The entry of coronaviruses is initiated by spike recognition of host cellular receptors, involving proteinaceous and/or glycan receptors. Recently, TMPRSS2 was identified as the proteinaceous receptor for HCoV-HKU1 alongside sialoglycan as a glycan receptor. However, the underlying mechanisms for viral entry remain unknown. Here, we investigated the HCoV-HKU1C spike in the inactive, glycan-activated, and functionally anchored states, revealing that sialoglycan binding induces a conformational change of the NTD and promotes the neighboring RBD of the spike to open for TMPRSS2 recognition, exhibiting a synergistic mechanism for the entry of HCoV-HKU1. The RBD of HCoV-HKU1 features an insertion subdomain that recognizes TMPRSS2 through three previously undiscovered interfaces. Furthermore, structural investigation of HCoV-HKU1A in combination with mutagenesis and binding assays confirms a conserved receptor recognition pattern adopted by HCoV-HKU1. These studies advance our understanding of the complex viral-host interactions during entry, laying the groundwork for developing new therapeutics against coronavirus-associated diseases.

The human coronavirus HKU1 spike (S) glycoprotein engages host cell surface sialoglycans and transmembrane protease serine 2 (TMPRSS2) to initiate infection. The molecular basis of HKU1 binding to TMPRSS2 and determinants of host receptor tropism remain elusive. We designed an active human TMPRSS2 construct enabling high-yield recombinant production in human cells of this key therapeutic target. We determined a cryo-electron microscopy structure of the HKU1 RBD bound to human TMPRSS2, providing a blueprint of the interactions supporting viral entry and explaining the specificity for TMPRSS2 among orthologous proteases. We identified TMPRSS2 orthologs from five mammalian orders promoting HKU1 S-mediated entry into cells along with key residues governing host receptor usage. Our data show that the TMPRSS2 binding motif is a site of vulnerability to neutralizing antibodies and suggest that HKU1 uses S conformational masking and glycan shielding to balance immune evasion and receptor engagement.

Dexamethasone is a life-saving treatment for severe COVID-19, yet its mechanism of action is unknown, and many patients deteriorate or die despite timely treatment initiation. Here, we identify dexamethasone treatment-induced cellular and molecular changes associated with improved survival in COVID-19 patients. We observed a reversal of transcriptional hallmark signatures in monocytes associated with severe COVID-19 and the induction of a monocyte substate characterized by the expression of glucocorticoid-response genes. These molecular responses to dexamethasone were detected in circulating and pulmonary monocytes, and they were directly linked to survival. Monocyte single-cell RNA sequencing (scRNA-seq)-derived signatures were enriched in whole blood transcriptomes of patients with fatal outcome in two independent cohorts, highlighting the potential for identifying non-responders refractory to dexamethasone. Our findings link the effects of dexamethasone to specific immunomodulation and reversal of monocyte dysregulation, and they highlight the potential of single-cell omics for monitoring in vivo target engagement of immunomodulatory drugs and for patient stratification for precision medicine approaches.

The human seasonal coronavirus HKU1-CoV, which causes common colds worldwide, relies on the sequential binding to surface glycans and transmembrane serine protease 2 (TMPRSS2) for entry into target cells. TMPRSS2 is synthesized as a zymogen that undergoes autolytic activation to process its substrates. Several respiratory viruses, in particular coronaviruses, use TMPRSS2 for proteolytic priming of their surface spike protein to drive membrane fusion upon receptor binding. We describe the crystal structure of the HKU1-CoV receptor binding domain in complex with TMPRSS2, showing that it recognizes residues lining the catalytic groove. Combined mutagenesis of interface residues and comparison across species highlight positions 417 and 469 as determinants of HKU1-CoV host tropism. The structure of a receptor-blocking nanobody in complex with zymogen or activated TMPRSS2 further provides the structural basis of TMPRSS2 activating conformational change, which alters loops recognized by HKU1-CoV and dramatically increases binding affinity.

The ability of mitochondria to coordinate stress responses across tissues is critical for health. In C. elegans, neurons experiencing mitochondrial stress elicit an inter-tissue signaling pathway through the release of mitokine signals, such as serotonin or the Wnt ligand EGL-20, which activate the mitochondrial unfolded protein response (UPRMT) in the periphery to promote organismal health and lifespan. We find that germline mitochondria play a surprising role in neuron-to-periphery UPRMT signaling. Specifically, we find that germline mitochondria signal downstream of neuronal mitokines, Wnt and serotonin, and upstream of lipid metabolic pathways in the periphery to regulate UPRMT activation. We also find that the germline tissue itself is essential for UPRMT signaling. We propose that the germline has a central signaling role in coordinating mitochondrial stress responses across tissues, and germline mitochondria play a defining role in this coordination because of their inherent roles in germline integrity and inter-tissue signaling.

Hypothalamic neural circuits regulate instinctive behaviors such as food seeking, the fight/flight response, socialization, and maternal care. Here, we identified microdeletions on chromosome Xq23 disrupting the brain-expressed transient receptor potential (TRP) channel 5 (TRPC5). This family of channels detects sensory stimuli and converts them into electrical signals interpretable by the brain. Male TRPC5 deletion carriers exhibited food seeking, obesity, anxiety, and autism, which were recapitulated in knockin male mice harboring a human loss-of-function TRPC5 mutation. Women carrying TRPC5 deletions had severe postpartum depression. As mothers, female knockin mice exhibited anhedonia and depression-like behavior with impaired care of offspring. Deletion of Trpc5 from oxytocin neurons in the hypothalamic paraventricular nucleus caused obesity in both sexes and postpartum depressive behavior in females, while Trpc5 overexpression in oxytocin neurons in knock-in mice reversed these phenotypes. We demonstrate that TRPC5 plays a pivotal role in mediating innate human behaviors fundamental to survival, including food seeking and maternal care.

A number of species have recently recovered from near-extinction. Although these species have avoided the immediate extinction threat, their long-term viability remains precarious due to the potential genetic consequences of population declines, which are poorly understood on a timescale beyond a few generations. Woolly mammoths (Mammuthus primigenius) became isolated on Wrangel Island around 10,000 years ago and persisted for over 200 generations before becoming extinct around 4,000 years ago. To study the evolutionary processes leading up to the mammoths' extinction, we analyzed 21 Siberian woolly mammoth genomes. Our results show that the population recovered quickly from a severe bottleneck and remained demographically stable during the ensuing six millennia. We find that mildly deleterious mutations gradually accumulated, whereas highly deleterious mutations were purged, suggesting ongoing inbreeding depression that lasted for hundreds of generations. The time-lag between demographic and genetic recovery has wide-ranging implications for conservation management of recently bottlenecked populations.

Cellular homeostasis is intricately influenced by stimuli from the microenvironment, including signaling molecules, metabolites, and pathogens. Functioning as a signaling hub within the cell, mitochondria integrate information from various intracellular compartments to regulate cellular signaling and metabolism. Multiple studies have shown that mitochondria may respond to various extracellular signaling events. However, it is less clear how changes in the extracellular matrix (ECM) can impact mitochondrial homeostasis to regulate animal physiology. We find that ECM remodeling alters mitochondrial homeostasis in an evolutionarily conserved manner. Mechanistically, ECM remodeling triggers a TGF-β response to induce mitochondrial fission and the unfolded protein response of the mitochondria (UPRMT). At the organismal level, ECM remodeling promotes defense of animals against pathogens through enhanced mitochondrial stress responses. We postulate that this ECM-mitochondria crosstalk represents an ancient immune pathway, which detects infection- or mechanical-stress-induced ECM damage, thereby initiating adaptive mitochondria-based immune and metabolic responses.

Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.

The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.

Interleukin (IL)-23 and IL-17 are well-validated therapeutic targets in autoinflammatory diseases. Antibodies targeting IL-23 and IL-17 have shown clinical efficacy but are limited by high costs, safety risks, lack of sustained efficacy, and poor patient convenience as they require parenteral administration. Here, we present designed miniproteins inhibiting IL-23R and IL-17 with antibody-like, low picomolar affinities at a fraction of the molecular size. The minibinders potently block cell signaling in vitro and are extremely stable, enabling oral administration and low-cost manufacturing. The orally administered IL-23R minibinder shows efficacy better than a clinical anti-IL-23 antibody in mouse colitis and has a favorable pharmacokinetics (PK) and biodistribution profile in rats. This work demonstrates that orally administered de novo-designed minibinders can reach a therapeutic target past the gut epithelial barrier. With high potency, gut stability, and straightforward manufacturability, de novo-designed minibinders are a promising modality for oral biologics.

Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.

Most mammalian genes have multiple polyA sites, representing a substantial source of transcript diversity regulated by the cleavage and polyadenylation (CPA) machinery. To better understand how these proteins govern polyA site choice, we introduce CPA-Perturb-seq, a multiplexed perturbation screen dataset of 42 CPA regulators with a 3' scRNA-seq readout that enables transcriptome-wide inference of polyA site usage. We develop a framework to detect perturbation-dependent changes in polyadenylation and characterize modules of co-regulated polyA sites. We find groups of intronic polyA sites regulated by distinct components of the nuclear RNA life cycle, including elongation, splicing, termination, and surveillance. We train and validate a deep neural network (APARENT-Perturb) for tandem polyA site usage, delineating a cis-regulatory code that predicts perturbation response and reveals interactions between regulatory complexes. Our work highlights the potential for multiplexed single-cell perturbation screens to further our understanding of post-transcriptional regulation.

Mammalian blastocyst formation involves the specification of the trophectoderm followed by the differentiation of the inner cell mass into embryonic epiblast and extra-embryonic primitive endoderm (PrE). During this time, the embryo maintains a window of plasticity and can redirect its cellular fate when challenged experimentally. In this context, we found that the PrE alone was sufficient to regenerate a complete blastocyst and continue post-implantation development. We identify an in vitro population similar to the early PrE in vivo that exhibits the same embryonic and extra-embryonic potency and can form complete stem cell-based embryo models, termed blastoids. Commitment in the PrE is suppressed by JAK/STAT signaling, collaborating with OCT4 and the sustained expression of a subset of pluripotency-related transcription factors that safeguard an enhancer landscape permissive for multi-lineage differentiation. Our observations support the notion that transcription factor persistence underlies plasticity in regulative development and highlight the importance of the PrE in perturbed development.

Spatial transcriptomics (ST) methods unlock molecular mechanisms underlying tissue development, homeostasis, or disease. However, there is a need for easy-to-use, high-resolution, cost-efficient, and 3D-scalable methods. Here, we report Open-ST, a sequencing-based, open-source experimental and computational resource to address these challenges and to study the molecular organization of tissues in 2D and 3D. In mouse brain, Open-ST captured transcripts at subcellular resolution and reconstructed cell types. In primary head-and-neck tumors and patient-matched healthy/metastatic lymph nodes, Open-ST captured the diversity of immune, stromal, and tumor populations in space, validated by imaging-based ST. Distinct cell states were organized around cell-cell communication hotspots in the tumor but not the metastasis. Strikingly, the 3D reconstruction and multimodal analysis of the metastatic lymph node revealed spatially contiguous structures not visible in 2D and potential biomarkers precisely at the 3D tumor/lymph node boundary. All protocols and software are available at https://rajewsky-lab.github.io/openst.

Fewer than 200 proteins are targeted by cancer drugs approved by the Food and Drug Administration (FDA). We integrate Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomics data from 1,043 patients across 10 cancer types with additional public datasets to identify potential therapeutic targets. Pan-cancer analysis of 2,863 druggable proteins reveals a wide abundance range and identifies biological factors that affect mRNA-protein correlation. Integration of proteomic data from tumors and genetic screen data from cell lines identifies protein overexpression- or hyperactivation-driven druggable dependencies, enabling accurate predictions of effective drug targets. Proteogenomic identification of synthetic lethality provides a strategy to target tumor suppressor gene loss. Combining proteogenomic analysis and MHC binding prediction prioritizes mutant KRAS peptides as promising public neoantigens. Computational identification of shared tumor-associated antigens followed by experimental confirmation nominates peptides as immunotherapy targets. These analyses, summarized at https://targets.linkedomics.org, form a comprehensive landscape of protein and peptide targets for companion diagnostics, drug repurposing, and therapy development.

In aging, physiologic networks decline in function at rates that differ between individuals, producing a wide distribution of lifespan. Though 70% of human lifespan variance remains unexplained by heritable factors, little is known about the intrinsic sources of physiologic heterogeneity in aging. To understand how complex physiologic networks generate lifespan variation, new methods are needed. Here, we present Asynch-seq, an approach that uses gene-expression heterogeneity within isogenic populations to study the processes generating lifespan variation. By collecting thousands of single-individual transcriptomes, we capture the Caenorhabditis elegans "pan-transcriptome"-a highly resolved atlas of non-genetic variation. We use our atlas to guide a large-scale perturbation screen that identifies the decoupling of total mRNA content between germline and soma as the largest source of physiologic heterogeneity in aging, driven by pleiotropic genes whose knockdown dramatically reduces lifespan variance. Our work demonstrates how systematic mapping of physiologic heterogeneity can be applied to reduce inter-individual disparities in aging.

Insufficient telomerase activity, stemming from low telomerase reverse transcriptase (TERT) gene transcription, contributes to telomere dysfunction and aging pathologies. Besides its traditional function in telomere synthesis, TERT acts as a transcriptional co-regulator of genes pivotal in aging and age-associated diseases. Here, we report the identification of a TERT activator compound (TAC) that upregulates TERT transcription via the MEK/ERK/AP-1 cascade. In primary human cells and naturally aged mice, TAC-induced elevation of TERT levels promotes telomere synthesis, blunts tissue aging hallmarks with reduced cellular senescence and inflammatory cytokines, and silences p16INK4a expression via upregulation of DNMT3B-mediated promoter hypermethylation. In the brain, TAC alleviates neuroinflammation, increases neurotrophic factors, stimulates adult neurogenesis, and preserves cognitive function without evident toxicity, including cancer risk. Together, these findings underscore TERT's critical role in aging processes and provide preclinical proof of concept for physiological TERT activation as a strategy to mitigate multiple aging hallmarks and associated pathologies.

Developmental biology-the study of the processes by which cells, tissues, and organisms develop and change over time-has entered a new golden age. After the molecular genetics revolution in the 80s and 90s and the diversification of the field in the early 21st century, we have entered a phase when powerful technologies provide new approaches and open unexplored avenues. Progress in the field has been accelerated by advances in genomics, imaging, engineering, and computational biology and by emerging model systems ranging from tardigrades to organoids. We summarize how revolutionary technologies have led to remarkable progress in understanding animal development. We describe how classic questions in gene regulation, pattern formation, morphogenesis, organogenesis, and stem cell biology are being revisited. We discuss the connections of development with evolution, self-organization, metabolism, time, and ecology. We speculate how developmental biology might evolve in an era of synthetic biology, artificial intelligence, and human engineering.

The gut microbiota influences the clinical responses of cancer patients to immunecheckpoint inhibitors (ICIs). However, there is no consensus definition of detrimental dysbiosis. Based on metagenomics (MG) sequencing of 245 non-small cell lung cancer (NSCLC) patient feces, we constructed species-level co-abundance networks that were clustered into species-interacting groups (SIGs) correlating with overall survival. Thirty-seven and forty-five MG species (MGSs) were associated with resistance (SIG1) and response (SIG2) to ICIs, respectively. When combined with the quantification of Akkermansia species, this procedure allowed a person-based calculation of a topological score (TOPOSCORE) that was validated in an additional 254 NSCLC patients and in 216 genitourinary cancer patients. Finally, this TOPOSCORE was translated into a 21-bacterial probe set-based qPCR scoring that was validated in a prospective cohort of NSCLC patients as well as in colorectal and melanoma patients. This approach could represent a dynamic diagnosis tool for intestinal dysbiosis to guide personalized microbiota-centered interventions.