Biology subjects

Liu, Y.

Publications and source records attributed to Liu, Y..

At least 397 records · Page 22Linked to original sources

bioRxiv2026

Cholesterol Biosynthesis is a Targetable Vulnerability of CEBPA-mutant Acute Myeloid Leukemia

Bi-allelic CEBPA mutations occur in 5-15% of acute myeloid leukemia (AML) patients. The precise molecular consequences of CEBPA mutations, especially in combination with frequently co-occurring mutations in TET2, WT1, and GATA2, remain incompletely understood. Here, we present a robust human model of CEBPA-mutant AML through gene editing of healthy bone marrow-derived hematopoietic stem cells. Loss of the CEBPA-p42 isoform expressed in healthy cells with concomitant upregulation of the leukemic CEBPA-p30 isoform resulted in a myeloproliferative phenotype. Concurrent loss-of-function mutations in TET2 or WT1 drove full leukemic transformation, while GATA2 haploinsufficiency promoted erythroid precursor accumulation without overt AML. Single-cell transcriptomics and low-input proteomics revealed enhanced myeloid output, increased interferon signaling and elevated cholesterol biosynthesis in leukemic cells. Targeting cholesterol synthesis enhanced chemosensitivity, highlighting a potential therapeutic vulnerability, particularly relevant for CEBPA-mutant patients harboring co-mutations in TET2 or WT1, which have poor outcomes. Statement of significanceInduction of CEBPA-p30 by CRISPR/Cas gene editing in healthy human BM HSCs drives overt AML in vivo. TET2 and WT1 loss accelerate leukemogenesis, while GATA2 haploinsufficiency redirects differentiation toward erythroid precursors potentially driving acute erythroid leukemia. CEBPA-p30 AML exhibits cholesterol biosynthesis dependency, revealing a therapeutic vulnerability to statins.

cancer biology↗

bioRxiv2026

Histone H2BK120 ubiquitination modulates PRC1 activity and H2AK119ub deposition on nucleosomes

Polycomb group and Trithorax group complexes antagonistically regulate developmental gene expression, yet the mechanisms coordinating their interplay remain poorly understood. Here, we uncover a synergistic histone crosstalk in which H2BK120ub (histone H2B ubiquitination at lysine 120), a Trithorax-associated active mark, enhances Polycomb repressive complex 1 (PRC1) activity by directly binding specific PCGF subunits of PRC1 on the nucleosome surface, as shown in cryo-electron microscopy (cryo-EM) analysis. Beyond regulating enzymatic activity, H2BK120ub also modulates the competitive binding between the catalytic RNF2-BMI1 module and the RYBP subunit of noncanonical PRC1.4 complexes to nucleosomes, potentially restricting H2AK119ub (histone H2A ubiquitination at lysine 119) spreading to neighboring nucleosomes. In mouse embryonic stem cells, H2BK120ub directs the deposition of H2AK119ub within the gene bodies of a subset of developmental genes, which exhibit more rapid activation compared to canonical H3K4me3/H3K27me3 bivalent genes. These findings reveal a previously unrecognized mechanism that links active and repressive histone modifications, providing new insights into the intricate chromatin regulation of gene expression during development.

molecular biology↗

bioRxiv2026

Inducible Impairment of Polymerase Gamma Activity in Cardiomyocytes Promotes Severe Cardiomyopathy with Cardiac Hepatopathy

Mitochondrial dysfunction is a hallmark feature of heart failure (HF) and cardiomyopathy, with substantial human and preclinical evidence suggesting that congenital mitochondrial defects can directly drive these conditions. Despite these strong links, not all individuals with mitochondrial disease develop cardiomyopathy, and therefore the precise mitochondrial defects that initiate cardiac pathology in this setting remain incompletely understood. Here, we describe a novel mouse model that induces progressive deterioration in mtDNA integrity specifically in cardiomyocytes. This model was generated through a post developmental, cardiomyocyte specific deletion of the exonuclease domain of Polymerase Gamma (PolG), impairing its ability to repair mtDNA. Strikingly, from just 16 weeks post-induction these mice displayed progressive worsening of cardiac output, ejection fraction, global strain and blood pressure culminating in premature death at 28-30 weeks post-mutation. Morphologically, these mice displayed cardinal features of hypertrophic cardiomyopathy with enlarged hearts, ventricles and cardiac fibrosis - but little evidence of congestive HF. We also demonstrate using various transcriptional and proteomic readouts, that signalling pathways characteristic of cardiomyopathy were activated. Interestingly, prior to substantial declines in cardiac function, we detect robust activation of the integrated stress response (ISR) in PolGMut mice, and major rewiring of mitochondrial folate metabolism pathways, suggesting that these pathways underpin the developing pathology. Lastly, we also observed a striking hepatopathy phenotype in mutant mice reminiscent of that observed in patients with HF, a condition not robustly recapitulated previously in other mouse models. Thus, our data establish a direct link between mtDNA instability leading to chronic activation of the ISR and rewiring of folate metabolism in cardiomyocytes, subsequently driving a severe cardiac phenotype associated with hepatopathy.

physiology↗

bioRxiv2026

A novel TaNF-YC10-TaNF-YB1-TabHLH95 module coordinates starch biosynthesis in wheat endosperm

Wheat grain weight and flour quality largely depend on starch biosynthesis, yet the mechanisms by which transcription factors coordinate this process remain poorly understood. In this study, using an integrative strategy that combines genome-wide association analysis with yeast two-hybrid library screening, we identify TaNF-YC10, a Nuclear Factor Y transcription factor, as a positive regulator of starch accumulation in the wheat endosperm. Loss of TaNF-YC10 reduces starch content and alters starch granule size distribution, whereas overexpression enhances starch accumulation and increases grain weight. TaNF-YC10 binds and activates core starch biosynthetic-related genes, including AGPL1, GBSS1, YUC11, and NF-YB7, and forms higher-order transcriptional complexes with TaNF-YB1 and TabHLH95 to coordinate multiple regulatory pathways. TaNF-YC10-A1-Hap2 is associated with higher starch content and thousand grain weight and has been selected during wheat breeding in China. Collectively, our findings establish TaNF-YC10 as a pivotal transcriptional hub in starch regulation and highlight its potential as a target for genetic improvement of grain yield in wheat.

plant biology↗

bioRxiv2026

Distinct Chiral Nanostructures of Graphene Quantum Dots Govern Divergent Passive and Active Enantioselective Transport across Biological Membranes

Chirality in two-dimensional nanomaterials provides a powerful lever to control biological interfaces, yet the structural origins of nanoscale chirality remain poorly understood. Here, we systematically investigate chiral ligand modulated graphene quantum dots (GQDs) and reveal how ligand stereochemistry and edge chemistry modulate the formation of diverse chiral or achiral nanostructures. Spectroscopy, microscopy, and density functional theory with ring-puckering analysis identified six structural motifs, twisted-, twisted-boat, saddle-shaped, hybrid, unbuckled, and random. Among these, twisted-, twisted-boat, and saddle-shaped GQDs exhibited genuine nanoscale structural chirality, while unbuckled, hybrid, and random conformations lacked organized distortion. Importantly, structural chirality governed passive permeation into biological membrane (e.g. lipid membrane of extracellular vesicles), whereas achiral variants relied mainly on hydrophobic interactions. In contrast, active transport across biological membrane (e.g. endocytosis) is insensitive to nanoscale structural chirality but strongly influenced by chiral ligand identity and transporter recognition. Collectively, these results establish chiral ligand conjugation as a modular route to program both chiral and achiral motifs in graphene nanostructures and highlight nanoscale structural chirality as a design principle for engineering bio-nano interactions.

bioengineering↗

bioRxiv2026

Unwinding of RNA G-quadruplexes induces mouse and human totipotency

Totipotent stem cells (TotiSCs) have significant application prospects in regenerative medicine and assisted reproductive technologies. The mRNAs of totipotency-related genes are rich in G-quadruplex (G4) structures, while the G4 density of pluripotency-related genes mRNAs is relatively low, suggesting a potential functional link between RNA G4s and the totipotent state. Nevertheless, how G4s are involved in the establishment or maintenance of mouse and human totipotency remains poorly understood. Here, we demonstrate that FC3, a small-molecule RNA G4 unwinder, enables efficient induction and maintenance of mouse and human TotiSCs (mG4TotiSCs and hG4TotiSCs). These FC3-induced TotiSCs closely recapitulate the molecular features of 2-cell-stage blastomeres via transcriptomic and epigenomic profiling. Functionally, both mG4TotiSCs and hG4TotiSCs exhibit totipotent capacity: they differentiate into embryonic and extraembryonic lineages in vitro and in chimeric embryos; moreover, they autonomously self-organize into blastocyst-like structures without exogenous signalling. Mechanistically, we identify that Gata2 mRNA, a G4-containing transcript, is essential for FC3-mediated reprogramming. Genetic disruption of Gata2 would abolish totipotency induction. Collectively, RNA G4 unwinding is a new regulatory axis governing totipotency acquisition and maintenance, providing insight into the functional interplay between nucleic acid secondary structures and cell fate plasticity.

cell biology↗

bioRxiv2026

Evolutionary dynamics of insect odorant receptors reveal ecological tuning shaping olfactory perception

Insect olfaction is facilitated by a heterotetrameric odorant receptor-odorant receptor co-receptor (OR-Orco) complex, which is distinct from that of vertebrate ORs. However, extreme sequence divergence among insect ORs has hindered a unified understanding of their evolutionary history and ecological importance. In this study, we present a multiscale analysis of OR genes across 115 insect species. We overcome the limitations of traditional phylogenetic approaches by applying a protein similarity network-based strategy and introduce a "trunk-branch" framework to systematically describe the evolutionary trajectories of insect ORs across sequence, structural, and predicted olfactory potential levels. Although they possess different sequences and structural communities, all sampled insect orders contained ORs assigned to all six docking-derived functional communities. Notably, extant insect lineages with order-level origins spanning the end-Permian mass extinction (EPME) differed in docking-derived functional OR repertoires, and early-and late-diverging lineages differed in OR sequence-community diversity. The emergence of Orco represents a key evolutionary transition point, marking the shift from a homomeric to a heteromeric complex accompanied by specialization of the extracellular domain and binding pocket. Furthermore, we established robust associations between potential olfactory recognition breadth and ecological variables, including diet, circadian rhythm, and habitat. Our findings provide a class-level framework for investigating insect OR evolution and generate testable hypotheses about how receptor repertoire diversification may relate to ecological adaptation.

evolutionary biology↗

bioRxiv2026

Elimination of intramuscular immunoglobulin accumulation alleviates Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disorder due to loss of dystrophin, a cytoskeletal protein critical for muscle integrity and functionality. Despite recent therapeutic advances, there remains a significant unmet need for more effective and accessible therapeutics. Here, we discovered an early accumulation of immunoglobulin G (IgG) in the sarcolemma, which exacerbates tissue inflammation and disease progression. The IgG accumulation primarily resulted from ectopic localization of Fc{gamma}R1, a high-affinity IgG Fc receptor, on dystrophin-deficient myofibers. In two independent murine models, eliminating IgG accumulation via B cell depletion provided sustained benefit to alleviate DMD progression. Our findings uncover a novel disease accelerator in DMD and demonstrate the potential to target this mechanism as therapeutics for broader population of patients with DMD.

immunology↗

bioRxiv2026

Functional activation reveals a repressor-gated fourth type VI secretion system in clinical Pseudomonas aeruginosa

Pseudomonas aeruginosa is a major opportunistic pathogen in which the type VI secretion system (T6SS) contributes to interbacterial competition and virulence. While most strains encode three T6SSs, we here functionally characterize a fourth system (H4-T6SS) in a clinical P. aeruginosa isolate. The H4-T6SS gene cluster is transcriptionally silent under laboratory conditions. By promoter rewiring, we demonstrate that the H4-T6SS assembles a contractile machine and mediates effector secretion and robust inter- and intraspecific antagonism. We identify a dominant pore-forming effector and its secretion pathway and a silent yet conserved nuclease effector. Genetic and biochemical analyses show that H4-T6SS expression is activated by an H4-encoded transcriptional regulator and repressed by H-NS family proteins. Analysis of over 1,200 clinical isolates reveals that the H4-T6SS is distributed across lineages and geographic regions. Finally, we observe functional crosstalk between H4- and H2-T6SSs. Together, these findings establish H4-T6SS as a functional and repressor-gated secretion system that expands the competitive arsenal of clinical P. aeruginosa.

microbiology↗

bioRxiv2026

VC-RDAgent: An efficient rare disease diagnosis agent via virtual case construction informed by hybrid statistical-metric and hyperbolic-semantic prioritization

While Large Language Models (LLMs) have shown promise in clinical decision support, current Retrieval-Augmented Generation (RAG) paradigms face a fundamental bottleneck in rare disease diagnosis: the scarcity, privacy restrictions, and extreme heterogeneity of real-world patient records. This reliance on sparse or inaccessible data leads to a severe "retrieval mismatch," where the lack of high-quality reference cases causes diagnostic performance to degrade sharply. To break this deadlock, we propose VC-RDAgent, a privacy-preserving and offline-capable framework that decouples diagnostic reasoning from sensitive real-world records by synthesizing virtual standardized cases. The system is powered by VC-Ranker, a multi-dimensional engine that integrates statistical-metric measures with hyperbolic-semantic embeddings to capture deep hierarchical ontology relationships. This approach allows for the dynamic generation of high-fidelity virtual references directly from authoritative knowledge bases. Extensive benchmarking across four diverse datasets demonstrates that VC-RDAgent effectively functions as a "performance equalizer." It boosts average Top-1 hit rates by 8.7% to 85.9% over zero-case baselines, enabling lightweight open-source models to rival frontier commercial systems. Notably, VC-Ranker alone achieved an aggregate Top-10 hit rate of 0.819, outperforming prior state-of-the-art methods by 6%. By eliminating the dependency on real-time web retrieval and private case sharing, VC-RDAgent provides a scalable, robust, and clinically deployable solution to shorten the diagnostic odyssey, which is made accessible through an intuitive, chat-based web application https://rarellm.service.bio-it.tech/rdagent/.

bioinformatics↗

bioRxiv2026

Artificial Recombination of Nonribosomal Peptide Synthetases Rapidly Evolves Natural Products

Efficient engineering of nonribosomal peptide synthetases (NRPSs) is a key strategy for expanding valuable peptide natural products. Current bioinformatics-guided approaches are often constrained by a set of predefined fusion sites, remaining experimentally challenging in most NRPS systems. Here we present the Recombineering Accelerated Evolution (RACE), which harnesses Red/ET recombineering mediated partially matched homologous recombination to recapitulate recombination-driven NRPS evolution on a highly accelerated timescale. Application of RACE to six known NRPS gene clusters generated 830 recombinants and yielded over 600 novel peptides including novel bis-lipopeptides. These recombinants reveal 112 previously unrecognized recombination fusion sites, providing an extensive landscape for NRPS evolution and more useful resources for guiding NRPS engineering. The RACE establishes a new paradigm for programmable NRPS evolution and enables rapid discovery of bioactive peptides.

synthetic biology↗

bioRxiv2026

PlantMDCS: A code-free, modular toolkit for rapid deployment of plant multi-omics databases

With the rapid accumulation of diverse omics datasets, achieving efficient management and integrative analysis of plant multi-omics data remains a major challenge. Conventional solutions rely on constructing web-based databases, which often demand substantial programming expertise and long-term financial support. To address these limitations, we developed the Plant Multi-omics Database Construction System (PlantMDCS)-a locally deployable, user-friendly, and collaborative platform that unifies database construction and downstream multi-omics analysis within a graphical environment. PlantMDCS adopts a decoupled front-end/back-end architecture. The back end serves as the core engine for data management and computation, and is responsible for the storage, preprocessing, integration, and hierarchical association of multi-omics data. Once initialized, the front end supports the complete research workflow, including data import, querying, integrative analysis and visualization. All operations can be performed without programming, while local resource usage is dominated by disk storage required for user-provided datasets rather than sustained computational overhead. Benchmarking across plant species ranging from Arabidopsis to hexaploid wheat demonstrated that database construction can be completed within minutes, independent of genome size or data complexity. PlantMDCS is designed for local deployment to ensure data security, while allowing multi-user collaboration within local networks and supporting controlled remote access for teams distributed across different regions. Overall, PlantMDCS offers a secure and sustainable framework that integrates data management and analysis within a unified system. This design shifts multi-omics research away from fragmented file-based processing toward persistent, database-driven exploration, thereby enhancing analytical efficiency and reproducibility.

bioinformatics↗

bioRxiv2026

Large-scale quantum computing framework enhances drug discovery in multiple stages

Coherent Ising machines (CIMs) excel at solving large-scale combinational optimization problems (COPs), but their insufficient long-term stability has hindered their applications in compute-intensive tasks like computer-aided drug discovery (CADD). By improving fiber vibration isolation and temperature control system, we have implemented a 2000-node CIM named QBoson-CPQC-3Gen achieving stable solutions over one hour on large-scale COPs. Graph-based encoding schemes were further introduced to realize a CIM-based CADD workflow including allosteric site detection, protein-peptide docking and intermolecular similarity calculation. CIM-based methods demonstrated superior speed and accuracy than heuristic algorithms. Especially, QBoson-CPQC-3Gen identified 2 novel druggable sites and bioactive compounds for 6 targets, which were further validated in vitro, in-cell and by crystal structures. Our contributions established a quantum-computing framework for multi-stage drug discovery, representing a significant advancement in both quantum computing applications and pharmaceutical research.

bioinformatics↗

bioRxiv2026

M1 protein expression determines niche-specific spread of Streptococcus pyogenes

Invasive infections with Streptococcus pyogenes account for significant mortality. Cell surface M protein is believed to be essential for S. pyogenes virulence, with critical roles in dissemination and immune evasion. In contrast, the role of secreted M protein in infection is less clear. Here, we assessed the role of M protein in streptococcal colonisation, persistence, and invasion in respiratory and soft tissue murine infection models, and report that while cell-surface M protein is required to establish nasopharyngeal infection, it does not drive invasive infection. Cell-surface M protein was important for binding of major host matrix proteins and growth in immune human blood, but soluble M protein was the predominant determinant of lymphatic survival, tissue spread, and systemic infection in mice. A mutant emm1 strain which only expresses secreted M protein exhibited enhanced survival in the lymph node niche and overall competitive advantage over the wildtype parent strain in co-infections. Absence of M protein at the bacterial cell surface led to enhanced hyaluronan binding, providing explanation for homing to the draining lymph node. Together, these findings highlight potential nuance in the role of M protein that may impact vaccine approaches targeting M protein. Author SummaryS. pyogenes is a significant cause of illness, both mild and lethal, worldwide. Despite this, there are currently no approved vaccines. Many vaccine candidates are based on the M protein, a surface expressed anti-phagocytic factor that is thought to be critical for the ability of S. pyogenes to evade the host immune system. The unexpected selection of an M1 protein mutant strain following in vivo soft tissue infection raised many questions for us. If M protein is meant to be the critical factor in avoiding immune killing, how was this strain able to persist? One explanation could be that, although the strain was negative for M1 protein on the bacterial surface, it still secreted a truncated version of the protein with potential inflammatory effects. Importantly, the strain was selected in the draining lymph node pointing to a niche-specific advantage. We showed that cell surface-associated M1 protein, although required to establish nasopharyngeal infection, was not required in soft tissue infection, and that expression of a soluble, truncated form of M1 protein gave a survival advantage over the wild-type strain expressing cell wall-associated M1 protein. Furthermore, the absence of cell surface M protein led to enhanced hyaluronan binding providing explanation for lymphatic niche homing.

microbiology↗

bioRxiv2026

Metabolic Perturbation Exacerbates Sinoatrial Node Dysfunction in Heart Failure

Heart failure (HF) affects approximately 6.2 million people in the United States, with a 5-year mortality exceeding 50%. Bradyarrhythmia, a known complication in HF due to sinoatrial node (SAN) dysfunction (SAND), increases the morbidity and mortality of HF patients. Insights into the mechanistic underpinnings of SAND in HF could therefore uncover vital therapeutic targets to improve clinical outcomes. The SAN cells are endowed with a dense mitochondrial network crucial for sustaining their pacemaking function on a beat-to-beat basis. We have previously demonstrated significant disruptions in the mitochondrial-sarcoplasmic reticulum connectomics, resulting in abnormal mitochondrial Ca2+ handling and impaired mitochondrial function in HF. Here, we hypothesize that the metabolic perturbation is one of the critical mechanisms underlying SAND. To this end, we took advantage of a multi-omics approach combined with ultra-resolution imaging and functional analyses to decipher the metabolic shift that transpires in the HF SAN. Our findings revealed significant metabolic remodeling within the SAN mitochondria in HF, with a diminished reliance on fatty acid {beta}-oxidation, enhanced utilization of ketone bodies, and heightened dependence on carbohydrate catabolism. Notably, metabolomics analyses identified the pronounced increase of glucosylceramides and ceramides as one of the mechanisms leading to mitochondrial dysfunction. We directly test this hypothesis and demonstrate that ceramides induce a dose-dependent metabolic shift from oxidative phosphorylation to glycolysis. Importantly, these alterations lead to a significant impairment in SAN automaticity in a dose-dependent manner. Collectively, the findings support the notion that ceramides are not only markers of metabolic derangement, but also active mediators of mitochondrial and metabolic dysfunction in the SAN. Overall, the study provides evidence that ceramides may be a potential therapeutic target for mitigating SAND in HF.

physiology↗

bioRxiv2026

Mitochondrial Retrograde Signaling in Arabidopsis thaliana: heterogenous, spatial and polarised aspects

Organelle retrograde signaling studies have exclusively relied on bulk tissues, obscuring cell-type specificity, spatial organization, and heterogeneity within cell types. Here, we use single-nucleus and spatial RNA sequencing to define the cellular and spatial architecture of mitochondrial stress signaling in Arabidopsis thaliana. Inhibition of the mitochondrial electron transport chain by antimycin A and myxothiazol rapidly redistributed nuclei among transcriptional states within epidermal, leaf pavement, and mesophyll lineages, revealing that mitochondrial dysfunction reshapes cell identity trajectories rather than eliciting a uniform response. Stress-associated clusters were already present at low frequency in untreated samples, but the numbers of cells increased following stress treatments, indicating that stress increases the proportion of nuclei occupying pre-existing, primed transcriptional states. Gene-level analyses revealed distinct temporal dynamics and variable cell-state penetrance of the canonical mitochondrial stress markers and identified broadly responsive genes absent from earlier marker sets. Spatial transcriptomics resolved tissue-scale responses, spanning from pan-tissue induction to cell-type- and cluster-restricted activation, and uncovered pronounced adaxial-abaxial polarity in gene expression. Cell identity and spatial inferences were supported by extensive experimental validation using a custom 465-probe 10x Xenium panel and 52 promoter-GFP reporter lines. Together, these data provide a high-resolution framework for organelle-to-nucleus signaling and a resource of cell-type markers and spatial maps to dissect mitochondrial stress signaling and its integration with developmental and environmental pathways.

plant biology↗

bioRxiv2026

ATLAS: Population-Level Disease Locus Discovery via Differential Attention in Genomic Language Models

Identifying disease-associated genetic variants remains a key challenge in genomics, especially in small cohorts or for rare and complex mutation types where genome-wide association studies (GWAS) often fall short. We introduce ATLAS, a population-level framework that leverages attention signals from pretrained genomic language models (gLMs) to detect disease-associated genes and loci directly from raw sequences--without requiring explicit variant calls or supervised training. ATLAS first performs gene-level differential attention analysis to prioritize candidate genes, followed by base-level analysis to localize disease-associated regions at single-haplotype resolution. We validate ATLAS on synthetic and {beta}-thalassemia datasets, demonstrating robust performance across diverse allele frequencies (down to 10%), cohort sizes (below 200 individuals per group), and genomic scales. Compared to GWAS, ATLAS achieves higher recall of known loci and captures haplotype-specific signals missed by traditional methods. Cross-model benchmarking further shows that precise localization depends on both model size and pretraining on diverse human genomes. In summary, ATLAS offers a scalable, sequence-native alternative to traditional statistical genetics.

genetics↗

bioRxiv2026

A spatiotemporal immune atlas of subarachnoid hemorrhage from single-cell and spatial transcriptomics

Background and PurposeSubarachnoid hemorrhage (SAH) triggers a complex immune response that critically influences early brain injury (EBI) and long-term outcomes. However, the precise spatiotemporal dynamics and heterogeneity of immune cell infiltration and microglial reprogramming remain poorly understood. We aimed to construct a high-resolution immune atlas to delineate cell states, lineage trajectories, and spatial niches following SAH. MethodsWe integrated single-cell RNA sequencing (scRNA-seq) of CD45+ immune cells with spatial transcriptomics (ST) in a murine endovascular perforation SAH model. Immune landscapes were profiled at 24 hours (acute phase) and 72 hours (subacute phase) post-injury, compared with sham controls. Advanced bioinformatics integrated transcriptional signatures with spatial localization to map macrophage, neutrophil, and microglial dynamics. ResultsOur atlas reveals a coordinated immune transition from acute inflammation to reparative processing. We identified five macrophage, four neutrophil, and eight microglial subsets with distinct spatiotemporal patterns. Notably, we discovered a SAH-specific inflammatory microglial population (MG_03; Spp1+/Lpl+) that clusters at the rupture site during the acute phase. This subset is transcriptionally distinct from disease-associated microglia (DAM) in other neurodegenerative conditions. Trajectory analysis suggests MG_03 acts as a signaling hub for immune recruitment before transitioning toward proliferative and reparative states (MG_06-08) that disperse into the parenchyma by 72 hours. ConclusionsThis study provides the first comprehensive spatiotemporal immune atlas of SAH, highlighting the distinct role of the Spp1+ MG_03 subpopulation in early injury sensing. These findings offer a roadmap for identifying precise therapeutic windows and targeting specific immune subsets to mitigate EBI. Graphical AbstractExperimental workflow for single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) in a mouse SAH model induced by endovascular perforation. Brain tissue from the ipsilateral (injured) hemisphere was collected from sham and at 24 h and 72 h post-SAH. For scRNA-seq, CD45 immune cells were isolated prior to library preparation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/703421v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@4a1bc9org.highwire.dtl.DTLVardef@16664c4org.highwire.dtl.DTLVardef@1619725org.highwire.dtl.DTLVardef@a0d34_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗