Cellular homeostasis requires the robust control of biomolecule concentrations, but how do millions of mRNAs coordinate their stoichiometries in the face of dynamic translational changes? Here, we identified a two-tiered mechanism controlling mRNA:mRNA and mRNA:protein stoichiometries where mRNAs super-assemble into condensates with buffering capacity and sorting selectivity through phase-transition mechanisms. Using C. elegans oogenesis arrest as a model, we investigated the transcriptome cytosolic reorganization through the sequencing of RNA super-assemblies coupled with single mRNA imaging. Tightly repressed mRNAs self-assembled into same-sequence nanoclusters that further co-assembled into multiphase condensates. mRNA self-sorting was concentration dependent, providing a self-buffering mechanism that is selective to sequence identity and controls mRNA:mRNA stoichiometries. The cooperative sharing of limiting translation repressors between clustered mRNAs prevented the disruption of mRNA:repressor stoichiometries in the cytosol. Robust control of mRNA:mRNA and mRNA:protein stoichiometries emerges from mRNA self-demixing and cooperative super-assembly into multiphase multiscale condensates with dynamic storage capacity.

Endometrial decidualization connecting embryo implantation and placentation is transient but essential for successful pregnancy, which, however, is not systematically investigated. Here, we use a scStereo-seq technology to spatially visualize and define the dynamic functional decidual hubs assembled by distinct immune, endothelial, trophoblast, and decidual stromal cells (DSCs) in early pregnant mice. We unravel the DSC transdifferentiation trajectory and surprisingly discover a dual-featured type of immune-featured DSCs (iDSCs). We find that immature DSCs attract immune cells and induce decidual angiogenesis at the mesenchymal-epithelial transition hub during decidualization initiation. iDSCs enable immune cell recruitment and suppression, govern vascularization, and promote cytolysis at immune cell assembling and vascular hubs, respectively, to establish decidual homeostasis at a later stage. Interestingly, dysfunctional and spatially disordered iDSCs cause abnormal accumulation of immune cells in the vascular hub, which disrupts decidual hub specification and eventually leads to pregnancy complications in DBA/2-mated CBA/J mice.

Macrophages are heterogeneous and play critical roles in development and disease, but their diversity, function, and specification remain inadequately understood during human development. We generated a single-cell RNA sequencing map of the dynamics of human macrophage specification from PCW 4-26 across 19 tissues. We identified a microglia-like population and a proangiogenic population in 15 macrophage subtypes. Microglia-like cells, molecularly and morphologically similar to microglia in the CNS, are present in the fetal epidermis, testicle, and heart. They are the major immune population in the early epidermis, exhibit a polarized distribution along the dorsal-lateral-ventral axis, and interact with neural crest cells, modulating their differentiation along the melanocyte lineage. Through spatial and differentiation trajectory analysis, we also showed that proangiogenic macrophages are perivascular across fetal organs and likely yolk-sac-derived as microglia. Our study provides a comprehensive map of the heterogeneity and developmental dynamics of human macrophages and unravels their diverse functions during development.

Natural killer (NK) cells play indispensable roles in innate immune responses against tumor progression. To depict their phenotypic and functional diversities in the tumor microenvironment, we perform integrative single-cell RNA sequencing analyses on NK cells from 716 patients with cancer, covering 24 cancer types. We observed heterogeneity in NK cell composition in a tumor-type-specific manner. Notably, we have identified a group of tumor-associated NK cells that are enriched in tumors, show impaired anti-tumor functions, and are associated with unfavorable prognosis and resistance to immunotherapy. Specific myeloid cell subpopulations, in particular LAMP3+ dendritic cells, appear to mediate the regulation of NK cell anti-tumor immunity. Our study provides insights into NK-cell-based cancer immunity and highlights potential clinical utilities of NK cell subsets as therapeutic targets.

Menopause is the cessation of ovarian function, with loss of reproductive hormone production and irreversible loss of fertility. It is a natural part of reproductive aging. The physiology of the menopause is complex and incompletely understood. Globally, menopause occurs around the age of 49 years, with geographic and ethnic variation. The hormonal changes of the menopause transition may result in both symptoms and long-term systemic effects, predominantly adverse effects on cardiometabolic and musculoskeletal health. The most effective treatment for bothersome menopausal symptoms is evidence-based, menopausal hormone therapy (MHT), which reduces bone loss and may have cardiometabolic benefits. Evidence-based non-hormonal interventions are also available for symptom relief. Treatment should be individualized with shared decision-making. Most MHT regimens are not regulator approved for perimenopausal women. Studies that include perimenopausal women are needed to determine the efficacy and safety of treatment options. Further research is crucial to improve menopause care, along with research to guide policy and clinical practice.

Aging is the key risk factor for cognitive decline, yet the molecular changes underlying brain aging remain poorly understood. Here, we conducted spatiotemporal RNA sequencing of the mouse brain, profiling 1,076 samples from 15 regions across 7 ages and 2 rejuvenation interventions. Our analysis identified a brain-wide gene signature of aging in glial cells, which exhibited spatially defined changes in magnitude. By integrating spatial and single-nucleus transcriptomics, we found that glial aging was particularly accelerated in white matter compared with cortical regions, whereas specialized neuronal populations showed region-specific expression changes. Rejuvenation interventions, including young plasma injection and dietary restriction, exhibited distinct effects on gene expression in specific brain regions. Furthermore, we discovered differential gene expression patterns associated with three human neurodegenerative diseases, highlighting the importance of regional aging as a potential modulator of disease. Our findings identify molecular foci of brain aging, providing a foundation to target age-related cognitive decline.

Tn7-like transposons have co-opted CRISPR-Cas systems to facilitate the movement of their own DNA. These CRISPR-associated transposons (CASTs) are promising tools for programmable gene knockin. A key feature of CASTs is their ability to recruit Tn7-like transposons to nuclease-deficient CRISPR effectors. However, how Tn7-like transposons are recruited by diverse CRISPR effectors remains poorly understood. Here, we present the cryo-EM structure of a recruitment complex comprising the Cascade complex, TniQ, TnsC, and the target DNA in the type I-B CAST from Peltigera membranacea cyanobiont 210A. Target DNA recognition by Cascade induces conformational changes in Cas6 and primes TniQ recruitment through its C-terminal domain. The N-terminal domain of TniQ is bound to the seam region of the TnsC spiral heptamer. Our findings provide insights into the diverse mechanisms for the recruitment of Tn7-like transposons to CRISPR effectors and will aid in the development of CASTs as gene knockin tools.

Selective clearance of organelles, including endoplasmic reticulum (ER) and mitochondria, by autophagy plays an important role in cell health. Here, we describe a developmentally programmed selective ER clearance by autophagy. We show that Parkinson's disease-associated PINK1, as well as Atl, Rtnl1, and Trp1 receptors, regulate ER clearance by autophagy. The E3 ubiquitin ligase Parkin functions downstream of PINK1 and is required for mitochondrial clearance while having the opposite function in ER clearance. By contrast, Keap1 and the E3 ubiquitin ligase Cullin3 function downstream of PINK1 to regulate ER clearance by influencing Rtnl1 and Atl. PINK1 regulates a change in Keap1 localization and Keap1-dependent ubiquitylation of the ER-phagy receptor Rtnl1 to facilitate ER clearance. Thus, PINK1 regulates the selective clearance of ER and mitochondria by influencing the balance of Keap1- and Parkin-dependent ubiquitylation of substrates that determine which organelle is removed by autophagy.

Social preference, the decision to interact with one member of the same species over another, is critical to optimize social interactions. Thus, adult rodents favor interacting with novel conspecifics over familiar ones, but whether this social preference stems from neural circuits facilitating interactions with novel individuals or suppressing interactions with familiar ones remains unknown. Here, we identify neurons in the infra-limbic area (ILA) of the mouse prefrontal cortex that express the neuropeptide corticotropin-releasing hormone (CRH) and project to the dorsal region of the rostral lateral septum (rLS). We show how release of CRH during familiar encounters disinhibits rLS neurons, thereby suppressing social interactions with familiar mice and contributing to social novelty preference. We further demonstrate how the maturation of CRH expression in ILA during the first 2 post-natal weeks enables the developmental shift from a preference for littermates in juveniles to a preference for novel mice in adults.

Changes in an animal's behavior and internal state are accompanied by widespread changes in activity across its brain. However, how neurons across the brain encode behavior and how this is impacted by state is poorly understood. We recorded brain-wide activity and the diverse motor programs of freely moving C. elegans and built probabilistic models that explain how each neuron encodes quantitative behavioral features. By determining the identities of the recorded neurons, we created an atlas of how the defined neuron classes in the C. elegans connectome encode behavior. Many neuron classes have conjunctive representations of multiple behaviors. Moreover, although many neurons encode current motor actions, others integrate recent actions. Changes in behavioral state are accompanied by widespread changes in how neurons encode behavior, and we identify these flexible nodes in the connectome. Our results provide a global map of how the cell types across an animal's brain encode its behavior.

The CD1 system binds lipid antigens for display to T cells. Here, we solved lipidomes for the four human CD1 antigen-presenting molecules, providing a map of self-lipid display. Answering a basic question, the detection of >2,000 CD1-lipid complexes demonstrates broad presentation of self-sphingolipids and phospholipids. Whereas peptide antigens are chemically processed, many lipids are presented in an unaltered form. However, each type of CD1 protein differentially edits the self-lipidome to show distinct capture motifs based on lipid length and chemical composition, suggesting general antigen display mechanisms. For CD1a and CD1d, lipid size matches the CD1 cleft volume. CD1c cleft size is more variable, and CD1b is the outlier, where ligands and clefts show an extreme size mismatch that is explained by uniformly seating two small lipids in one cleft. Furthermore, the list of compounds that comprise the integrated CD1 lipidome supports the ongoing discovery of lipid blockers and antigens for T cells.

Inflammasomes are critical sentinels of the innate immune system that respond to threats to the host through recognition of distinct molecules, known as pathogen- or damage-associated molecular patterns (PAMPs/DAMPs), or disruptions of cellular homeostasis, referred to as homeostasis-altering molecular processes (HAMPs) or effector-triggered immunity (ETI). Several distinct proteins nucleate inflammasomes, including NLRP1, CARD8, NLRP3, NLRP6, NLRC4/NAIP, AIM2, pyrin, and caspases-4/-5/-11. This diverse array of sensors strengthens the inflammasome response through redundancy and plasticity. Here, we present an overview of these pathways, outlining the mechanisms of inflammasome formation, subcellular regulation, and pyroptosis, and discuss the wide-reaching effects of inflammasomes in human disease.

Systematic evaluation of the impact of genetic variants is critical for the study and treatment of human physiology and disease. While specific mutations can be introduced by genome engineering, we still lack scalable approaches that are applicable to the important setting of primary cells, such as blood and immune cells. Here, we describe the development of massively parallel base-editing screens in human hematopoietic stem and progenitor cells. Such approaches enable functional screens for variant effects across any hematopoietic differentiation state. Moreover, they allow for rich phenotyping through single-cell RNA sequencing readouts and separately for characterization of editing outcomes through pooled single-cell genotyping. We efficiently design improved leukemia immunotherapy approaches, comprehensively identify non-coding variants modulating fetal hemoglobin expression, define mechanisms regulating hematopoietic differentiation, and probe the pathogenicity of uncharacterized disease-associated variants. These strategies will advance effective and high-throughput variant-to-function mapping in human hematopoiesis to identify the causes of diverse diseases.

Enabling and constraining immune activation is of fundamental importance in maintaining cellular homeostasis. Depleting BAK1 and SERK4, the co-receptors of multiple pattern recognition receptors (PRRs), abolishes pattern-triggered immunity but triggers intracellular NOD-like receptor (NLR)-mediated autoimmunity with an elusive mechanism. By deploying RNAi-based genetic screens in Arabidopsis, we identified BAK-TO-LIFE 2 (BTL2), an uncharacterized receptor kinase, sensing BAK1/SERK4 integrity. BTL2 induces autoimmunity through activating Ca2+ channel CNGC20 in a kinase-dependent manner when BAK1/SERK4 are perturbed. To compensate for BAK1 deficiency, BTL2 complexes with multiple phytocytokine receptors, leading to potent phytocytokine responses mediated by helper NLR ADR1 family immune receptors, suggesting phytocytokine signaling as a molecular link connecting PRR- and NLR-mediated immunity. Remarkably, BAK1 constrains BTL2 activation via specific phosphorylation to maintain cellular integrity. Thus, BTL2 serves as a surveillance rheostat sensing the perturbation of BAK1/SERK4 immune co-receptors in promoting NLR-mediated phytocytokine signaling to ensure plant immunity.

A functional network of blood vessels is essential for organ growth and homeostasis, yet how the vasculature matures and maintains homeostasis remains elusive in live mice. By longitudinally tracking the same neonatal endothelial cells (ECs) over days to weeks, we found that capillary plexus expansion is driven by vessel regression to optimize network perfusion. Neonatal ECs rearrange positions to evenly distribute throughout the developing plexus and become positionally stable in adulthood. Upon local ablation, adult ECs survive through a plasmalemmal self-repair response, while neonatal ECs are predisposed to die. Furthermore, adult ECs reactivate migration to assist vessel repair. Global ablation reveals coordinated maintenance of the adult vascular architecture that allows for eventual network recovery. Lastly, neonatal remodeling and adult maintenance of the skin vascular plexus are orchestrated by temporally restricted, neonatal VEGFR2 signaling. Our work sheds light on fundamental mechanisms that underlie both vascular maturation and adult homeostasis in vivo.

T cell responses play an important role in protection against beta-coronavirus infections, including SARS-CoV-2, where they associate with decreased COVID-19 disease severity and duration. To enhance T cell immunity across epitopes infrequently altered in SARS-CoV-2 variants, we designed BNT162b4, an mRNA vaccine component that is intended to be combined with BNT162b2, the spike-protein-encoding vaccine. BNT162b4 encodes variant-conserved, immunogenic segments of the SARS-CoV-2 nucleocapsid, membrane, and ORF1ab proteins, targeting diverse HLA alleles. BNT162b4 elicits polyfunctional CD4+ and CD8+ T cell responses to diverse epitopes in animal models, alone or when co-administered with BNT162b2 while preserving spike-specific immunity. Importantly, we demonstrate that BNT162b4 protects hamsters from severe disease and reduces viral titers following challenge with viral variants. These data suggest that a combination of BNT162b2 and BNT162b4 could reduce COVID-19 disease severity and duration caused by circulating or future variants. BNT162b4 is currently being clinically evaluated in combination with the BA.4/BA.5 Omicron-updated bivalent BNT162b2 (NCT05541861).

Hybrid potato breeding will transform the crop from a clonally propagated tetraploid to a seed-reproducing diploid. Historical accumulation of deleterious mutations in potato genomes has hindered the development of elite inbred lines and hybrids. Utilizing a whole-genome phylogeny of 92 Solanaceae and its sister clade species, we employ an evolutionary strategy to identify deleterious mutations. The deep phylogeny reveals the genome-wide landscape of highly constrained sites, comprising ∼2.4% of the genome. Based on a diploid potato diversity panel, we infer 367,499 deleterious variants, of which 50% occur at non-coding and 15% at synonymous sites. Counterintuitively, diploid lines with relatively high homozygous deleterious burden can be better starting material for inbred-line development, despite showing less vigorous growth. Inclusion of inferred deleterious mutations increases genomic-prediction accuracy for yield by 24.7%. Our study generates insights into the genome-wide incidence and properties of deleterious mutations and their far-reaching consequences for breeding.

Ribonuclease HII (RNaseHII) is the principal enzyme that removes misincorporated ribonucleoside monophosphates (rNMPs) from genomic DNA. Here, we present structural, biochemical, and genetic evidence demonstrating that ribonucleotide excision repair (RER) is directly coupled to transcription. Affinity pull-downs and mass-spectrometry-assisted mapping of in cellulo inter-protein cross-linking reveal the majority of RNaseHII molecules interacting with RNA polymerase (RNAP) in E. coli. Cryoelectron microscopy structures of RNaseHII bound to RNAP during elongation, with and without the target rNMP substrate, show specific protein-protein interactions that define the transcription-coupled RER (TC-RER) complex in engaged and unengaged states. The weakening of RNAP-RNaseHII interactions compromises RER in vivo. The structure-functional data support a model where RNaseHII scans DNA in one dimension in search for rNMPs while "riding" the RNAP. We further demonstrate that TC-RER accounts for a significant fraction of repair events, thereby establishing RNAP as a surveillance "vehicle" for detecting the most frequently occurring replication errors.

Exposures to fine particulate matter (PM2.5) concentrations above the WHO guidelines affect 99% of the world population. In a recent issue of Nature, Hill et al. dissect the tumor promotion paradigm orchestrated by PM2.5 inhalation exposures in lung carcinogenesis, supporting the hypothesis that PM2.5 can increase your risk of lung carcinoma without ever smoking.

Holistic understanding of physio-pathological processes requires noninvasive 3D imaging in deep tissue across multiple spatial and temporal scales to link diverse transient subcellular behaviors with long-term physiogenesis. Despite broad applications of two-photon microscopy (TPM), there remains an inevitable tradeoff among spatiotemporal resolution, imaging volumes, and durations due to the point-scanning scheme, accumulated phototoxicity, and optical aberrations. Here, we harnessed the concept of synthetic aperture radar in TPM to achieve aberration-corrected 3D imaging of subcellular dynamics at a millisecond scale for over 100,000 large volumes in deep tissue, with three orders of magnitude reduction in photobleaching. With its advantages, we identified direct intercellular communications through migrasome generation following traumatic brain injury, visualized the formation process of germinal center in the mouse lymph node, and characterized heterogeneous cellular states in the mouse visual cortex, opening up a horizon for intravital imaging to understand the organizations and functions of biological systems at a holistic level.