as task specifications, PocketXMol supports various molecular tasks, including structure prediction and de novo design of small molecules and peptides, without task-specific fine-tuning. PocketXMol achieved strong performance on 11 of 13 computational benchmarks and remained competitive on the remaining two, outperforming 55 baseline models. We applied PocketXMol to design caspase-9-inhibiting small molecules, achieving efficacy comparable with commercial pan-caspase inhibitors. We also adopted PocketXMol to generate PD-L1-binding peptides, resulting in a success rate that largely exceeds library screening. Three representative peptides underwent further experiments, which validated their cellular specificity and confirmed their potential for molecular probing and therapeutics. PocketXMol provides a general platform for AI-aided drug discovery and enables a wide range of future applications.

Insulating sheaths of myelin accelerate neuronal communication in the mammalian brain. Oligodendrocytes that produce myelin are generated throughout life to gradually increase myelin coverage, but these dynamics have not been defined brain-wide across the lifespan. We developed a cellular mapping pipeline involving tissue clearing, lightsheet microscopy, and AI-assisted analysis to identify the precise location of millions of oligodendrocytes and assess regional myelin density in the mouse brain. These atlases revealed the diversity of oligodendrocyte patterning, which was consistent between brain hemispheres, individuals, and sexes but displayed both age- and region-specific differences. Integration of these atlases with transcriptomic and ultrastructural datasets highlighted underlying mechanisms that may control this patterning. In models of demyelination and disease, we identified regions of enhanced oligodendrocyte resilience and vulnerability and white matter injury near β-amyloid plaques, demonstrating the utility of this pipeline for defining brain-wide oligodendrocyte dynamics in both health and disease.

Blood factors transfer the benefits of exercise to the aged brain independent of physical activity. Here, we show that the liver-derived exercise factor (exerkine) glycosylphosphatidylinositol (GPI)-specific phospholipase D1 (GPLD1), a GPI-degrading enzyme, reverses aging- and Alzheimer's-related memory loss by targeting the brain vasculature. GPLD1 has the potential to cleave over 100 putative GPI-anchored proteins, necessitating the identification of downstream targets that mediate cognitive rejuvenation for translational application. We identified GPI-anchored tissue-nonspecific alkaline phosphatase (TNAP) on the brain vasculature as a GPLD1 substrate. Mimicking age-related increases in cerebrovascular TNAP impaired blood-brain transport and cognition in young mice and mitigated GPLD1-induced cognitive benefits in aged mice. Inhibiting TNAP recapitulated the benefits of GPLD1 in old age, restoring youthful hippocampal transcriptional signatures and rescuing cognition. In an Alzheimer's disease model, increasing GPLD1 or inhibiting TNAP ameliorated Aβ pathology and improved cognitive deficits. We thus identify brain vasculature as a mediator of the cognitive benefits of a liver-to-brain exercise axis.

In contrast to living organisms, viruses were long thought to lack protein synthesis machinery and instead depend on host factors to translate viral transcripts. Here, we discover that giant DNA viruses encode a distinct and functional IF4F translation-initiation complex to drive protein synthesis, thereby blurring the line between cellular and acellular biology. During infection, eukaryotic IF4F on host ribosomes is replaced by an essential viral IF4F that regulates viral translation, virion formation, and replication plasticity during altered host states. Structural dissection of viral IF4F reveals that the mRNA cap-binding subunit mediates exclusive interactions with viral mRNAs, constituting a molecular switch from translating host to viral proteins. Thus, our study establishes that viruses express a eukaryotic translation-initiation complex for protein synthesis, illuminating a series of evolutionary innovations in a core process of life.

Endothelial cells (ECs) are essential components of the vertebrate circulatory system; however, a comprehensive atlas characterizing how ECs acquire organ-specific transcriptomic heterogeneity has not been established. Here, we generated a time-series endothelial resource covering the entirety of mouse embryonic development, including 26 time points and 8 organs. Time-series multi-organ comparison revealed emergence timing and lineage trajectory of organotypic ECs together with organ-specific genes and pathways. Using these resources, we found that most ECs showed distinguishable organ specificity before late gestation. The organotypic EC-enriched genes were associated with vascular function in the organs. Human and mouse pulmonary ECs underwent an evolutionarily conserved transcriptional transition. Endothelial-specific knockout of Casz1, a pulmonary EC-enriched transcription factor, resulted in impaired vascular growth, disturbed pulmonary endothelial organotypic differentiation, and deficient epithelial-EC crosstalk. Our work provides a powerful endothelial resource that reveals fundamental principles of organ-specific EC differentiation and uncovers previously unknown molecular mechanisms governing lung-specific vascular development.

A recent first-in-human clinical trial demonstrated that survival in glioblastoma (GBM) patients following rQNestin34.5v.2 oncolytic virus treatment was associated with immune activation signatures. This study was registered at ClinicalTrials.gov (NCT03152318). Here, we provide in situ evidence of ongoing T cell-mediated cytotoxicity against tumor cells at late time points following single treatment, with deep and persistent T cell infiltration into tumor regions. Shorter distances between cleaved caspase-3+ tumor cells and granzyme B+ T cells were associated with longer progression-free survival following treatment. Pre-existing tumor-infiltrating T cells expanded locally upon treatment, correlating with longer overall patient survival. T cells with an early activation program closely interacted with tumor cells and were strongly enriched upon treatment. Viral remnants were restricted to necrotic regions, while T cells infiltrated deeply into live tumor regions. These data demonstrate that single oncolytic virus treatment can expand pre-existing T cell clones and trigger persistent T cell-mediated immunity against GBM.

Microbes are ubiquitous on Earth, forming microbiomes that sustain macroscopic life and biogeochemical cycles. Microbial dispersal, driven by natural processes and human activities, interconnects microbiomes across habitats, yet most comparative studies focus on specific ecosystems. To study planetary microbiome structure, function, and inter-habitat interactions, we systematically integrated 85,604 public metagenomes spanning diverse habitats worldwide. Using species-based unsupervised clustering and parameter modeling, we delineated 40 habitat clusters and quantified their ecological similarity. Our framework identified key drivers shaping microbiome structure, such as ocean temperature and host lifestyle. Regardless of biogeography, microbiomes were structured primarily by host-associated or environmental conditions, also reflected in genomic and functional traits inferred from 2,065,975 genomes. Generalists emerged as vehicles thriving and facilitating gene flow across ecologically disparate habitat types, illustrated by generalist-mediated horizontal transfer of an antibiotic resistance island across human gut and wastewater, further dispersing to environmental habitats, exemplifying human impact on the planetary microbiome.

The small intestinal epithelium represents the most rapidly self-renewing adult mammalian tissue, with a turnover time of 1-2 weeks. It contains ∼12 easily recognizable cell types with a wide diversity of functions, including nutrient absorption, mucus production, antimicrobial defense, and the regulation of metabolism by incretins like Glp1. The simple and repetitive crypt-villus architecture allows for easily interpretable experimentation in transgenic mice in vivo, while the human stem cell hierarchy is experimentally accessible in epithelial organoids in vitro. This review aims to comprehensively describe the design, the cellular constituents, and the molecular regulation of crypt-villus epithelial self-renewal. More generally, it highlights deviations from commonly held views on tissue stem cell biology: gut stem cells divide continually and symmetrically. They can be expanded indefinitely in vitro, while the plasticity of daughter cells can recreate stem cells during regeneration.

Human dermal sleeping nociceptors display ongoing activity in neuropathic pain, affecting 10% of the population. Despite advances in rodents, a molecular marker for these mechano-insensitive C-fibers (CMis) in human skin remains elusive, preventing targeted therapy. Using a Patch-seq approach, we combined single-cell transcriptomics, following electrophysiological characterization, with single-nucleus and spatial transcriptomics from pigs and integrated our findings with cross-species and human transcriptomic data. We functionally identified CMis in pig sensory neurons with patch clamp, using adapted protocols from human microneurography. We identified oncostatin M receptor (OSMR) and somatostatin (SST) as marker genes for CMis. Following dermal injection in healthy human volunteers, oncostatin M, the ligand of OSMR, exclusively modulates CMis. Our findings characterize the molecular architecture of human dermal sleeping nociceptors, providing a framework for mechanistic insight into neuropathic pain and potential therapeutic strategies.

Biomarkers of obsessive-compulsive disorder (OCD) symptom dynamics and related behavior could advance personalized interventions. Aberrant activity in the orbitofrontal cortex (OFC) has been implicated in symptom exacerbation in OCD. We conducted an intracranial monitoring assay to identify high-resolution neurophysiologic correlates of OCD symptoms in the human OFC. We found that low-gamma power in the anteromedial OFC was consistently elevated during high symptom states in a symptom provocation task. Furthermore, electrical stimulation of the ventral basal ganglia that reduced OCD symptoms also reduced anteromedial OFC gamma power. These results link OFC gamma activity to moment-to-moment expression of OCD symptoms, providing mechanistic insights to guide therapeutic strategies such as deep brain stimulation.

In Alzheimer's disease (AD), pathological tau protein shows a progressive accumulation of post-translational modifications (PTMs), reflecting disease severity, progression, and prion-like activity. Although many neurodegenerative diseases with dementia display tau aggregates, the pathological proteoforms of tau protein from each disease type remain unknown. Here, using a quantitative mass spectrometry-based proteomics platform, FLEXITau, deep characterization of pathological tau protein isolated from the brains of 203 human subjects with AD, familial AD (fAD), chronic traumatic encephalopathy (CTE), corticobasal degeneration (CBD), Pick's disease (PiD), progressive supranuclear palsy (PSP), dementia with Lewy bodies (DLB)-a non-tauopathy symptomatic control-and healthy controls (CTR) is performed. Unsupervised data analyses and supervised machine learning identify distinct molecular features of pathological tau for each disease, enabling molecular disease stratification. This study identifies potential disease-specific biomarkers and therapeutic targets for tauopathies and provides critical quantitative information for pharmacokinetic modeling required for therapeutic and disease mechanism studies.

Cancer presents a remarkably instructive perturbation of mechanisms manifesting in our biology that have gone awry, eliciting a malady that is inexorably increasing in incidence and societal burden concomitant with healthier aging. The wealth of knowledge and data forthcoming from decades of cancer research can be organized into conceptually distinct but interconnected parametric dimensions that define the mechanistic foundation of the disease: aberrantly acquired functional capabilities (the hallmarks of cancer), enabling phenotypic characteristics, hallmark-conveying cells populating cancer microenvironments, and systemic interactions. Collectively, they provide a logical framework with which to illuminate the operating systems of these outlaw organs, from inception through multistage tumorigenesis to adaptive evolution. This review presents a concise synthesis of the hallmark conceptualization as it has been refined during the past 25 years, including a corollary hypothesis that mechanism-guided hallmark co-targeting could offer impactful new therapeutic strategies for treating human cancers.

The ring-shaped sliding clamp proliferating cell nuclear antigen (PCNA) enables DNA polymerases to perform processive DNA synthesis during replication and repair. The loading of PCNA onto DNA is catalyzed by the ATPase clamp-loader replication factor C (RFC). Using a single-molecule platform to visualize the dynamic interplay between PCNA and RFC on DNA, we unexpectedly discovered that RFC continues to associate with PCNA after loading, contrary to the conventional view. Functionally, this clamp-loader/clamp (CLC) complex is required for processive DNA synthesis by polymerase ẟ (Polẟ), as the PCNA-Polẟ assembly is inherently unstable. This architectural role of RFC is dependent on the BRCA1 C-terminal homology (BRCT) domain of Rfc1, and mutation of its DNA-binding residues causes sensitivity to genotoxic stress in vivo. We further showed that flap endonuclease I (FEN1) can also stabilize the PCNA-Polẟ interaction and mediate robust synthesis. Overall, our work revealed that, beyond their canonical enzymatic functions, PCNA-binding proteins harbor non-catalytic functions important for DNA replication and genome maintenance.

Although ATP-independent chaperones assist RNA folding, the mechanisms by which they function remain elusive. Here, we demonstrate how two RNA chaperones collaborate to unfold misfolded noncoding RNAs (ncRNAs). The ring-shaped Ro60 protein binds the ends of misfolded ncRNAs in its cavity, whereas La stabilizes nascent ncRNAs and assists their folding. Using cryo-electron microscopy to resolve the structure of a misfolded RNA complexed with Ro60 and La, we show that La cradles the Ro60 ribonucleoprotein (RNP), with its N-terminal domain binding the RNA 3' end after it passes through the Ro60 cavity, while its C-terminal domain destabilizes structures in the misfolded RNA body. Using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP), we show that La and Ro60 function synergistically to unfold non-native structures. As the RNAs bound by Ro60 and La include both ncRNA precursors and ncRNAs with oligouridine tails, this RNA chaperone machine may function widely to recognize misfolded and otherwise aberrant ncRNAs and assist their unfolding.

Aggregation of the protein tau defines tauopathies, the most common age-related neurodegenerative diseases, which include Alzheimer's disease and frontotemporal dementia. Specific neuronal subtypes are selectively vulnerable to tau aggregation, dysfunction, and death. However, molecular mechanisms underlying cell-type-selective vulnerability are unknown. To systematically uncover the cellular factors controlling the accumulation of tau aggregates in human neurons, we conducted a genome-wide CRISPRi screen in induced pluripotent stem cell (iPSC)-derived neurons. The screen uncovered both known and unexpected pathways, including UFMylation and GPI anchor biosynthesis, which control tau oligomer levels. We discovered that the E3 ubiquitin ligase CRL5SOCS4 controls tau levels in human neurons, ubiquitinates tau, and is correlated with resilience to tauopathies in human disease. Disruption of mitochondrial function promotes proteasomal misprocessing of tau, generating disease-relevant tau proteolytic fragments and changing tau aggregation in vitro. These results systematically reveal principles of tau proteostasis in human neurons and suggest potential therapeutic targets for tauopathies.

Myocardial infarction (MI) triggers adverse cardiac events, immune responses, and nervous system activation, but the neural and neuroimmune mechanisms remain understudied. Using single-cell RNA sequencing (scRNA-seq) and tissue clearing, we identified transient receptor potential vanilloid-1 (TRPV1)-expressing vagal sensory neurons (VSNs) that increase ventricular innervation post MI. Ablating these VSNs mitigated MI pathology, reducing infarct size, abnormal electrocardiograms, cardiac dysfunction, sympathetic innervation, and pro-inflammatory cytokine interleukin 1β (IL-1β). Single-nuclei RNA-seq (snRNA-seq) and spatial transcriptomics revealed reduced border zone expansion in MI hearts following VSN ablation. Tracing the effects to the brain, we found that MI activated angiotensin II receptor type 1 (AT1aR)-expressing neurons in the paraventricular nucleus (PVN), whose inhibition mirrored benefits of TRPV1 VSN ablation. Additionally, the superior cervical ganglia (SCGs) exhibited intensified post-MI sympathetic innervation and IL-1β signaling. Blocking IL-1β in the SCG significantly reduced complications post MI. This study reveals a triple-node heart-brain loop underlying MI and potential therapeutic targets.

Many proteins localize in membraneless organelles. However, understanding the steps along membraneless organelle formation-and the structural impact on granule constituents-has been hindered by limited resolution of intracellular data. We address these challenges through in situ cryo-electron tomography (cryo-ET) along with formation of yeast proteasome storage granules (PSGs). During the transition from proliferation to quiescence, doubly capped 26S proteasomes arrested in an inactive state arrange into ∼7.5 MDa trimeric units, dispersed in the nucleoplasm and congregated along the nuclear envelope near the nuclear pore. 9-Å-resolution cryo-ET structures reveal that cytoplasmic PSGs formed in various energy-limiting conditions are paracrystalline arrays of bundled fibers, assembled from stacking of proteasome trimers. The paracrystalline arrangement maintains a pool of fully assembled inactive 26S proteasomes that are released in energy-rich conditions. Overall, our data reveal structural steps along the assembly of an intracellular membraneless organelle in situ and quinary structure formation controlling a major eukaryotic regulatory machine.

Only some non-muscle-invasive bladder cancer (NMIBC) patients benefit from intravesical Bacillus Calmette-Guérin (BCG), and predictive biomarkers remain lacking. While urine tumor DNA (utDNA) analysis is promising, mutations in tumor-adjacent normal urothelium, namely the field effect, limit specificity. Here, we show that the prevalence of somatic mutations in the urine increases with age. We introduce an improved utDNA minimal residual disease (MRD) approach that increases specificity by removing field-effect mutations. Applying this field-effect-informed MRD approach to 261 samples from NMIBC patients undergoing surgery and adjuvant BCG, we identify three molecular response classes, including surgical responders, BCG responders, and non-responders. Molecular predictors of response to the two treatments differ, with pre-existing immune activation and higher mutation burden enriched in BCG but not surgery responders. These findings highlight the potential of field-effect-informed liquid biopsy methods for guiding personalized therapy and uncovering biomarkers for individual components of multimodal treatments.