The female Aedes aegypti mosquito's remarkable ability to hunt humans and transmit pathogens relies on her unique biology. Here, we present the Aedes aegypti Mosquito Cell Atlas, a comprehensive single-nucleus RNA sequencing dataset of more than 367,000 nuclei from 19 dissected tissues of adult female and male Aedes aegypti, providing cellular-level resolution of mosquito biology. We identify novel cell types and expand our understanding of sensory neuron organization of chemoreceptors across all sensory tissues. Our analysis uncovers male-specific cells and sexually dimorphic gene expression in the antenna and brain. In female mosquitoes, we find that glial cells, rather than neurons, undergo the most extensive transcriptional changes in the brain following blood feeding. Our findings provide insights into the cellular basis of mosquito behavior and sexual dimorphism. The Aedes aegypti Mosquito Cell Atlas resource enables systematic investigation of cell-type-specific expression across all mosquito tissues.

Symbiosis is widespread in nature and plays a fundamental role in organism adaptation and evolution. In nutritional endosymbiosis, host cells accommodate intracellular bacteria and act as a "metabolic factory," requiring extensive metabolic exchanges between host and endosymbiont. To investigate the mechanisms supporting these exchanges, we used the association between the bacterium Sodalis pierantonius and the insect Sitophilus spp. that thrives on an exclusive cereal diet. Volume electron microscopy uncovered that endosymbionts generate complex membranous tubular networks (tubenets) that connect bacteria and drastically increase their exchange surface with the host cytosol. In situ high spatial resolution chemical analysis indicated that tubenets are enriched in carbohydrates, which are the main substrate used by bacteria to generate nutrients for the host. Multiple membranous structures favoring nutrient absorption are described in multicellular organisms. This work demonstrates that bacteria have convergently evolved a similar "biostrategy" that enhances nutrient acquisition by increasing membrane interface.

The second trimester of pregnancy is a pivotal stage in human immune system development. Utilizing single-cell RNA sequencing and T cell receptor sequencing, we profiled 2,868,420 immune cells from 321 samples across 23 organs, including adult tissues as comparators. We identify an extrathymic CD4+ T cell subset mediating TOX2+ precursor cells' transition to mature naive CD4+ T cells. Contrary to the prevailing paradigm of fetal immune quiescence, we uncover widespread memory/activated T cells and tissue-resident memory clones shared across organs, indicating systemic immune activity beyond localized barrier defense. Cell-cell communication and functional assays indicate two tolerance mechanisms that suppress fetal T cell activation: ARG1+ neutrophils and a PTGES3/PTGER4 signaling pathway. We also find that hematopoietic stem cells (HSCs) disperse across multiple organs and show that HSCs from non-canonical hematopoietic organs differentiate into diverse immune lineages. These findings provide insights into human immune system maturation and tolerance in fetuses and adults.

Many species regenerate lost body parts following amputation. Most limb regeneration research has focused on the immediate injury site. Meanwhile, body-wide injury responses remain largely unexplored but may be critical for regeneration. Here, we discovered a role for the sympathetic nervous system in stimulating a body-wide stem cell activation response to amputation that drives enhanced limb regeneration in axolotls. This response is mediated by adrenergic signaling, which coordinates distant cellular activation responses via the α2Α-adrenergic receptor, and local regeneration responses via β-adrenergic receptors. Both α2A- and β-adrenergic signaling act upstream of mTOR signaling. Notably, systemically activated axolotls regenerate limbs faster than naive animals, suggesting a potential selective advantage in environments where injury from cannibalism or predation is common. This work challenges the predominant view that cellular responses underlying regeneration are confined to the injury site and argues instead for body-wide cellular priming as a foundational step that enables localized tissue regrowth.

Detection of DNA is a fundamental strategy for life to recognize non-self or abnormal-self to subsequently trigger the downstream responses. However, the mechanism underlying DNA sensing is incompletely understood. Here, we show that a key neural executioner, sterile alpha and Toll/interleukin-1 receptor (TIR) motif containing 1 (SARM1), senses double-stranded DNA (dsDNA) to promote cell death. dsDNA-bound and -activated SARM1 to degrade nicotinamide adenine dinucleotide (NAD+) in a sequence-independent manner. SARM1 bound dsDNA via the TIR domain, and lysine residues in the TIR domain contributed to dsDNA binding. In the cellular context, cytosolic dsDNA from dsDNA transfection or chemotherapy treatment was colocalized with SARM1 and activated SARM1 to elicit NAD+ degradation and cell death, which was abrogated by SARM1 knockout or DNA-binding residue mutation. Consistently, SARM1 knockout blocked chemotherapy-induced neuropathy (CIN) in mice. Our results reveal SARM1 as a DNA sensor, which might be targetable for therapeutic interventions.

T cell-mediated tumor killing underlies immunotherapy success. Here, we used long-term in vivo imaging and high-resolution spatial transcriptomics of zebrafish endogenous melanoma, as well as multiplex imaging of human melanoma, to identify domains facilitating the immune response during immunotherapy. We identified cancer regions of antigen presentation and T cell engagement and retention (CRATERs) as pockets at the stroma-melanocyte boundaries of zebrafish and human melanoma. CRATERs are rich in antigen-recognition molecules, harboring the highest density of CD8+ T cells in tumors. In zebrafish, CD8+ T cells formed prolonged interactions with melanoma cells within CRATERs, characteristic of antigen recognition. Following immunostimulatory treatment, CRATERs expanded, becoming the major sites of activated CD8+ T cell accumulation and tumor killing. In humans, elevation in CRATER density in biopsies following immune checkpoint blockade (ICB) therapy correlated with a clinical response to therapy. CRATERs are structures that show active tumor killing and may be useful as a diagnostic indicator for immunotherapy success.

The origins of the early medieval Magyars who appeared in the Carpathian Basin by the end of the 9th century CE remain incompletely understood. Previous archaeogenetic research identified the newcomers as migrants from the Eurasian steppe. However, genome-wide ancient DNA from putative source populations has not been available to test alternative theories of their precise source. We generated genome-wide ancient DNA data for 131 individuals from archaeological sites in the Ural region in northern Eurasia, which are candidates for the source based on historical, linguistic, and archaeological evidence. Our results tightly link the Magyars to people of the early medieval Karayakupovo archaeological horizon on both the European and Asian sides of the southern Urals. The ancestors of the people of the Karayakupovo archaeological horizon were established in the broader Urals by the Late Iron Age, and their descendants persisted in the Volga-Kama region until at least the 14th century.

Progressive multiple sclerosis (PMS), which is characterized by relentless disease progression, lacks effective treatment. While recent studies have highlighted the importance of B cells in driving compartmentalized central nervous system (CNS) inflammation in PMS pathogenesis, current B cell depletion therapies, such as CD20 monoclonal antibodies, face challenges in targeting plasma cells within the CNS. Here, we treated five patients with PMS (one primary PMS and four secondary PMS) with anti-B cell maturation antigen (BCMA) chimeric antigen receptor T (CAR-T) cell therapy in an ongoing phase 1 clinical trial (ClinicalTrials.gov: NCT04561557). Only grade 1 cytokine release syndrome was observed, and all grade ≥3 cytopenias occurred within 40 days post-infusion in all five patients. Meanwhile, we detected plasma cell depletion in CNS compartments, prolonged expansion and relieved exhaustion of CAR-T cells in the cerebrospinal fluid, and attenuation of microglial activation. These findings provided insights into the potential application of anti-BCMA CAR-T therapy for advancing clinical management of PMS.

Sugarcane is a major crop of unclear origins due to its complex polyploid interspecific genome. We analyzed genome ancestries using whole-genome sequence data from 390 representative accessions based on repeated k-mers and chloroplast phylogeny. The results provided evidence that Saccharum officinarum was domesticated in the New Guinea region from the S. robustum wild species and revealed that its genome is a mosaic involving different S. robustum subgroups. We discovered a wild Saccharum contributor to most modern cultivars, likely originating from East Melanesia. We highlighted two early centers of sugarcane diversification associated with human transport, one in continental Asia through hybridization with different S. spontaneum subgroups and one in the Melanesian and Polynesian islands via hybridization with the discovered ancestor and Miscanthus. Finally, we revealed the genome ancestry of modern cultivars, highlighting untapped wild Saccharum diversity as a source of alleles for breeding programs.

Parkinson's disease (PD) has long been considered an appropriate candidate for cell replacement therapy. We generated high-purity dopaminergic progenitors (A9-DPCs) from human embryonic stem cells and evaluated their safety and exploratory efficacy in a single-center, open-label, dose-escalation phase 1/2a trial (NCT05887466) for PD patients. Twelve patients with moderate-to-severe PD received bilateral putamen transplantation of low-dose (3.15 million cells; n = 6) or high-dose (6.30 million cells; n = 6) A9-DPC with immunosuppression. No dose-limiting toxicities or graft-related adverse events were observed. At 12 months, off-medication Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) part III scores and Hoehn and Yahr stage improved, with greater motor improvements in the high-dose group. Dopamine transporter positron emission tomography (PET) imaging showed increased posterior putamen uptake with greater uptake in the high-dose group after transplantation, supporting graft survival. These findings indicate that bilateral transplantation of A9-DPC is safe and may improve parkinsonian motor symptoms in patients with PD.

Rapid remodeling of actin filament (F-actin) networks is essential for the movement and morphogenesis of eukaryotic cells. The conserved actin-binding proteins coronin, cofilin, and actin-interacting protein 1 (AIP1) act in synergy to promote rapid F-actin network disassembly, but the underlying mechanisms have remained elusive. Here, using cryo-electron microscopy (cryo-EM), we uncover the concerted molecular actions of coronin, cofilin, and AIP1 that lead to actin filament aging and severing. We find that the cooperative binding of coronin allosterically promotes inorganic phosphate release from F-actin and induces filament undertwisting, thereby priming the filament for cofilin binding. Cofilin then displaces coronin from the filament via a strand-restricted cooperative binding mechanism. The resulting cofilactin serves as a high-affinity platform for AIP1, which induces severing by acting as a clamp that disrupts inter-subunit filament contacts. In this "molecular squeezing" mechanism, AIP1 and not cofilin is responsible for filament severing. Our work redefines the role of key disassembly factors in actin dynamics.

Variable expressivity of disease-associated variants implies a role for secondary variants that modify clinical features. We assessed the effects of modifier variants on the clinical outcomes of 2,455 individuals with primary variants. Among 124 families with the 16p12.1 deletion, distinct rare and common variant classes conferred risks for specific developmental features, including short tandem repeats for neurological defects. Network analysis suggested distinct mechanisms involving 16p12.1 genes and secondary variants specific to each proband. Within disease and population cohorts of 976 individuals with the 16p12.1 deletion, we found opposing effects of secondary variants on clinical features across ascertainments. Additional analysis of 1,479 probands with other primary variants, such as the 16p11.2 deletion and CHD8 variants, and 1,528 probands without primary variants showed that phenotypic associations differed by primary variant context and were influenced by synergistic interactions between primary and secondary variants. Our study provides a paradigm to dissect the personalized genomic architecture of complex disorders.

Phytohormone auxin and its directional transport mediate much of the remarkably plastic development of higher plants. Positive feedback between auxin signaling and transport is a prerequisite for (1) self-organizing processes, including vascular tissue formation, and (2) directional growth responses such as gravitropism. Here, we identify a mechanism by which auxin signaling directly targets PIN auxin transporters. Via the cell-surface AUXIN-BINDING PROTEIN1 (ABP1)-TRANSMEMBRANE KINASE 1 (TMK1) receptor module, auxin rapidly induces phosphorylation and thus stabilization of PIN2. Following gravistimulation, initial auxin asymmetry activates autophosphorylation of the TMK1 kinase. This induces TMK1 interaction with and phosphorylation of PIN2, stabilizing PIN2 at the lower root side, thus reinforcing asymmetric auxin flow for root bending. Upstream of TMK1 in this regulation, ABP1 acts redundantly with the root-expressed ABP1-LIKE 3 (ABL3) auxin receptor. Such positive feedback between cell-surface auxin signaling and PIN-mediated polar auxin transport is fundamental for robust root gravitropism and presumably for other self-organizing developmental phenomena.

Recent breakthroughs in spatial transcriptomics technologies have enhanced our understanding of diverse cellular identities, spatial organizations, and functions. Yet existing spatial transcriptomics tools are still limited in either transcriptomic coverage or spatial resolution, hindering unbiased, hypothesis-free transcriptomic analyses at high spatial resolution. Here, we develop reverse-padlock amplicon-encoding fluorescence in situ hybridization (RAEFISH), an image-based spatial transcriptomics method with whole-genome coverage and single-molecule resolution in intact tissues. We demonstrate the spatial profiling of transcripts from 23,000 human or 22,000 mouse genes in single cells and tissue sections. Our analyses reveal transcript-specific subcellular localization, cell-type-specific and cell-type-invariant zonation-dependent transcriptomes, and gene programs underlying preferential cell-cell interactions. Finally, we further develop our technology for the direct spatial readout of guide RNAs (gRNAs) in an image-based, high-content CRISPR screen. Overall, these developments offer a broadly applicable technology that enables high-coverage, high-resolution spatial profiling of both long and short, native and engineered RNAs in many biomedical contexts.

Mycobacterium tuberculosis (Mtb) has co-evolved with humans for thousands of years and is characterized by variation in virulence, transmissibility, and disease phenotypes. To identify bacterial contributors to phenotypic diversity, we developed new RNA sequencing (RNA-seq) and phylogenomic tools to capture hundreds of Mtb isolate transcriptomes, link transcriptional and genetic variation, and find associations between variants and epidemiologic traits. Across 274 Mtb clinical isolates, we uncovered unexpected diversity in virulence gene expression, which we linked to known and unknown regulators. Surprisingly, we found that many isolates harbor variants associated with decreased expression of EsxA (Esat6) and EsxB (Cfp10), which are virulence effectors, dominant T cell antigens, and immunodiagnostic targets. Across >55,000 isolates, these variants associate with increased transmissibility, especially in drug-resistant Mtb strains. Our data suggest expression of Mtb virulence genes is evolving in response to drug-linked pressure, raising concerns about use of these targets in immunodiagnostics and next-generation vaccines.

The molecular mechanisms underlying the pathogenesis of Alzheimer's disease (AD), the most common form of dementia, remain poorly understood. Proteomics offers a crucial approach to elucidating AD pathogenesis, as alterations in protein expression are more directly linked to phenotypic outcomes than changes at the genetic or transcriptomic level. In this study, we develop multiscale proteomic network models for AD by integrating large-scale matched proteomic and genetic data from brain regions vulnerable to the disease. These models reveal detailed protein interaction structures and identify putative key driver proteins (KDPs) involved in AD progression. Notably, the network analysis uncovers an AD-associated subnetwork that captures glia-neuron interactions. AHNAK, a top KDP in this glia-neuron network, is experimentally validated in human induced pluripotent stem cell (iPSC)-based models of AD. This systematic identification of dysregulated protein regulatory networks and KDPs lays down a foundation for developing innovative therapeutic strategies for AD.

Bispecific antibodies and chimeric antigen receptor T cells are some of the most potent cancer immunotherapeutics in clinical use, yet most cancers remain poorly targetable. High-affinity antibodies required to maximize killing detect low antigen expression in normal tissue, risking "on-target, off-cancer" toxicity. This compels identification of cancer-restricted cell-surface protein antigens, which are rare. Tumor-associated carbohydrate antigens (TACAs) are the most abundant and widespread cancer antigens known but are poorly targetable by antibodies. Here, we describe glycan-dependent T cell recruiter (GlyTR) pan-cancer immunotherapeutics that utilize high-avidity "velcro-like" lectin binding to kill cells with high but not low TACA expression. GlyTR1 and GlyTR2 bind immunosuppressive β1,6GlcNAc-branched N-glycans or multiple TACAs (Tn, sialyl-Tn, LacDiNAc, and GD2), respectively, overcome immunosuppressive mechanisms in the tumor microenvironment and trigger target-density-dependent T cell-mediated pan-cancer killing, yet they lack toxicity in mice with human-like TACA expression. Density-dependent lectin binding to TACAs provides highly potent and safe pan-cancer immunotherapeutics.

Primary cilia are critical organelles found on most human cells. Their dysfunction is linked to hereditary ciliopathies with a wide phenotypic spectrum. Despite their significance, the specific roles of cilia in different cell types remain poorly understood due to limitations in analyzing ciliary protein composition. We employed antibody-based spatial proteomics to expand the Human Protein Atlas to primary cilia. Our analysis identified the subciliary locations of 715 proteins across three cell lines, examining 128,156 individual cilia. We found that 69% of the ciliary proteome is cell-type specific, and 78% exhibited single-cilia heterogeneity. Our findings portray cilia as sensors tuning their proteome to effectively sense the environment and compute cellular responses. We reveal 91 cilia proteins and found a genetic candidate variant in CREB3 in one clinical case with features overlapping ciliopathy phenotypes. This open, spatial cilia atlas advances research on cilia and ciliopathies.

Astrocyte Ca2+ dynamics control synaptic circuits and behavior, yet the underlying biology remains poorly understood. By combining volumetric high-resolution electron microscopy and two-photon Ca2+ imaging, we characterize astrocyte leaflets that interface with synapses. These convoluted structures with ≤250 nm diameter originate from astrocytic shafts or cell bodies, contain minuscule endoplasmic reticulum saccules expressing IP3 receptors but not mitochondria, and are often interconnected via gap junctions forming domains with cytosolic continuity. Leaflets enwrap 90% of synapses in clusters and only 10% individually. By fast imaging of astrocyte peripheral microvolumes, we identify leaflet-specific Ca2+ events that were synaptically induced, IP3R1-mediated, and often displayed separate originations merging into large, long-lasting Ca2+ elevations. Using combined axon-leaflet Ca2+ imaging, we show that these complex events reflect integration of incoming inputs from different neurons. The astrocyte leaflet organization may thus coordinate, via Ca2+ signals, multiple synapses and circuits active at different spatiotemporal scales, executing computations distinct from neurons.