Biology subjects

Xue, C.

Publications and source records attributed to Xue, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Development of an HK-fbp1-based vaccine platform against Cryptococcus neoformans

Cryptococcal meningitis remains a leading cause of death among HIV/AIDS patients, and no antifungal vaccine is available in clinical use. We previously showed that mice immunized with a heat-killed preparation of Cryptococcus neoformans (Cn) fbp1{Delta} mutant (HK-fbp1) are protected against cryptococcosis and other invasive mycoses, suggesting that HK-fbp1 is an intriguing vaccine candidate for further study. Recent studies have reported that heat-killed wild type H99 (HK-H99) can also offer some protection in other vaccination models. Here we set out to decipher the differential protective capacity of HK-fbp1 versus HK-H99 across multiple conditions. We observed that HK-fbp1 confers superior protection in both prophylactic and therapeutic intranasal immunization settings than HK-H99, which was associated with greater activation of pulmonary CD4+ T cells and stronger airway IFN-{gamma} production. In addition, HK-fbp1 preparations were highly stable, retaining full protective efficacy even after 5 years of frozen storage. Mice intranasally immunized with two-dose HK-fbp1 were protected against challenge by up to 106 H99 cells, a 100-fold increase over the commonly used lethal inoculum. Addition of a CpG adjuvant ODN to HK-fbp1 (HK-bp1/ODN) led to a 5-fold reduction of the intranasal vaccine inoculum required for protection, and its single-dose intramuscular vaccination induced protection persisted for at least 3 months post vaccination. Together, these findings highlight the robust protective capacity and formulation versatility of HK-fbp1, supporting its further development as a stable, dose-sparing antifungal vaccine candidate.

microbiology↗

bioRxiv2026

Cytokinesis Arrest-induced Binucleation of Macrophages ProducesHighly Efficient Efferocytes

Efferocytosis, the phagocytic clearance of dying cells and debris, supports tissue homeostasis, immune tolerance, and inflammation resolution, whereas its failure contributes to autoimmunity, atherosclerosis, aging, and impaired tissue repair. Although many molecular regulators of efferocytosis have been defined, less is known about whether macrophages can be reprogrammed into a distinct cellular state with intrinsically enhanced efferocytosis capacity. Guided by a CRISPR screen, we found that Pdcd6ip loss induces cytokinesis arrest and binucleation, creating macrophages with superior efferocytic function. Binucleated Pdcd6ip-/- bone marrow-derived macrophages demonstrate a coordinately enhanced multi-corpse capture and processing, and resolution response, and acquired a distinct transcriptomic signature. In vivo, Pdcd6ip deletion enhanced splenic macrophage efferocytosis, reduced autoimmune responses after repeated apoptotic cell challenge, and promoted plaque stability without metabolic or hematologic changes. PDCD6IP perturbation similarly increased binucleation and engulfment in human macrophage-like cells. Thus, incomplete cytokinesis represents an unrecognized route to macrophage specialization with enhanced efferocytosis capacity.

cell biology↗

bioRxiv2026

Sound-shape correspondences in macaques reveal evolutionary roots of sound symbolism

Humans reliably associate certain speech-like sounds with visual shapes, most notably in the Bouba-Kiki Effect, where rounded and spiky shapes are matched with the sounds Bouba and Kiki, respectively. Although often linked to language and culture, evidence from preverbal infants and domestic chickens suggests that sound-shape correspondences may reflect an experience-independent perceptual bias. However, studies on great apes failed to detect such bias, leaving open the question on its phylogenetic origin. Using a free-choice task, we showed the Bouba-Kiki effect in nine macaques, thus suggesting that such correspondences represent a conserved feature of vertebrate perception that may have provided a scaffold for the later emergence of symbolic communication systems in our species.

animal behavior and cognition↗

bioRxiv2026

Lipid Flippase Mediated Membrane Asymmetry Governs Extracellular Vesicles Biogenesis and Host Interactions in Cryptococcus neoformans

Cryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised patients. Alveolar macrophages are the first line of defense against Cryptococcus infection. Our previous study showed that deletion of Cdc50, the regulatory subunit of P4-ATPase (lipid flippase) complex, results in increased phagocytosis and macrophage killing, and avirulence in animal models. However, how fungal flippase dysfunction modulates Cryptococcus-macrophage interaction remains unknown. Here we identify Cdc50 as a central determinant of membrane lipid homeostasis, extracellular vesicle (EV) biogenesis and macrophage responses in C. neoformans. Our whole cell lipidomic analysis revealed that loss of Cdc50 disrupted membrane lipid homeostasis leading to phospholipid enrichment in cdc50{Delta} mutant, and a reduction in fatty acid production accompanied by pronounced ultrastructural defects in membrane architecture. Loss of Cdc50 also induced a hyper-vesiculating phenotype, with cdc50{Delta} producing significantly more extracellular vesicles (EVs) than wild type H99 cells. Lipidomic profiling of cdc50{Delta} EVs revealed enrichment of phospholipids, including phosphatidylserine (PS), indicating active lipid sorting during vesicle biogenesis. Functional analysis showed that EVs from the wildtype H99 suppress phagocytosis whereas cdc50{Delta} EVs enhance phagocytosis, indicating a differential macrophage priming. Despite increased PS externalization in cdc50{Delta} cells and EVs, macrophage recognition and uptake occur independent of PS-mediated efferocytosis pathways, including PS receptor MertK. Following macrophage uptake, cdc50{Delta} were intrinsically vulnerable to macrophage killing due to rapid phagosome acidification. Together, we demonstrate that Cdc50 dependent lipid homeostasis regulates EV production, lipid composition, membrane architecture and drives the intracellular fate of C. neoformans. IMPORTANCECryptococcus neoformans is the leading cause of fungal meningitis in immunocompromised individuals. Understanding how this pathogen evades host immune mediated clearance is essential for developing new treatment strategies. Here, we demonstrated that Cdc50, the regulatory subunit of fungal lipid flippase complex, regulates membrane lipid homeostasis that governs extracellular vesicles (EV) biogenesis and macrophage immune responses. Loss of Cdc50 drives global membrane lipid remodeling, hyper-production of phospholipid enriched EVs that enhance macrophage phagocytosis, while the wild-type EV reduce macrophage phagocytosis. Contrary to the prevailing assumption that phosphatidylserine (PS) externalization on the fungal surfaces mimics the mammalian "eat-me signal", we show fungal PS does not engage canonical PS receptor MertK, revealing a fundamental difference between fungal and mammalian PS biology. Furthermore, cdc50{Delta} cells are unable to resist phagosomal acidification, rendering them susceptible to macrophage killing. These findings establish how phospholipid homeostasis contributes to early host-pathogen interactions and serves as a compelling antifungal target in cryptococcosis.

microbiology↗

bioRxiv2026

Sequence-Defined Digital Bottlebrush Polymers for Programmable Oligonucleotide Delivery

Oligonucleotide therapeutics hold transformative potential, yet their clinical translation is hindered by delivery barriers, including rapid renal/hepatic clearance and poor organ specificity. Bottlebrush polymers conjugates have emerged as a promising vector to address these limitations, but conventional architectures with uniform backbones can only achieve an unmodifiable, rigid biodistribution profile. Here, we report a library of sequence-defined "digital" bottlebrush polymers, precisely engineered with controlled placements of chemical motifs that modify physiochemical properties - including lipids, cholesterol, and cationic groups - along a polyphosphodiester backbone. Systematic evaluation of the digital bottlebrush polymer library reveals distinct structure-property relationships and enables organ-biased systemic delivery to several traditionally difficult-to-reach tissues, including muscle and skin. In a mouse model of rheumatoid arthritis, a single dose of a spleen-homing polymer-conjugated antisense oligonucleotide targeting TNF- achieves potent knockdown and drives full functional recovery. These findings establish a versatile design framework for tailoring bottlebrush polymers to specific therapeutic applications.

bioengineering↗

bioRxiv2026

A SuperSelective primer-based real-time PCR Platform for hypersensitive detection of azole heteroresistance in Cryptococcus neoformans

Cryptococcus neoformans is the leading cause of fungal meningitis with limited treatment options, making early and accurate diagnosis critical for improved patient outcome. Current diagnostic methods for cryptococcosis rely largely on capsule antigen detection and fungal culture, which are time-consuming and unable to identify mutation-driven antifungal heteroresistance. In this study, we have developed a SuperSelective primer-based PCR (SSP-PCR) platform for the rapid and specific detection of azole resistance-associated single-nucleotide polymorphisms (SNPs). We demonstrate that SSP-PCR reliably detected a single copy mutant allele in the presence of excess wild-type (WT) DNA, with sensitivity reaching a 1:104 mutant-to-WT ratio. Incorporating molecular beacon (MB) probes with our SSP-PCR platform further enhanced amplification specificity, enabling selective detection of the ERG11 Y145F (A434T) mutation that is known to cause high azole resistance. Using genomic DNA from in vitro cultures and mouse lung tissues infected with either WT strain H99 strain or a fluconazole-hyper-resistant mrl1 clinical isolate that carries the ERG11(A434T) mutation, or both strains, we successfully detected the A434T mutant allele in both settings. Moreover, our SSP-PCR simultaneously identified ERG11(A434T) and the multi-azole resistance-associated ERG11(G1885A) mutant alleles in a single-tube duplex reaction. Collectively, the SSP-PCR platform provides a robust and ultrasensitive molecular approach for the detection of azole resistance and heteroresistance in C. neoformans, with strong potential for high-throughput clinical screening applications. ImportanceInvasive fungal infections are a growing public health threat, causing over 1.5 million deaths annually, with cryptococcal meningitis accounting for over 15% HIV/AIDS related mortality. The problem is aggravated by limited treatment options and emerging of drug resistance. Long-term use of fungistatic azoles like fluconazole promotes the emergence of azole heteroresistance, contributing to clinical treatment failure. Current diagnostic assays often fail to detect resistance-associated mutations within heterogeneous fungal populations, limiting their clinical utility. In this study, we developed a SuperSelective primer-based PCR (SSP-PCR) platform for the rapid and ultrasensitive detection of azole resistance associated single-nucleotide polymorphisms (SNPs) in Cryptococcus neoformans. By integrating molecular beacon probes, this assay achieves high specificity and enables simultaneously detection of multiple SNP mutations in a single reaction. Our SSP-PCR platform offers a powerful molecular approach for identifying azole resistance and heteroresistance, with strong potential to improve diagnostic precision and guide antifungal therapy in clinical settings.

microbiology↗

bioRxiv2026

IgG Propels Atherosclerosis by Noncanonically Activating Macrophages

Despite being a central component of adaptive immunity and a highly abundant serum protein, the contribution of IgG to the milieu of atherosclerosis remains unappreciated. Here, we identify a pro-atherogenic role for IgG as it activates an innate immune cascade, independent of its classical antigen-neutralizing function. Analyses of human coronary artery plaques reveal a positive correlation between IgG and cardiovascular and cerebrovascular disease severity. Integrated single-cell plaque analyses localize IgG, coinciding with its recycling receptor FcRn, to pro-inflammatory and foamy macrophages. Genetic ablation of FcRn in myeloid cells prevents IgG from accumulating in mouse atherosclerotic lesions, diminishing plaque size and inflammation. Mechanistically, IgG acts as an endogenous ligand for TLR4, triggering NF-{kappa}B-NLRP3 inflammasome signaling without requiring its antigen-binding domain. Additionally, IgG accelerates macrophage foam cell formation through upregulation of downstream effector LCN2. Our work uncovers a role for previously overlooked adaptive immune molecules in the pathogenesis of atherosclerosis through a noncanonical mechanism linked with innate immunity.

immunology↗

bioRxiv2026

An Inositol Receptor Orchestrates Carbon Utilization and Fungal Virulence

Nutrient sensing and utilization are critical for microbes to adapt to their environment and evade predators. The encapsuled yeast Cryptococcus neoformans has a unique ability to sense and utilize the sugar inositol as both a signaling molecule, and as a carbon source. This trait is advantageous not only in the natural environment, but to promote its pathogenesis, including invasion of the central nervous system. Unlike many other fungal species, which utilize the canonical carbon catabolite repression (CCR) mechanism to prioritize glucose metabolism, we have identified a novel inositol transporter-like receptor (transceptor) Itr4 that also regulates CCR genes to maintain inositol metabolism activity even under high glucose conditions. Itr4 binds inositol and regulates the function of other inositol transporters (ITRs) and its loss leads to a significant decrease in inositol uptake activity, resulting in a lack of mating, reduced capsule size, reduced blood brain barrier (BBB) penetration, and virulence attenuation. Mutagenesis analysis of Itr4 protein identified an essential N-terminal tail and two amino acids, Q388 and Q389, as sites of functional importance. The ITR4Q388A, Q389A allele showed a dominant phenotype with increased inositol uptake, enlarged capsule and significantly attenuated virulence. In summary, we identify a new C. neoformans inositol transceptor, Itr4, the first such example in eukaryotes, with novel regulatory roles in both inositol and glucose metabolism and fungal virulence.

microbiology↗

bioRxiv2026

SMECT: a framework for benchmarking post-GWAS methods for spatial mapping of cells associated with human complex traits

Spatially resolving the cellular basis of complex human traits is essential for elucidating disease mechanisms, yet the comparative performance of computational methods for this task has not been systematically evaluated. Here, we present SMECT (Spatial Mapping Evaluation of Complex Traits), the first comprehensive framework for systematically evaluating methods that integrate genetic data with spatial transcriptomics. SMECT combines a biologically realistic simulation engine, a curated resource of 21 diverse real-world datasets, and a multi-faceted assessment toolkit. Using this framework, we benchmarked three state-of-the-art methods--DESE, S-LDSC, and scDRS--across 19 complex traits. Our analysis reveals a fundamental trade-off between detection sensitivity and biological specificity. We demonstrate that while S-LDSC identifies extensive spatial signals, it suffers from inflated non-specific significant associations. Conversely, scDRS is highly specific but conservative, performing well only in tissues with strong biological signals while missing subtle associations in sparser datasets. DESE overcomes these limitations, consistently achieving high power and robust specificity across both simulated and real-world scenarios. SMECT provides critical guidelines for method selection and serves as a foundational resource for developing robust spatial analyses of human complex traits. The framework is publicly available at https://github.com/pmglab/smect.

bioinformatics↗

bioRxiv2026

Mismatch type impacts interference and priming activities in the type I-E CRISPR-Cas system

Type I-E CRISPR-Cas systems direct RNA-guided interference against foreign nucleic acids using the CRISPR RNA (crRNA)-guided Cascade complex and Cas3 helicase-nuclease. DNA targeting by Cascade-Cas3 promotes priming, a mechanism that allows for rapid acquisition of new spacers within the CRISPR array. Target mutations in the PAM and PAM-proximal seed region can block interference but may still allow priming. Previous studies have suggested that target mutations to T and A are tolerated, but that C and G substitutions are deleterious to interference and priming, respectively. However, the contributions of the crRNA spacer sequence to mutational tolerance remain unclear. Here, we systematically tested the effects of crRNA seed sequences on mutational tolerance. We engineered four E. coli strains with variable spacer sequences and tested CRISPR interference and priming against a plasmid library for each strain. Consistent with prior studies, we observe that mutations to C or G in the seed can be highly deleterious, especially at positions 1, 2 and 4. However, the corresponding crRNA sequence also strongly impacts the level of defect, with rC-dC and rA/G-dG causing the largest defects in our plasmid library experiments. Using in vitro biochemistry, we observe that mismatch type at the first position of the seed affects Cas8 conformation, and results in reduction in the rates of both Cascade-target binding and Cas3 recruitment. Overall, our results reveal that although nucleotide identity of target mutations is an important determinant of type I-E CRISPR immunity, the crRNA sequence also strongly impacts immune outcomes upon target mutation.

biochemistry↗

bioRxiv2025

Multimodal AI Decodes Extreme Environment Functional Dark Matter Beyond Homology

Functional annotation of proteins from extreme environments represents a major bottleneck for bioresource discovery, as a vast reservoir of functional dark matter defies existing homology-based methods. We demonstrate that environmental pressures impart conserved physicochemical energy signatures that co-determine protein function with sequence and structure. Here we developed ACCESS, a multimodal graph neural network employing hierarchical contrastive learning with a tailored label-sample co-embedding to fuse energy, sequence, and structural information and overcome homology scarcity. ACCESS surpasses state-of-the-art methods including BLASTp and CLEAN in annotating low-identity enzymes. Applied to extreme environmental metagenomics, we constructed a function map of extremophile enzymes to expand the biocatalyst library, pinpointed functionally critical residues to guide rational design, and enabled large-scale, function-based macro-evolutionary analyses. This paradigm transcends the limitations of homology, illuminating protein dark matter and accelerating the exploration of the biospheres functional diversity for applications in biotechnology and therapeutic development.

bioinformatics↗

bioRxiv2025

IS110 transposon utilizes two mechanistically distinct RNA-guided transposition pathways

The recently discovered IS110 elements employ RNA-guided transposases that use bridge-RNA to coordinate donor and target DNA recognition and integration via donor- and target-binding loops, offering promise for precise, DSB-free genome editing. However, their transposition mechanism remains unclear, with prior inconsistent observations. Using a IS110 element from Caloranaerobacter azorensis, we show that IS110 transposons utilize two independent transposition pathways. The first depends on bridge-RNA and excises the element via copy-out to form a circular intermediate, revisiting the earlier cut-out model. Integration occurs predominantly through top-strand insertion, suggesting an alternative to the earlier Holliday junction-mediated dual-strand mechanism. The second involves cooperation between a truncated target-binding loop RNA and bridge-RNA, enabling direct top-strand transfer to a new target via two sequential reactions, bypassing circular intermediates. Both pathways preserve the original transposon copy. These findings redefine the mechanistic understanding of IS110 transposition and reconcile prior discrepancies, offering insight into programmable, RNA-guided genome modification.

molecular biology↗

bioRxiv2025

Neuronal signatures of successful one-shot memory in mid-level visual cortex

High-capacity, one-shot visual recognition memory challenges theories of learning and neural coding because it requires rapid, robust, and durable representations. Most studies have focused on the hippocampus and other higher areas. However, behavioral evidence demonstrating links between image properties and memorability and revealing image specificity of visual memory suggests an important role for mid-level visual cortex. We tested the hypothesis that area V4 contains signals that could support recognition memory. Our task increased difficulty, allowing comparisons of neuronal population responses on correct and error trials. We observed signatures of several proposed memory mechanisms including magnitude coding, repetition suppression, sparse coding, and population response consistency, but only sparse coding and population response consistency predicted behavior. Familiar images also evoked faster dynamics, consistent with pattern completion. These findings demonstrate that the building blocks of fast, high-capacity memory are present in mid-level sensory cortex, highlighting its role in distributed memory networks.

neuroscience↗

bioRxiv2025

Developmental expression of risk genes implicates the age of onset for neuropsychiatric disorders

The functional effects of genetic variants associated with complex diseases exhibit pronounced spatiotemporal specificity. Although spatially resolved studies have advanced, their temporal dynamics remain poorly characterized. Here, we present an analytical framework integrating developmental gene expression with genome-wide association studies to decipher age-specific windows during which genetic variants exert their effects and to elucidate underlying mechanisms. Applying this framework to five major neuropsychiatric disorders, we uncover a fundamental principle: the peak incidence of a disease precisely coincides with the developmental window of peak expression of its associated risk genes in the prefrontal cortex. These risk windows are characterized by distinct biological processes; for instance, childhood risk for attention-deficit/hyperactivity disorder aligns with a peak in presynaptic machinery gene expression, whereas late-life risk for Alzheimers disease corresponds to heightened immune-related gene activity. Leveraging this principle of temporal convergence significantly improves the prioritization of disease genes. Our work establishes the developmental basis for the age of onset of complex diseases, providing a temporal roadmap for understanding disease mechanisms and developing age-appropriate therapeutic strategies.

genetics↗

bioRxiv2025

Towards a mental programming neural circuit: Insights from working memory sequence manipulation

Cognitive reasoning, also known as mental programming, is fundamental to our intelligence. A central function in mental programming is working memory (WM), involving both temporarily maintaining the information and manipulating it based on rules. While the neural mechanisms of WM maintenance have been extensively studied, those governing WM manipulation remain largely unknown. To bridge this gap, the present study focuses on an elementary operation in WM manipulation: re-ordering items in WM sequences. We propose a functional, biologically plausible neural circuit model that consists of two interconnected modules: a memory module composed of continuous attractor-based memory slots that store item features, and a control module sending gain-modulating commands to orchestrate specific operations in the memory module. The model successfully implements two-item swapping in a WM sequence, generating neuronal responses similar to recent primate experiments of WM sequence manipulation. By incorporating principles from the algebraic permutation group, we generalize the circuit model to accommodate more complex sequence manipulations. This math foundation reveals how arbitrary permutations can be decomposed into sequences of elementary swapping operations, which can be generated by a hierarchical tree-structured control circuit module. And the mutual inhibition within the control tree ensures that only one program is being executed at the same time. Our study establishes overarching connections among mental programming neural circuit models, neuro-science experiments, and abstract algebraic structure. These insights enhance our understanding of the neural underpinnings of cognitive reasoning and inspire the design of artificial cognitive systems.

neuroscience↗

bioRxiv2025

Hornbills Adjust Response Speed According to Solvability of Patterned-Strings Problems

Cognitive adaptations for processing causal information are fundamental to flexible problem-solving across species. Patterned-string tasks offer a classic means-end problem, requiring subjects to identify the functional connection between an action and its distal outcome. This study investigated patterned-string pulling in East Asian hornbills, a cognitively underexplored avian taxon, to assess their problem-solving abilities and underlying cognitive mechanisms. Nine hornbills were presented with three string configurations of increasing complexity: parallel (baseline), broken (contact/no-contact), and crossed strings. Subjects reliably selected the correct string in the baseline and contact conditions, demonstrating sensitivity to physical continuity cues. In contrast, accuracy declined significantly in the crossed condition, which required recognition of spatial displacement between the baited and accessible string ends. Reaction time (RT) analyses revealed prolonged latencies in the crossed condition, likely reflecting increased cognitive demands or limited habituation. Individuals who failed often adopted suboptimal strategies, such as proximity or side biases. We also observed lateralisation effects, with a left-side RT advantage, suggesting hemispheric specialisation. These findings support the hypothesis that task complexity modulates both decision accuracy and response speed, highlight RT as a useful proxy for interim cognitive processing, and establish hornbills as a valuable comparative model for studying causal cognition in birds.

animal behavior and cognition↗

bioRxiv2025

Mannoprotein Cig1 Contributes to the Immunogenicity of a Heat-Killed F-box Protein Fbp1 Cryptococcus neoformans Vaccine Model

Currently, no fungal vaccine exists for clinical use while fungal infections are responsible for over 1.5 million deaths every year. Our previous studies identified a Cryptococcus neoformans mutant strain fbp1{Delta} as a potential vaccine candidate. This mutant strain contains a deletion of the F-box protein Fbp1, a key subunit of the SCF E3 ligase complex necessary for ubiquitin-mediated proteolysis. Vaccination with heat-killed fbp1{Delta} (HK-fbp1) can elicit protection against C. neoformans parental strain and its sibling species C. gattii in an interferon gamma (IFN-{gamma}) dependent Type 1 immune response. However, we have yet to decipher the immunogenic factor(s) expressed by the fbp1{Delta} mutant that are responsible for the induction of the protective immune response. In this study, we have identified that capsule plays an important role in HK-fbp1 vaccine mediated protection, as acapsular HK-fbp1 cells showed diminished protection against wild type challenge. Additionally, our studies have shown that Cytokine Inducing Glycoprotein 1 (Cig1), a GPI anchored mannoprotein, is regulated by Fbp1 and contributes to the immunogenicity of HK-fbp1. Deletion of Cig1 in the fbp1{Delta} background resulted in decreased recruitment of anti-fungal effector T cells and diminished production of protective inflammatory cytokines by the host. Furthermore, loss of Cig1 in the fbp1{Delta} mutant resulted in reduced protection in vaccination survival studies at lower vaccine inoculum doses compared to HK-fbp1. In aggregate, these findings demonstrate Cig1 is an antigen contributing to the immunogenicity of HK-fbp1 that may be utilized to further optimize the HK-fbp1 fungal vaccine as a tool in the arsenal against invasive fungal infections.

microbiology↗

bioRxiv2025

Comprehensive Multimodal Profiling of Atherosclerosis Reveals Bhlhe40 as a Potential Regulator of Vascular Smooth Muscle Cell Phenotypic Modulation

BackgroundVascular smooth muscle cells (VSMCs) play a central role in atherosclerosis by undergoing phenotypic modulation from a quiescent, contractile state to a range of synthetic phenotypes, including fibroblast-like, macrophage-like, and lipid-laden foam cell-like states. However, a comprehensive multimodal characterization and understanding of the transcriptional programs driving these transitions remain incomplete. MethodsTo comprehensively define the phenotypic diversity of VSMCs during atherosclerosis progression, we performed in-depth profiling using cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and bulk RNA sequencing in a VSMC lineage-tracing atherosclerotic mouse model. Insights from these datasets guided the design of targeted in vitro experiments to investigate candidate regulatory mechanisms. ResultsSingle-cell multi-omics revealed extensive cellular heterogeneity within atherosclerotic plaques, including a rare population of VSMC-derived macrophage-like cells, whose presence was confirmed by histological analysis. These studies also identified a substantial population of VSMC-derived foam cells, comprising approximately 70% of all foam cells in the lesions. These cells exhibited activation of gene programs associated with lipid metabolism, proliferation, and tumor-like features. The transcription factor Bhlhe40 emerged as a key regulator of this phenotypic transition, with elevated expression in VSMC-derived foam cells during disease progression. Functional knockdown of Bhlhe40 suppressed VSMC phenotypic switching and foam cell characteristics, underscoring its potential role as a driver of VSMC modulation. ConclusionsThese findings advance our understanding of VSMC phenotypic modulation in atherosclerosis and highlight Bhlhe40 as a key regulator of this process. Elucidating the mechanisms governing VSMC plasticity may offer new therapeutic opportunities to reduce cardiovascular risk by targeting disease-driving cellular transitions.

molecular biology↗