Science

Miniaturization, the reduction of adult body size to an extreme degree, has evolved repeatedly across vertebrates. Yet its genomic underpinnings remain poorly understood. Cypriniformes, the most species-rich order of freshwater fishes, contains multiple miniaturized lineages that have evolved contrasting developmental processes. Proportioned dwarfs are tiny-bodied but otherwise morphologically similar to larger relatives, while progenetic miniatures exhibit developmental truncation thus retaining larval-like anatomical features into adulthood. Using a new time-calibrated phylogeny of 309 cypriniform species and comparative genomic analyses of 33 high-quality genome assemblies, we investigated the evolutionary history and genomic correlates of miniaturization across this order. Ancestral state reconstruction revealed multiple independent origins of both miniature types, with transitions predominantly unidirectional and non-randomly distributed across the phylogeny. The origins of the two types of miniatures differed in their timing. Progenetic miniatures arose predominantly as early as the Eocene while proportioned dwarfs arose mainly within the Miocene period. Genome size variation across Cypriniformes has been overwhelmingly driven by polyploidy. However, progenetic miniatures but not proportioned dwarfs showed consistent genome size reduction. Comparative genomic analyses revealed that all three independently-evolved progenetic miniature lineages share convergent signatures of repeat loss alongside genome-wide intron shortening, patterns absent in proportioned dwarfs. Our study provides the broadest evidence to date that progenetic miniaturization, despite independent origins, is underpinned by predictable structural genomic changes, revealing a fundamental link between developmental truncation and genome architecture in vertebrates.

Reconstructing complete and accurate lineage trees remains a long-standing challenge in biology. Here, we introduce PALINCODE (Palindromic Coding and Decoding), a system that utilizes ternary CRISPR bits (cBits) to stochastically write one of three possible states over time, permanently embedding lineage relationships in the genome. We demonstrate PALINCODEs lineage-recording potential through simulations and establish palindromic CRISPR editing in cell culture models. We show that truncated Cas9 guide sequences yield ternary outcomes at high efficiency when compared to conventional guides. Using PALINCODE, we derived lineage-recording cell lines with a theoretical coding capacity of up to 10^25 bits, enabling the generation of lineage trees 32 cell divisions deep in single-cell sequencing of 293T cells. Furthermore, we applied PALINCODE using an in vivo melanoma model to jointly read out lineage history and gene expression, enabling in vivo reconstruction of clonal evolution within tumor cell clonal populations. PALINCODE circumvents several limitations of prior CRISPR-based systems while increasing the information potential at individual CRISPR sites, creating a lineage-recording platform with higher density than many competing approaches.

How conserved stress responses are across the plant kingdom remains poorly understood. Here, we present a kingdom-wide stress transcriptome atlas of 36 Viridiplantae species, from chlorophytes to angiosperms, across nine abiotic and biotic stresses. The atlas integrates reanalyzed public RNA-seq data with new in-house stress experiments on three species representing basal lineages, yielding 13.6 million differential expression calls from over 3,200 manually curated control-treatment comparisons. We find that ancient gene families respond broadly but moderately, while lineage-specific families respond narrowly but intensely, revealing a division of labor in stress gene deployment. Stress response conservation decays with phylogenetic distance yet remains detectable across more than 700 million years of divergence, with upregulated genes diverging faster than downregulated genes. Functional co-occurrence analysis uncovers a deeply conserved growth-defence tradeoff alongside stress-specific transcriptional rewiring. Conserved stress co-expression modules undergo regulatory subfunctionalization through duplication, with whole-genome duplicate pairs preferentially retained within modules. Finally, DNA and RNA foundation models predict stress responsiveness from sequence alone (auROC 0.755), suggesting a partially conserved cis-regulatory code underlying stress responses across the kingdom.

A large fraction of marine protists, particularly the smallest ones, belong to uncultured lineages that lack genomic data, limiting insights into their ecological roles and evolutionary strategies. Here, we generated 325 single-cell amplified genomes (SAGs) from 2-5 {micro}m planktonic protists, which resulted in 147 genomes from dominant marine species at varying levels of completeness (40 of them >50%). These genomes matched the in situ community, with Prymnesiophyceae, Mamiellophyceae and Chrysophyceae dominating pigmented cells and MAST, Choanoflagellata and Picozoa prevailing among heterotrophic colourless cells. This resource allowed us to describe the genomic architecture of marine protist species, and revealed a pronounced genome streamlining in ecologically successful lineages. Comparative analyses highlighted unique functions enriched in photosynthetic and heterotrophic taxa (including motility, signal transduction, digestion and secondary metabolism), and revealed a broad distribution of gene families with adaptive traits such as polyketide synthases and rhodopsins. This large-scale single-cell genomics dataset provides a mechanistic foundation for investigating functional diversity, ecological strategies and genome evolution in the ocean.

The Roman province of Dacia, located north of the Danube frontier, represented a key zone of cultural and demographic interaction during the Imperial period. However, the biological impact of Roman colonization in this region has not been characterized using genomic data. Here, we analyze genome-wide data from 34 individuals recovered from the Apulum-Dealul Furcilor necropolis, one of the largest funerary complexes in Roman Dacia. The genome-wide data reveal pronounced genetic heterogeneity within this population, reflecting its position at the intersection of Eastern Europe, the Mediterranean, and West Asia. Notably, we observe a sex-biased pattern of ancestry. Female individuals show stronger affinities to Eastern European, Steppe, and Caucasus-associated populations, suggesting the persistence of local or regionally connected genetic lineages. In contrast, male individuals display closer genetic relationships with Mediterranean and North African groups, including populations associated with Roman and Punic contexts, indicating male-mediated gene flow linked to long-distance mobility. These findings highlight the complex demographic processes shaping Roman frontier communities, where local and incoming populations were integrated through asymmetric social dynamics. Our results provide genomic evidence consistent with sex-biased admixture in Roman Dacia and underscore the role of frontier regions as hubs of genetic and cultural interaction within the Roman Empire.

Awareness of other individuals is a foundational element of social behavior. Here we examined how specific neural systems detect and signal the visual presence of conspecifics related to social arousal and motivation. We found that visual exposure to videos of other mice can activate hypothalamic oxytocin neurons and promote onset of pup retrieval behavior in naive virgin female mice. A range of social videos depicting conspecifics in diverse contexts, including but not limited to parental behavior, could accelerate onset of pup retrieval compared to non-social controls. Animals would elect to watch social videos over non-social videos. We made photo-tagged recordings from oxytocin neurons of the paraventricular nucleus (PVN), which were preferentially activated during social video viewing. Optogenetic silencing of PVN oxytocin neurons during exposure prevented this behavioral enhancement of pup retrieval onset. We also made photo-tagged recordings from a population of PVN-projecting neurons of the superficial superior colliculus (sSC[->]PVN units). Compared to other sSC neurons, the sSC[->]PVN neurons had specialized horizontal direction tuning with robust and sustained responses to social videos. sSC[->]PVN neurons differentiated visual scenes based on social content, responding most strongly to pup retrieval and less to scenes with increasing numbers of animals. Our results identify a subcortical visual pathway that signals the presence and salience of conspecifics to the oxytocin system, providing a circuit mechanism by which social visual awareness drives neuroendocrine arousal and the acquisition of parental behavior.

Selecting a diet containing all essential amino acids (EAAs) is critical for health. Following EAA deprivation, animals can select a nutritiously complete food source; however, the underlying mechanisms in vertebrates remain unclear. In mice, we show that leucine deficiency activates hypothalamic agouti-related protein (AgRP) neurons, which project to the paraventricular thalamus (PVT) via {gamma}-aminobutyric acid and are required for EAA deficiency-induced leucine appetite in mice. Furthermore, the peripheral tongue amino acid sensor general control nonderepressive-2 (GCN2) mediates acute EAA appetite via AgRP neurons. Together, these findings identify a tongue-AgRP-PVT circuit underlying EAA appetite, which is important for the rapid and accurate selection of essential nutrients.

Cis-regulatory elements (CREs) such as enhancers play a central role in orchestrating mammalian development, yet how they have gained, lost, maintained or changed function over the course of mammalian evolution remains poorly understood. To address this gap, we densely mapped the functional evolution of five mouse developmental enhancers by testing orthologous sequences from 480 extant and ancestrally reconstructed mammalian genomes (Zoonomia1, Cactus2) with massively parallel reporter assays (MPRAs). This phylogenetic dissection revealed diverse modes of evolution, from lineage-restricted activity to deep functional conservation despite extensive sequence divergence. To pinpoint causal changes, we developed a model-driven reconstitution strategy that uses deep learning-based predictions of chromatin accessibility to re-introduce a succession of mutations into ancestral orthologs; this revealed critical transcription factor binding site (TFBS) changes and pervasive context-dependent epistasis, including instances where mutational effects were strongly contingent on the order of their introduction. When we extended this strategy to tune the activity of extant orthologs, we found that ablation of enhancer function required as few as one to seven mutations, whereas enhancement was constrained by element-specific activity ceilings--a striking asymmetry in the predictability of model-guided enhancer editing. Together, these results shed light on how the plasticity of mammalian enhancers intersects with their evolution, and advance a framework for reprogramming the activity of endogenous CREs at nucleotide resolution.

Human Fc receptor-like 5 (FCRL5) is a low-affinity IgG Fc receptor expressed on various B cell subsets and a potential therapeutic target. We discovered that commonly used Fc-silencing mutations, designed to prevent interactions between the Fc{gamma} receptors on immune cells and the Fc domain of therapeutic IgG, do not prevent binding to FCRL5. As a result, unintended interactions between Fc-silent therapeutic IgG and human B cells may occur. We isolated a well-expressed variant of the Fc-binding portion of human FCRL5 by directed evolution and used structural modeling to guide the engineering of a human IgG1 Fc variant with approximately 100-fold higher affinity for FCRL5, enabling us to produce FCRL5:Fc complexes in solution. Native mass spectrometry, size exclusion chromatography, and the crystal structure of the FCRL5- IgG1 Fc complex solved at 3.4 [A] indicate that the two proteins bind in a 1:1 stoichiometry. Furthermore, the structure revealed that FCRL5 binds to IgG1 Fc in a manner completely distinct from that of previously characterized Fc-binding proteins, such as Fc{gamma} receptors, explaining why most Fc-silencing mutations do not disrupt FCRL5 binding. We demonstrate that selective cross-linking of FCRL5 with the B cell receptor (BCR) in cis, using Fc-engineered antibodies with either physiological or enhanced FCRL5 affinity, inhibits Ca2+ flux in FCRL5-expressing B cells. We compare this effect with the selective co-ligation of Fc{gamma}RIIb with the BCR. Our work demonstrates that FCRL5 interacts with human IgG Fc in a distinctive manner and that engagement of FCRL5 by Fc-silent therapeutic IgG could influence B cell function.

Since 2021, highly pathogenic avian influenza viruses (HPAIVs) belonging to H5N1 clade 2.3.4.4b have circulated widely in North American wild birds and repeatedly spilled over into mammals. In 2025, the first H5N1-associated deaths in humans were recorded in the Western hemisphere, raising questions about how the ongoing evolution of the virus in wild birds impacts spillover risk. Here, our analysis of 21,471 H5N1 genomes identified an evolutionary shift in mid-2024, driven by interhemispheric migration from Asia and reassortment with new antigens. The genotypes that dominated the early years of North Americas H5N1 epizootic traced their ancestry back to Europe, but Asia was the source of new "D1.1" genotype viruses that (a) spread faster, (b) have higher reassortment potential, (c) a broader host range, (d) repeatedly spill over to bovines, and (e) cause severe disease in humans, including non-farm workers.

The theory of island biogeography and its trophic extensions predict that both species richness and food-web complexity should increase with increasing ecosystem surface area. Accordingly, Species-Area Relationships (SARs) and Network-Area Relationships (NARs) are often observed to be positively-sloped, an observation that came to be considered as a law, and on which rest many area-based conservation plans for biodiversity. However, our mechanistic understanding of the driving mechanisms of SARs and NARs slopes remains limited, undermining our ability to predict how biodiversity will respond to habitat gain or loss. We show in 180 rural ponds sampled across five years that invasive alien predators reversed the SAR and NARs from positive in invader-free ponds, to negative in invaded ponds. Relationship reversal resulted from a higher prevalence of invasive alien predators driving magnified prey extinctions and simplified food webs in larger ponds. The ability of invasive alien predators to reverse SAR and NARs presumably reflected disproportionately high predation rates combined with a low sensitivity to prey extinction conferred by a wide trophic generalism. In a world where virtually all ecosystems face biological invasions, omnipresent invasive alien predators stress the pivotal role played by predation in shaping biocomplexity-area relationships, and highlight a growing need to preserve small ecosystems where invasive alien predators are less prevalent.

Protein folding is fast relative to mRNA translation and nascent polypeptides begin to fold during their synthesis on the ribosome. The resulting cotranslational folding intermediates are accessible to diverse molecular chaperones that recognise incompletely folded client proteins. Here, we sought to understand how specific chaperone:client complexes are established during protein synthesis, using the chaperone-dependent tumor suppressor protein p53 as a model. By capturing interactomes at different points during p53 synthesis, we show that Hsp70, Hsp90 and TRiC bind competitively to nascent p53. While Hsp70 and Hsp90 bind promiscuously, TRiC discriminates between subtly different partially-folded states. TRiC recruitment is dictated by local cotranslational folding and exposure of specific sequence motifs, and this is tuned by cancer-associated mutations in the p53 DNA-binding domain. Nascent chain interactions with the ribosome surface disfavor TRiC binding, demonstrating that the ribosome imposes unique constraints on chaperone recruitment during protein synthesis. Our results establish molecular principles underlying chaperone prioritization during protein biogenesis.

Successful chromosome segregation depends on robust kinetochore-microtubule attachments. The outer kinetochore load-bearers Ndc80 and Ska complexes functionally cooperate, but the molecular basis of their interaction remains elusive. Here, we combine cryo-EM and functional investigations of Ndc80:Ska on microtubules. Ndc80 forms longitudinal arrays along single protofilaments using two modules. The HEC1 N-terminal tail stabilizes interactions between microtubule-binding heads regulated by Aurora B. The HEC1 loop, away from microtubules, organizes Ndc80 coiled-coils into stacks matching the periodicity of tubulin subunits. SkaC binds to a previously unknown interface of Ndc80 as well as to microtubules, simultaneously stapling tubulin dimers longitudinally and neighboring protofilaments laterally. Our work demonstrates how several weak interactions of a small number of individual complexes are harnessed to generate a robust and regulated kinetochore coupler.

We have curated and annotated the topologic determinants for all human membrane proteins made at the endoplasmic reticulum (ER). This census of 4,863 proteins allowed us to systematically analyze the physical properties of their 20,546 TMDs and flanking soluble regions. Single-pass proteins house the majority of large exoplasmic and cytosolic domains, whereas multipass proteins overwhelmingly contain short loops and tails. All classes of transmembrane domains (TMDs) have positively charged cytosolic flanks, but negatively charged exoplasmic flanks feature primarily on TMDs inserted by Oxa1-family insertases. The TMD-pair, a topologic unit of two TMDs with a short exoplasmic loop, is the dominant building block of multipass proteins. TMD-pairs accommodate high-hydrophilicity and charge-containing TMDs crucial for multipass protein functions. We interpret these context-dependent TMD features in light of current mechanistic models for membrane protein biogenesis and function. Our findings have implications for the evolution of membrane proteomes and for engineering new membrane proteins.

Serological diagnostics for tuberculosis rely on pathogen-derived antigens to detect infection-specific antibodies. Whether chronic TB infection also reshapes the global topology of the antibody repertoire remains largely unexplored. Here we profile serum antibody binding across 6,936 peptides in 105 individuals from three countries using two complementary libraries: Mycobacterium tuberculosis peptides (TBC) and a resemblance-ranking library representing the human self-proteome (RRL). We construct a five-dimensional immune state vector from distributional binding properties and map individual sera into an immune phase space. A remodeling classifier achieves virtually identical performance on pathogen-derived and host-derived peptides (AUC 0.63-0.73), demonstrating that the diagnostic signal arises from global repertoire restructuring rather than antigen-specific recognition. HIV co-infection partially masks this signal; restricting analysis to HIV-negative individuals increases AUC to 0.73 (permutation p = 0.005) and enables detection of smear-negative TB (AUC = 0.83, specificity 0.95 with three peptides). Phase-space projections reveal that TB severity maps onto a continuous remodeling gradient, with smear-negative patients occupying intermediate positions between healthy controls and smear-positive cases. These findings position high-density peptide arrays as sensors of antibody repertoire topology, enabling detection of chronic immune states beyond antigen-specific recognition.

Nitric oxide (NO) signaling is pivotal in numerous physiological processes and is implicated in a spectrum of human diseases. Nitric oxide synthases (NOS) initiate NO signaling and govern its magnitude and duration, making them key drug targets. Despite decades of investigation, the structural mechanism by which NOS enzymes transfer electrons from NADPH to haem remains incompletely understood. Here, we report cryo-electron microscopy studies of the inducible NOS (iNOS) homodimer in complex with calmodulin captured under the catalytic turnover condition, resolving two important functional states: the electron input state and output state. In the input state, the FMN-binding subdomain (FMND) docks onto the FAD/NADPH-binding subdomain (FNR), positioning the FMN cofactor to accept electrons from FAD. The FMND then undergoes a large rotational movement to engage the oxygenase domain of the other protomer, adopting the output state, which enables electron transfer from FMN to the haem center via W366. This dynamic movement of the FMND shuttles electrons from the reductase domain to the oxygenase active site in iNOS. A point mutation (S594E) that disrupts the FMND-oxygenase interface markedly reduces catalytic activity of iNOS and traps the enzyme in a non-productive intermediate conformation. Together, these findings elucidate the structural mechanism of FMND-mediated electron transfer in the iNOS catalytic cycle.

AO_SCPLOWBSTRACTC_SCPLOWThe biology that governs progression and therapeutic response in autoimmune disease is organized in affected tissue, but direct molecular readout of that biology requires invasive biopsy and is rarely repeated during clinical trials or routine care. Using paired blood-skin single-cell RNA-sequencing from a systemic sclerosis (SSc) cohort of 74 individuals (57 patients and 17 matched controls, 192,809 cells across 53 annotated cell states), we show that peripheral blood carries a recoverable projection of tissue-resident molecular state. Across 63 pathways scored in both compartments, 43 same-pathway blood-skin associations reach FDR < 0.05; at cell-type resolution, 212 cross-compartment associations survive residualization for disease status and sex. Per-patient classifiers recover tissue-defined molecular states out of fold with AUCs between 0.62 and 0.79, with the strongest recoveries on fibroblast subtype programs that have no direct circulating analog: fibroblast COMP at 0.79, COCH at 0.75, MYOC2 at 0.74, POSTN at 0.74. Tissue programs route through different blood compartments at different representational levels: fibroblast programs resolve through T-cell, Treg, monocyte and B-cell axes at compositional and distributional levels, while interferon resolves through expression state across multiple cell types. Within SSc alone, a cross-validated partial least squares model learns a shared blood-skin latent axis at r = 0.486 (permutation p = 0.006); the induced patient ranking recovers tissue-interferon-high patients at 86% precision at the top-20% screening threshold against a 50% base rate. A paired multiview autoencoder, trained on module-level dependency structure under contrastive alignment, paired reconstruction, neighborhood preservation and tissue-target supervision, learns a shared latent geometry in which blood-only projections land in the same tissue-state region as their matched tissue samples and supports recovery of held-out tissue targets above simpler baselines and above two permutation null families. These results map the empirical geometry of cross-compartment inference in autoimmune disease and position peripheral blood as a substrate for tissue-state inference at trial and clinical scale.

The superior colliculus integrates retinal input to drive rapid, adaptive visual behavior, yet how the functional diversity of retinal ganglion cell types is represented in superior colliculus remains poorly understood. Using chronic two-photon calcium imaging of retinal ganglion cell axonal boutons in awake mice, we recorded over 200,000 boutons across superficial superior colliculus layers -- a scale that enabled systematic comparison with large-scale ex vivo retinal datasets. This revealed that the superior colliculus receives a near-complete sampling of retinal ganglion cell functional diversity. Functionally distinct response types were organized in systematic laminar gradients: not only response properties such as direction selectivity and contrast suppression, but retinal response types themselves varied systematically with depth. To probe how this organized input encodes natural scenes, we trained a "digital twin" deep network model on natural movie responses and validated its generalization to parametric stimuli, including cell type identification. Leveraging this model to generate predicted responses to looming stimuli, we identify a discrete subset of retinal response types tuned for collision detection at low angular thresholds -- a specialization embedded within a broader, non-specialized retinal population. The digital twin is made publicly available as a community resource. Together, these findings provide a comprehensive functional map of retinal drive to the superior colliculus and an in silico platform for linking retinal cell types to behaviorally relevant superior colliculus computations.

Survival during infection depends on both pathogen clearance and the ability to tolerate infection-induced physiological changes. Metabolic adaptations are a central component of this tolerance, but the mechanisms underlying these responses remain incompletely defined. Here, we identify white adipose tissue (WAT) lipolysis as a central regulator of metabolic tolerance to infection. In patients with sepsis, higher circulating non-esterified fatty acid (NEFA) levels were associated with reduced mortality. In mouse models of polymicrobial sepsis, infection induced robust adipose lipolysis and increased circulating NEFAs. Genetic ablation of adipose triglyceride lipase (ATGL) in adipose tissue impaired lipolysis, leading to hypothermia, bradycardia, and increased mortality without altering immune cell populations or pathogen burden, consistent with a defect in tolerance rather than resistance. Mechanistically, lipolysis-derived NEFAs, but not glycerol, were required for protection, as restoring circulating NEFAs rescued autonomic stability and survival in adipose tissue ATGL-deficient mice. Infection-induced lipolysis was redundantly regulated and did not depend on any single upstream signaling pathway. Both pharmacologic activation of lipolysis using a {beta}3-adrenergic agonist and exogenous fatty acid supplementation increased circulating NEFAs, improved survival, and promoted tolerance in mice. Consistent with these findings, analysis of real-world electronic health record data demonstrated that septic patients receiving FDA-approved {beta}3-adrenergic agonists had reduced mortality or hospice discharge in a propensity-matched cohort. Together, these results identify WAT lipolysis and circulating fatty acids as key mediators of tolerance to infection and support a therapeutic strategy based on repurposing clinically available {beta}3-adrenergic agonists to improve outcomes in sepsis. One Sentence SummaryWhite adipose tissue lipolysis promotes metabolic tolerance to infection through circulating fatty acids and is associated with improved survival in sepsis

Sensorimotor associative learning enables animals to adaptively link sensory cues with motor actions, a process that is critical for survival and everyday behavior. While Hebbian mechanisms explain associations formed through temporally overlapping neural activity, a fundamental challenge arises when sensory stimuli and motor responses are separated by a delay, because sensory and motor neurons are rarely coactive. Here, we identify the rostro-lateral posterior parietal cortex (PPC-rl) as a cortical hub that bridges tactile stimuli and temporally delayed licking actions during sensorimotor associative learning. Using cortex-wide calcium imaging with single-cell resolution to track [~]16,000 neurons simultaneously across sensory, motor, and association cortices, we find that PPC-rl uniquely exhibits sustained neural activity during the temporal delay early in learning, a signature that diminishes with expertise. Optogenetic silencing of this activity slows learning without impairing sensorimotor execution in expert mice. Learning strengthens the coupling of population dynamics within and between somatosensory and motor cortices. PPC-rl mediates this process by amplifying a low-dimensional communication subspace that synchronizes co-fluctuations across the somatosensory and motor cortices to facilitate linking. This PPC-rl dependent co-fluctuation dissolves post learning, underscoring PPC-rls role in bridging sensation to distal action. A biologically plausible network indicates that Hebbian plasticity with an eligibility trace gated by reward, PPC-rl persistent activity and PPC-rl dependent sensorimotor subspace communication synergize to support delayed association. Together, our findings uncover a PPC-rl based circuit mechanism that maintains temporal continuity to guide associative learning when sensory and motor events are separated in time.