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

Meningeal lymphatics serve as an outlet for cerebrospinal fluid, and their dysfunction is associated with various neurodegenerative conditions. Previous studies have demonstrated that dysfunctional meningeal lymphatics evoke behavioral changes, but the neural mechanisms underlying these changes have remained elusive. Here, we show that prolonged impairment of meningeal lymphatics alters the balance of cortical excitatory and inhibitory synaptic inputs, accompanied by deficits in memory tasks. These synaptic and behavioral alterations induced by lymphatic dysfunction are mediated by microglia, leading to increased expression of the interleukin 6 gene (Il6). IL-6 drives inhibitory synapse phenotypes via a combination of trans- and classical IL-6 signaling. Restoring meningeal lymphatic function in aged mice reverses age-associated synaptic and behavioral alterations. Our findings suggest that dysfunctional meningeal lymphatics adversely impact cortical circuitry through an IL-6-dependent mechanism and identify a potential target for treating aging-associated cognitive decline.

Microtubules are a hallmark of eukaryotes. Archaeal and bacterial homologs of tubulins typically form homopolymers and non-tubular superstructures. The origin of heterodimeric tubulins assembling into microtubules remains unclear. Here, we report the discovery of microtubule-forming tubulins in Asgard archaea, the closest known relatives of eukaryotes. These Asgard tubulins (AtubA/B) are closely related to eukaryotic α/β-tubulins and the enigmatic bacterial tubulins BtubA/B. Proteomics of Candidatus Lokiarchaeum ossiferum showed that AtubA/B were highly expressed. Cryoelectron microscopy structures demonstrate that AtubA/B form eukaryote-like heterodimers, which assembled into 5-protofilament bona fide microtubules in vitro. The additional paralog AtubB2 lacks a nucleotide-binding site and competitively displaced AtubB. These AtubA/B2 heterodimers polymerized into 7-protofilament non-canonical microtubules. In a sub-population of Ca. Lokiarchaeum ossiferum cells, cryo-tomography revealed tubular structures, while expansion microscopy identified AtubA/B cytoskeletal assemblies. Our findings suggest a pre-eukaryotic origin of microtubules and provide a framework for understanding the fundamental principles of microtubule assembly.

The organs of many female animals are remodeled by reproduction. Using the mouse intestine, a striking and tractable model of organ resizing, we find that reproductive remodeling is anticipatory and distinct from diet- or microbiota-induced resizing. Reproductive remodeling involves partially irreversible elongation of the small intestine and fully reversible growth of its epithelial villi, associated with an expansion of isthmus progenitors and accelerated enterocyte migration. We identify induction of the SGLT3a transporter in a subset of enterocytes as an early reproductive hallmark. Electrophysiological and genetic interrogations indicate that SGLT3a does not sustain digestive functions or enterocyte health; rather, it detects protons and sodium to extrinsically support the expansion of adjacent Fgfbp1-positive isthmus progenitors, promoting villus growth. Our findings reveal unanticipated specificity to physiological organ remodeling. We suggest that organ- and state-specific growth programs could be leveraged to improve pregnancy outcomes or prevent maladaptive consequences of such growth.

The relationships of human diversity with biomedical phenotypes are pervasive yet remain understudied, particularly in a single-cell genomics context. Here, we present the Asian Immune Diversity Atlas (AIDA), a multi-national single-cell RNA sequencing (scRNA-seq) healthy reference atlas of human immune cells. AIDA comprises 1,265,624 circulating immune cells from 619 donors, spanning 7 population groups across 5 Asian countries, and 6 controls. Though population groups are frequently compared at the continental level, we found that sub-continental diversity, age, and sex pervasively impacted cellular and molecular properties of immune cells. These included differential abundance of cell neighborhoods as well as cell populations and genes relevant to disease risk, pathogenesis, and diagnostics. We discovered functional genetic variants influencing cell-type-specific gene expression, which were under-represented in non-Asian populations, and helped contextualize disease-associated variants. AIDA enables analyses of multi-ancestry disease datasets and facilitates the development of precision medicine efforts in Asia and beyond.

Jumbo bacteriophages of the ϕKZ-like family assemble a lipid-based early phage infection (EPI) vesicle and a proteinaceous nucleus-like structure during infection. These structures protect the phage from nucleases and may create selective pressure for immunity mechanisms targeting this specific phage family. Here, we identify "jumbo phage killer" (Juk), a two-component immune system that terminates infection of ϕKZ-like phages, suppressing the expression of early phage genes and preventing phage DNA replication and phage nucleus assembly while saving the cell. JukA (formerly YaaW) rapidly senses the EPI vesicle by binding to an early-expressed phage protein, gp241, and then directly recruits JukB. The JukB effector structurally resembles a pore-forming toxin and destabilizes the EPI vesicle. Functional anti-ϕKZ JukA homologs are found across bacterial phyla, associated with diverse effectors. These findings reveal a widespread defense system that specifically targets early events executed by ϕKZ-like jumbo phages prior to phage nucleus assembly.

Insulin gene (INS) variation and beta-cell stress are associated with the risk of development of type 1 diabetes (T1D) and autoimmunity against insulin. The unfolded protein response alleviating endoplasmic reticulum (ER) stress involves activation of inositol-requiring enzyme 1α (IRE1α) that impedes translation by mRNA decay. We discover that the IRE1α digestion motif is present in insulin mRNA carrying SNP rs3842752 (G>A). This SNP in the 3' untranslated region of INS associates with protection from T1D (INSP). ER stress in beta cells with INSP led to accelerated insulin mRNA decay compared with the susceptible INS variant (INSS). Human islets with INSP showed improved vitality and function and reversed diabetes more rapidly when transplanted into diabetic mice than islets carrying INSS only. Surrogate beta cells with INSP expressed less ER stress and INS-DRiP neoantigen. This explanation for genetic protection from T1D may act instead of or in concert with the previously proposed mechanism attributed to INS promoter polymorphism.

Lymphocyte activation gene 3 (LAG3) has emerged as a promising cancer immunotherapy target, but the mechanism underlying LAG3 activation upon ligand engagement remains elusive. Here, LAG3 was found to undergo robust non-K48-linked polyubiquitination upon ligand engagement, which promotes LAG3's inhibitory function instead of causing degradation. This ubiquitination could be triggered by the engagement of major histocompatibility complex class II (MHC class II) and membrane-bound (but not soluble) fibrinogen-like protein 1 (FGL1). LAG3 ubiquitination, mediated redundantly by the E3 ligases c-Cbl and Cbl-b, disrupted the membrane binding of the juxtamembrane basic residue-rich sequence, thereby stabilizing the LAG3 cytoplasmic tail in a membrane-dissociated conformation enabling signaling. Furthermore, LAG3 ubiquitination is crucial for the LAG3-mediated suppression of antitumor immunity in vivo. Consistently, LAG3 therapeutic antibodies repress LAG3 ubiquitination, correlating with their checkpoint blockade effects. Moreover, patient cohort analyses suggest that LAG3/CBL coexpression could serve as a biomarker for response to LAG3 blockade. Collectively, our study reveals an immune-checkpoint-triggering mechanism with translational potential in cancer immunotherapy.

Pooled optical screens have enabled the study of cellular interactions, morphology, or dynamics at massive scale, but they have not yet leveraged the power of highly plexed single-cell resolved transcriptomic readouts to inform molecular pathways. Here, we present a combination of imaging spatial transcriptomics with parallel optical detection of in situ amplified guide RNAs (Perturb-FISH). Perturb-FISH recovers intracellular effects that are consistent with single-cell RNA-sequencing-based readouts of perturbation effects (Perturb-seq) in a screen of lipopolysaccharide response in cultured monocytes, and it uncovers intercellular and density-dependent regulation of the innate immune response. Similarly, in three-dimensional xenograft models, Perturb-FISH identifies tumor-immune interactions altered by genetic knockout. When paired with a functional readout in a separate screen of autism spectrum disorder risk genes in human-induced pluripotent stem cell (hIPSC) astrocytes, Perturb-FISH shows common calcium activity phenotypes and their associated genetic interactions and dysregulated molecular pathways. Perturb-FISH is thus a general method for studying the genetic and molecular associations of spatial and functional biology at single-cell resolution.

Reference genomes of root microbes are essential for metagenomic analyses and mechanistic studies of crop root microbiomes. By combining high-throughput bacterial cultivation with metagenomic sequencing, we constructed comprehensive bacterial and viral genome collections from the roots of wheat, rice, maize, and Medicago. The crop root bacterial genome collection (CRBC) significantly expands the quantity and phylogenetic diversity of publicly available crop root bacterial genomes, with 6,699 bacterial genomes (68.9% from isolates) and 1,817 undefined species, expanding crop root bacterial diversity by 290.6%. The crop root viral genome collection (CRVC) contains 9,736 non-redundant viral genomes, with 1,572 previously unreported genus-level clusters in crop root microbiomes. From these, we identified conserved bacterial functions enriched in root microbiomes across soils and host species and uncovered previously unexplored bacteria-virus connections in crop root ecosystems. Together, the CRBC and CRVC serve as valuable resources for investigating microbial mechanisms and applications, supporting sustainable agriculture.

Brassinosteroid hormones are positive regulators of plant organ growth, yet their function in proliferating tissues remains unclear. Here, through integrating single-cell RNA sequencing with long-term live-cell imaging of the Arabidopsis root, we reveal that brassinosteroid activity fluctuates throughout the cell cycle, decreasing during mitotic divisions and increasing during the G1 phase. The post-mitotic recovery of brassinosteroid activity is driven by the intrinsic polarity of the mother cell, resulting in one daughter cell with enhanced brassinosteroid signaling, while the other supports brassinosteroid biosynthesis. The coexistence of these distinct daughter cell states during the G1 phase circumvents a negative feedback loop to facilitate brassinosteroid production while signaling increases. Our findings uncover polarity-guided, uneven mitotic divisions in the meristem, which control brassinosteroid hormone activity to ensure optimal root growth.

The deep sea, especially hadal zones, characterized by high-hydrostatic pressure, low temperatures, and near-total darkness, present some of the most challenging environments for life on Earth. However, teleost fish have successfully colonized these extreme habitats through complex adaptations. We generated genome assemblies of 12 species, including 11 deep-sea fishes. Our findings reconstructed the teleost deep-sea colonization history and revealed the overall impact of the deep-sea environment on fishes. Interestingly, our results question the previously assumed linear correlation between trimethylamine oxide (TMAO) content and depth. By contrast, we observed a convergent aa replacement in the rtf1 gene in most deep-sea fishes under 3,000 m, and in vitro experiments suggest that this mutation can influence transcriptional efficiency, which is likely to be advantageous in the deep-sea environment. Moreover, our study underlines the pervasive impact of human activities, as we detected the presence of persistent organic pollutants in species from the Mariana Trench.

Systematic exploration of the hadal zone, Earth's deepest oceanic realm, has historically faced technical limitations. Here, we collected 1,648 sediment samples at 6-11 km in the Mariana Trench, Yap Trench, and Philippine Basin for the Mariana Trench Environment and Ecology Research (MEER) project. Metagenomic and 16S rRNA gene amplicon sequencing generated the 92-Tbp MEER dataset, comprising 7,564 species (89.4% unreported), indicating high taxonomic novelty. Unlike in reported environments, neutral drift played a minimal role, while homogeneous selection (HoS, 50.5%) and dispersal limitation (DL, 43.8%) emerged as dominant ecological drivers. HoS favored streamlined genomes with key functions for hadal adaptation, e.g., aromatic compound utilization (oligotrophic adaptation) and antioxidation (high-pressure adaptation). Conversely, DL promoted versatile metabolism with larger genomes. These findings indicated that environmental factors drive the high taxonomic novelty in the hadal zone, advancing our understanding of the ecological mechanisms governing microbial ecosystems in such an extreme oceanic environment.

The nervous system needs to balance the stability of neural representations with plasticity. It is unclear what the representational stability of simple well-rehearsed actions is, particularly in humans, and their adaptability to new contexts. Using an electrocorticography brain-computer interface (BCI) in tetraplegic participants, we found that the low-dimensional manifold and relative representational distances for a repertoire of simple imagined movements were remarkably stable. The manifold's absolute location, however, demonstrated constrained day-to-day drift. Strikingly, neural statistics, especially variance, could be flexibly regulated to increase representational distances during BCI control without somatotopic changes. Discernability strengthened with practice and was BCI-specific, demonstrating contextual specificity. Sampling representational plasticity and drift across days subsequently uncovered a meta-representational structure with generalizable decision boundaries for the repertoire; this allowed long-term neuroprosthetic control of a robotic arm and hand for reaching and grasping. Our study offers insights into mesoscale representational statistics that also enable long-term complex neuroprosthetic control.

Microbiome research has expanded significantly in the last two decades, yet translating findings into clinical applications remains challenging. This perspective discusses the persistent issue of correlational studies in microbiome research and proposes an iterative method leveraging in silico, in vitro, ex vivo, and in vivo studies toward successful preclinical and clinical trials. The evolution of research methodologies, including the shift from small cohort studies to large-scale, multi-cohort, and even "meta-cohort" analyses, has been facilitated by advancements in sequencing technologies, providing researchers with tools to examine multiple health phenotypes within a single study. The integration of multi-omics approaches-such as metagenomics, metatranscriptomics, metaproteomics, and metabolomics-provides a comprehensive understanding of host-microbe interactions and serves as a robust hypothesis generator for downstream in vitro and in vivo research. These hypotheses must then be rigorously tested, first with proof-of-concept experiments to clarify the causative effects of the microbiota, and then with the goal of deep mechanistic understanding. Only following these two phases can preclinical studies be conducted with the goal of translation into the clinic. We highlight the importance of combining traditional microbiological techniques with big-data approaches, underscoring the necessity of iterative experiments in diverse model systems to enhance the translational potential of microbiome research.

Long terminal repeat (LTR) retrotransposons belong to the transposable elements (TEs), autonomously replicating genetic elements that integrate into the host's genome. Among animals, Drosophila melanogaster serves as an important model organism for TE research and contains several LTR retrotransposons, including the Ty1-copia family, which is evolutionarily related to retroviruses and forms virus-like particles (VLPs). In this study, we use cryo-focused ion beam (FIB) milling and lift-out approaches to visualize copia VLPs in ovarian cells and intact egg chambers, resolving the in situ copia capsid structure to 7.7 Å resolution by cryoelectron tomography (cryo-ET). Although cytoplasmic copia VLPs vary in size, nuclear VLPs are homogeneous and form densely packed clusters, supporting a model in which nuclear import acts as a size selector. Analyzing flies deficient in the TE-suppressing PIWI-interacting RNA (piRNA) pathway, we observe copia's translocation into the nucleus during spermatogenesis. Our findings provide insights into the replication cycle and cellular structural biology of an active LTR retrotransposon.

Coronary arteries have a specific branching pattern crucial for oxygenating heart muscle. Among humans, there is natural variation in coronary anatomy with respect to perfusion of the inferior/posterior left heart, which can branch from either the right arterial tree, the left, or both-a phenotype known as coronary dominance. Using angiographic data for >60,000 US veterans of diverse ancestry, we conducted a genome-wide association study of coronary dominance, revealing moderate heritability and identifying ten significant loci. The strongest association occurred near CXCL12 in both European- and African-ancestry cohorts, with downstream analyses implicating effects on CXCL12 expression. We show that CXCL12 is expressed in human fetal hearts at the time dominance is established. Reducing Cxcl12 in mice altered coronary dominance and caused septal arteries to develop away from Cxcl12 expression domains. These findings indicate that CXCL12 patterns human coronary arteries, paving the way for "medical revascularization" through targeting developmental pathways.

Social interaction is integral to animal behavior. However, lacking tools to describe it in quantitative and rigorous ways has limited our understanding of its structure, underlying principles, and the neuropsychiatric disorders, like autism, that perturb it. Here, we present a technique for high-resolution 3D tracking of postural dynamics and social touch in freely interacting animals, solving the challenging subject occlusion and part-assignment problems using 3D geometric reasoning, graph neural networks, and semi-supervised learning. We collected over 110 million 3D pose samples in interacting rats and mice, including seven monogenic autism rat lines. Using a multi-scale embedding approach, we identified a rich landscape of stereotyped actions, interactions, synchrony, and body contacts. This high-resolution phenotyping revealed a spectrum of changes in autism models and in response to amphetamine not resolved by conventional measurements. Our framework and large library of interactions will facilitate studies of social behaviors and their neurobiological underpinnings.

Clear cell renal cell carcinoma (ccRCC), despite having a low mutational burden, is considered immunogenic because it occasionally undergoes spontaneous regressions and often responds to immunotherapies. The signature lesion in ccRCC is inactivation of the VHL tumor suppressor gene and consequent upregulation of the HIF transcription factor. An earlier case report described a ccRCC patient who was cured by an allogeneic stem cell transplant and later found to have donor-derived T cells that recognized a ccRCC-specific peptide encoded by a HIF-responsive endogenous retrovirus (ERV), ERVE-4. We report that ERVE-4 is one of many ERVs that are induced by HIF, translated into HLA-bound peptides in ccRCCs, and capable of generating antigen-specific T cell responses. Moreover, ERV expression can be induced in non-ccRCC tumors with clinical-grade HIF stabilizers. These findings have implications for leveraging ERVs for cancer immunotherapy.