Recent breakthroughs in the genetic manipulation of mitochondrial DNA (mtDNA) have enabled precise base substitutions and the efficient elimination of genomes carrying pathogenic mutations. However, reconstituting mtDNA deletions linked to mitochondrial myopathies remains challenging. Here, we engineered mtDNA deletions in human cells by co-expressing end-joining (EJ) machinery and targeted endonucleases. Using mitochondrial EJ (mito-EJ) and mito-ScaI, we generated a panel of clonal cell lines harboring a ∼3.5 kb mtDNA deletion across the full spectrum of heteroplasmy. Investigating these cells revealed a critical threshold of ∼75% deleted genomes, beyond which oxidative phosphorylation (OXPHOS) protein depletion, metabolic disruption, and impaired growth in galactose-containing media were observed. Single-cell multiomic profiling identified two distinct nuclear gene deregulation responses: one triggered at the deletion threshold and another progressively responding to heteroplasmy. Ultimately, we show that our method enables the modeling of disease-associated mtDNA deletions across cell types and could inform the development of targeted therapies.

Koala retrovirus-A (KoRV-A) is spreading through wild koalas in a north-to-south wave while transducing the germ line, modifying the inherited genome as it transitions to an endogenous retrovirus. Previously, we found that KoRV-A is expressed in the germ line, but unspliced genomic transcripts are processed into sense-strand PIWI-interacting RNAs (piRNAs), which may provide an initial "innate" form of post-transcriptional silencing. Here, we show that this initial post-transcriptional response is prevalent south of the Brisbane River, whereas KoRV-A expression is suppressed, promoters are methylated, and sense and antisense piRNAs are equally abundant in a subpopulation of animals north of the river. These animals share a KoRV-A provirus in the MAP4K4 gene's 3' UTR that is spreading through northern koalas and produces hybrid transcripts that are processed into antisense piRNAs, which guide transcriptional silencing. We speculate that this provirus triggers adaptive transcriptional silencing of KoRV-A and is sweeping to fixation.

Plants are composed of diverse secondary metabolites (PSMs), which are widely associated with human health. Whether and how the gut microbiome mediates such impacts of PSMs is poorly understood. Here, we show that discrete dietary and medicinal phenolic glycosides, abundant health-associated PSMs, are utilized by distinct members of the human gut microbiome. Within the Bacteroides, the predominant gram-negative bacteria of the Western human gut, we reveal a specialized multi-enzyme system dedicated to the processing of distinct glycosides based on structural differences in phenolic moieties. This Bacteroides metabolic system liberates chemically distinct aglycones with diverse biological functions, such as colonization resistance against the gut pathogen Clostridioides difficile via anti-microbial activation of polydatin to the stilbene resveratrol and intestinal homeostasis via activation of salicin to the immunoregulatory aglycone saligenin. Together, our results demonstrate generation of biological diversity of phenolic aglycone "effector" functions by a distinct gut-microbiome-encoded PSM-processing system.

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.

The amphipod Hirondellea gigas is a dominant species inhabiting the deepest part of the ocean (∼6,800-11,000 m), but little is known about its genetic adaptation and population dynamics. Here, we present a chromosome-level genome of H. gigas, characterized by a large genome size of 13.92 Gb. Whole-genome sequencing of 510 individuals from the Mariana Trench indicates no population differentiation across depths, suggesting its capacity to tolerate hydrostatic pressure across wide ranges. H. gigas in the West Philippine Basin is genetically divergent from the Mariana and Yap Trenches, suggesting genetic isolation attributed to the geographic separation of hadal features. A drastic reduction in effective population size potentially reflects glacial-interglacial changes. By integrating multi-omics analysis, we propose host-symbiotic microbial interactions may be crucial in the adaptation of H. gigas to the extremely high-pressure and food-limited environment. Our findings provide clues for adaptation to the hadal zone and population genetics.

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.

Here, we introduce the Mariana Trench Environment and Ecology Research (MEER) project, which provides the first systematic view of the ecosystem in the hadal zone.

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.

Bacterial immunotherapy holds promising cancer-fighting potential. However, unlocking its power requires a mechanistic understanding of how bacteria both evade antimicrobial immune defenses and stimulate anti-tumor immune responses within the tumor microenvironment (TME). Here, by harnessing an engineered Salmonella enterica strain with this dual proficiency, we unveil an underlying singular mechanism. Specifically, the hysteretic nonlinearity of interleukin-10 receptor (IL-10R) expression drives tumor-infiltrated immune cells into a tumor-specific IL-10Rhi state. Bacteria leverage this to enhance tumor-associated macrophages producing IL-10, evade phagocytosis by tumor-associated neutrophils, and coincidently expand and stimulate the preexisting exhausted tumor-resident CD8+ T cells. This effective combination eliminates tumors, prevents recurrence, and inhibits metastasis across multiple tumor types. Analysis of human samples suggests that the IL-10Rhi state might be a ubiquitous trait across human tumor types. Our study unveils the unsolved mechanism behind bacterial immunotherapy's dual challenge in solid tumors and provides a framework for intratumoral immunomodulation.

High-density probes allow electrophysiological recordings from many neurons simultaneously across entire brain circuits but fail to reveal cell type. Here, we develop a strategy to identify cell types from extracellular recordings in awake animals and reveal the computational roles of neurons with distinct functional, molecular, and anatomical properties. We combine optogenetics and pharmacology using the cerebellum as a testbed to generate a curated ground-truth library of electrophysiological properties for Purkinje cells, molecular layer interneurons, Golgi cells, and mossy fibers. We train a semi-supervised deep learning classifier that predicts cell types with greater than 95% accuracy based on the waveform, discharge statistics, and layer of the recorded neuron. The classifier's predictions agree with expert classification on recordings using different probes, in different laboratories, from functionally distinct cerebellar regions, and across species. Our classifier extends the power of modern dynamical systems analyses by revealing the unique contributions of simultaneously recorded cell types during behavior.

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.

NIH's abrupt decision to cap indirect cost reimbursement at 15% threatens the critical infrastructure supporting groundbreaking biomedical research in the United States. This policy jeopardizes America's global leadership in science and medicine. Urgent action is needed to advocate for its immediate and permanent reversal to protect the future of science.

Vitamin C (vitC) is essential for health and shows promise in treating diseases like cancer, yet its mechanisms remain elusive. Here, we report that vitC directly modifies lysine residues to form "vitcyl-lysine"-a process termed vitcylation. Vitcylation occurs in a dose-, pH-, and sequence-dependent manner in both cell-free systems and living cells. Mechanistically, vitC vitcylates signal transducer and activator of transcription-1 (STAT1)- lysine-298 (K298), impairing its interaction with T cell protein-tyrosine phosphatase (TCPTP) and preventing STAT1-Y701 dephosphorylation. This leads to enhanced STAT1-mediated interferon (IFN) signaling in tumor cells, increased major histocompatibility complex (MHC)/human leukocyte antigen (HLA) class I expression, and activation of anti-tumor immunity in vitro and in vivo. The discovery of vitcylation as a distinctive post-translational modification provides significant insights into vitC's cellular function and therapeutic potential, opening avenues for understanding its biological effects and applications in disease treatment.

The composition and organization of the cell surface determine how cells interact with their environment. Traditionally, glycosylated transmembrane proteins were thought to be the major constituents of the external surface of the plasma membrane. Here, we provide evidence that a group of RNA-binding proteins (RBPs) is present on the surface of living cells. These cell-surface RBPs (csRBPs) precisely organize into well-defined nanoclusters enriched for multiple RBPs and glycoRNAs, and their clustering can be disrupted by extracellular RNase addition. These glycoRNA-csRBP clusters further serve as sites of cell-surface interaction for the cell-penetrating peptide trans-activator of transcription (TAT). Removal of RNA from the cell surface, or loss of RNA-binding activity by TAT, causes defects in TAT cell internalization. Together, we provide evidence of an expanded view of the cell surface by positioning glycoRNA-csRBP clusters as a regulator of communication between cells and the extracellular environment.

Space habitation provides unique challenges in built environments isolated from Earth. We produced a 3D map of the microbes and metabolites throughout the United States Orbital Segment (USOS) of the International Space Station (ISS) with 803 samples collected during space flight, including controls. We find that the use of each of the nine sampled modules within the ISS strongly drives the microbiology and chemistry of the habitat. Relating the microbiology to other Earth habitats, we find that, as with human microbiota, built environment microbiota also align naturally along an axis of industrialization, with the ISS providing an extreme example of an industrialized environment. We demonstrate the utility of culture-independent sequencing for microbial risk monitoring, especially as the location of sequencing moves to space. The resulting resource of chemistry and microbiology in the space-built environment will guide long-term efforts to maintain human health in space for longer durations.