Biology subjects

Cao, W.

Publications and source records attributed to Cao, W..

At least 19 recordsLinked to original sources

bioRxiv2026

X-Y divergence of house fly (Musca domestica) proto-sex chromosomes follows distinct evolutionary trajectories despite residing in the same genome

Sex determination systems and sex chromosomes frequently differ between species. One cause of these differences is new sex determining genes that drive evolutionary turnover of sex chromosomes. At the earliest stages of this turnover, X and Y (or Z and W) chromosomes start out as nearly identical homologs, and they can diverge via chromosomal rearrangements (e.g., inversions) that suppress X-Y recombination. However, multiple examples from across animals and plants provide exceptions to this canonical model of sex chromosome evolution. For example, some X-Y pairs remain undifferentiated for long evolutionary time periods, while other sex chromosomes become differentiated without chromosomal rearrangements. How or why these non-canonical trajectories occur remains elusive, despite increasing evidence of their pervasiveness. The house fly, Musca domestica, is a well-suited system to address this gap because all six chromosomes can be a Y, providing multiple replicates of a natural experiment within a single genomic environment. To test for canonical and non-canonical evolutionary trajectories, we generated haplotype-resolved chromosome-level assemblies from five strains of the house fly, each of which carries a different Y chromosome (IM, IIM, IIIM, VM, and YM). We identified an inversion on only one of the sex chromosomes (IIM), which was associated with elevated X-Y divergence but did not capture the male-determining locus. In contrast, there was X-Y divergence across almost the entire length of the IM, IIIM, and VM sex chromosomes, despite no detectable inversions. YM was the only sex chromosome to contain substantial Y-specific sequences, which were limited to a segment on one end of the chromosome containing the male-determining gene. This YM chromosome, and its corresponding X, were highly diverged from the X chromosome found in many other flies (Muller element F), despite a strong cytological resemblance. This study highlights how multiple different canonical and non-canonical modes of sex chromosome evolution can co-exist within a single genome.

genomics↗

bioRxiv2026

Rice blast fungus hijacks a rice RALF peptide to initiate infection

Plant-derived peptides regulate immunity and host-microbe interactions, but whether fungal pathogens exploit endogenous plant peptides for infection-site selection remains largely unknown. Rapid alkalinization factors (RALFs) are a family of plant peptide hormones that contribute to plant development, reproduction, stress responses, and cell-wall organization. Here, we show that OsRALF7, which is an immune-activating peptide in rice (Oryza sativa), is co-opted by the rice blast fungus Magnaporthe oryzae as a host-derived cue for appressorium formation, specialized infection structures required for host penetration. While synthetic OsRALF7 peptide induced defense responses in rice suspension cells, the same peptide induced appressorium formation in M. oryzae. OsRALF7-induced appressorium formation was associated with activation of the cAMP/PKA and Pmk1 MAPK pathways and required a hydrophobic surface. M. oryzae inoculated onto Osralf7 knockout plants formed fewer appressoria, and the positioning of these appressoria relative to the boundaries between two adjacent plant epidermal cells was altered. These findings suggest that OsRALF7 provides positional information for infection initiation for M. oryzae. OsRALF7 localized to the plasma membrane and cell wall in rice, and its appressorium-inducing activity required a conserved YISY motif and cysteine residues. This activity was not conserved in Colletotrichum higginsianum, as RALF23 from Arabidopsis thaliana did not induce appressorium formation but instead showed antifungal activity. Together, these findings uncover a dual function of endogenous plant immune-modulating RALF peptides at the plant-pathogen interface, where they can be exploited by M. oryzae as a host-derived cue for infection initiation while also retaining antifungal activity against other fungal species.

plant biology↗

bioRxiv2026

High-Dimensional Multi-omic Mapping of Post-Mortem Human Brain Using Iterative Indirect Immunofluorescence Imaging on Xenium-Processed Tissues

Spatial transcriptomics approaches provide crucial insights into gene expression distribution within intact tissue architecture, but they encounter limitations in detecting morphologically complex cell types, assessing their spatial associations with pathology, and accurately annotating cell types using RNA data alone. Therefore, we developed a robust post-processing workflow integrating Xenium spatial technology with iterative indirect immunofluorescence imaging (4i) on formalin-fixed paraffin-embedded (FFPE) human brain tissue. The post-Xenium 4i protocol presented here allows multi-omic tissue mapping, enabling deeper investigation of pathological microenvironments defined by the spatial distribution of neuropathological hallmarks and enrichment of specific cell populations. We applied this workflow on calcarine cortex tissue sections where cerebral amyloid angiopathy (CAA) burden is present, in addition to amyloid plaques and tau pathology, generating a 15-plex image that captures the complexity of the pathological microenvironment.

neuroscience↗

bioRxiv2026

Decoding the Transcriptome Dark Matter: Construction of Single-Cell Whole-Transcriptome Regulatory Atlas by dropTotal

To profile unknown ncRNAs-"dark matter" in single cells, we developed dropTotal, a high-throughput droplet-based total RNA-seq method that uses dU-modified GAT primer with temperature-ramp hybridization and droplet merge barcoding to co-detect coding and non-coding transcripts with record sensitivity (>13,500 genes/cell, including >2,000 lncRNAs and >500 sncRNAs), compatible with fresh, frozen, fixed, and FFPE tissues. Applied to ~75,000 human glioma nuclei, it captured 60,313 genes (18,681 lncRNA, 19,859 mRNAs and 6,753 sncRNAs), enabling ncRNA-driven regulatory landscape construction. In oligodendroglioma, module analysis identified recurrence-associated ncRNA-centered modules linked to therapy resistance and invasion; in glioblastoma, six cellular states showed hundreds of state-specific unannotated ncRNAs with divergent functions, from MIR222HG-mediated immune modulation to SCIRT-driven hypoxia adaptation. Alternative splicing analysis identified 428 state-specific junction markers and mapped cell-state-specific alternative splicing regulation. dropTotal offers broad application for decoding the underlying ncRNA biology and single-cell whole transcriptome regulatory mechanisms in cellular identity and disease progression.

genomics↗

bioRxiv2026

rVSV-EBOV vaccination protects ferrets from lethal Bundibugyo virus disease

An uncontrolled and rapidly growing outbreak of Bundibugyo virus (BDBV) is currently gripping the Democratic Republic of the Congo and threatening health security across Central Africa. There are no available BDBV-specific vaccines, although emerging evidence suggests that the Ebola virus-specific vaccine, rVSV-EBOV (also known by its tradename ERVEBO), may offer cross-protective immunity. To directly address this question, we evaluated the efficacy of rVSV-EBOV in the uniformly lethal ferret model of BDBV infection. All vaccinated animals survived BDBV challenge and exhibited minimal clinical signs of infection, presumably as a result of a moderate--but protective--humoral immune response. These findings provide critical evidence further supporting the cross-protective efficacy of rVSV-EBOV, and they suggest a potential role for this vaccine in mitigating the ongoing BDBV outbreak.

microbiology↗

bioRxiv2026

Complex-valued representations of time-series gene expression profiles for network analysis

Time-series RNA sequencing provides a powerful framework for studying dynamic gene regulation, yet conventional analyses usually represent gene expression profiles as real-valued vectors in Euclidean space and quantify similarity using correlation or distance. Inspired by quantum information theory, we present a framework for encoding time-series gene expression profiles as complex-valued vectors comprising amplitude and phase components in Hilbert space. We designed multiple encoding models to represent gene expression in the amplitude of complex-valued vectors, encode temporal differences in the phase, and extend the phase representation to incorporate the direction of local expression changes. Gene-gene similarity was then quantified using fidelity, which measures the overlap between two encoded vectors. Evaluation using time-series RNA-seq datasets across diverse species and biological contexts showed that different encoding models produced distinct fidelity distributions that were related to, but distinct from, conventional correlation measures. We then constructed gene-gene networks using pairwise fidelity values and detected communities containing genes with similar temporal profiles. Although fidelity distributions differed across encoding models, the resulting communities captured major temporal expression programs, and functional annotations based on gene ontology and Kyoto encyclopedia of genes and genomes pathway analyses provided exploratory biological context. The detected communities were comparable to those obtained using conventional methods, including weighted correlation network analysis and fuzzy c-means clustering. Furthermore, as a proof-of-concept, we performed SWAP-test circuit simulations to mimic fidelity computation on a quantum computer; under noise-aware conditions, these simulations produced less accurate fidelity estimates with higher computational cost than classical computation. As a proof-of-concept, this study provides a complementary view of temporal transcriptome organization, rather than a uniformly superior alternative to conventional methods.

bioinformatics↗

bioRxiv2026

Lineage Memory and Convergent Adaptation Dictate the Single-Cell and Spatial Architecture of Brain Malignancies

How diverse cancer lineages navigate the draconian central nervous system environment--whether constrained by ancestral ontogeny or driven by convergent adaptation--remains a fundamental biological paradox. To decode this, we integrated extensive in-house and public resources to construct the largest single-cell atlas of primary and secondary brain malignancies (>550,000 cells). We demonstrate that malignant cells balance strict lineage imprinting with shared brain-adaptive programs, powerfully converging upon a dominant pan-cancer mesenchymal (MES)-like state alongside a SYT1+ pioneer subpopulation exploiting neuronal mimicry. Concurrently, the microenvironment undergoes lineage-constrained divergence. The myeloid landscape shifts from a resident-dominated architecture in gliomas to extensive blood-borne infiltration in metastases. Lymphoid responses parallel this, crowning laryngocarcinoma as an ultra-hot subtype and revealing that massive T cell influx in highly infiltrated niches is paradoxically driven into terminal exhaustion, contrasting with severe immune exclusion in cold tumors. Furthermore, stromal-vascular adaptation couples pro-angiogenic tip-endothelial activation with a dynamic opposition between structural extracellular matrix (ECM) deposition and focal proteolytic degradation. Anchored by the pan-cancer MES adaptation, we leveraged subcellular spatial transcriptomics to redefine the microenvironment around four MES-like architectural niches, mapping an evolutionary trajectory from perivascular entry to immunosuppressive stromal remodeling. Finally, projecting these architectures onto independent clinical cohort establishes the MES-S2 proliferative niche as the primary driver of severe clinical deterioration across both transcriptomic and proteomic dimensions.

Cancer Biology↗

bioRxiv2026

A Liver-Targeted Copper Supplement Reduces Metabolic Dysfunction-Associated Liver Steatosis by Increasing Lipolysis and Fatty Acid Oxidation

Metabolic-associated steatotic liver disease (MASLD) is a prevalent liver disease driven by complex dysregulation of hepatic lipid metabolism. Here we show that copper deficiency is a nutrient vulnerability in steatotic liver disease and that selective liver-targeted copper supplementation can reduce excess lipid accumulation. Analysis of steatotic patient and mouse tissues identify widespread alterations in hepatic copper homeostasis markers. Integrated multi-omics analyses reveal that copper induces lipolysis of PLIN2-containing lipid droplets while lipid importer CD36 is downregulated. We show that copper inhibits cAMP hydrolase activity of PDE3B, thus activating PKA-mediated HSL and AMPK activation upstream of lipolysis. Fatty acids liberated through lipolysis are subsequently degraded via enhanced mitochondrial fatty acid oxidation, supported by energetic rewiring toward oxidative phosphorylation (OXPHOS) with increased copper-dependent complex IV and SOD1 activity. Our findings establish a multi-pronged mechanism by which hepatic copper supplementation coordinately regulates lipid metabolism in response to steatosis and unveils a therapeutic metallomedicine strategy to rewire lipid regulation. SummaryLiver-targeted copper supplementation reduces diet-induced liver steatosis by dual activation of lipolysis and fatty acid degradation pathways. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/725917v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@982970org.highwire.dtl.DTLVardef@1c8adb5org.highwire.dtl.DTLVardef@4762dorg.highwire.dtl.DTLVardef@ea95cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2026

Population Structure and Antimicrobial Resistance Gene Transfer of Respiratory Escherichia coli Isolated from Swine in China

ABSTRACPorcine respiratory diseases caused by extraintestinal pathogenic Escherichia coli (ExPEC) pose a severe threat to swine production and public health; however, research on respiratory tract-isolated ExPEC remains limited. This study comprehensively analyzed the genomic characteristics and antibiotic resistance gene (ARG) transfer potential of 441 ExPEC strains isolated from porcine lungs across 21 Chinese provinces (including 53 newly isolated strains from 2022-2024 and 388 NCBI-deposited strains). Phylogenetic analysis revealed that 84% of the isolates belonged to phylogroups A, B1, and C, with ST410, ST101, and ST88 as the predominant STs. The strains exhibited extensive ARG diversity, harboring 111 distinct ARG subtypes, with sul2 (81.4%), floR (73.5%), and tet (A) (68.0%) being the most prevalent. Importantly, critical "last-resort" antibiotic resistance genes (e.g., blaNDM-1/5, the mcr family, and tet (X4)) were also detected. Notably, 77.2% of the ARGs presented horizontal transfer potential, with plasmids (especially IncF family replicons) serving as core vectors, followed by integrons and transposons. Cooccurrence network analysis identified aph (3)-Ib, aph (6)-Id, sul2, and floR as core subnetworks driving multidrug resistance dissemination. Pangenomic analysis confirmed an open genome architecture, with core genes accounting for only 6%, reflecting the strains capacity to acquire exogenous genetic material via horizontal transfer. From the One Health perspective, these transferable ARGs can spread to the environment and humans through fecal discharge and the food chain. These findings underscore the importance of monitoring and controlling ExPEC infections in swine, as such strains can as reservoirs of ARGs, pose potential risks to human health, and may even act as sources of pathogenic agents responsible for human infections. IMPORTANCEPorcine respiratory ExPEC-induced diseases threaten swine production and public health, yet respiratory tract-isolated ExPEC research remains scarce. This study comprehensively analyzed 441 porcine lung ExPEC strains across 21 Chinese provinces, uncovering their dominant phylogroups, high ARG diversity (111 subtypes) and the presence of "last-resort" antibiotic resistance genes. We identified 77.2% of ARGs with horizontal transfer potential, plasmids (especially IncF family) as core vectors, and a core ARG subnetwork driving multidrug resistance. The open pangenome (6% core genes) highlights ExPECs strong capacity to acquire exogenous genes. These findings fill the research gap of respiratory ExPEC, clarify ARG transmission mechanisms in swine ExPEC, and provide critical genomic data for One Health-based AMR surveillance and control, guiding targeted strategies to mitigate ARG spread from swine to humans and the environment.

microbiology↗

bioRxiv2026

Using Cryogenic Electron Tomography (cryoET) to Determine Rubisco Polymerization Constants in α-Carboxysomes

Bacteria microcompartments (BMCs) are pseudo-organelles comprised of a self-assembling, semi-permeable protein shell, most commonly enclosing components of enzymatic pathways. -Carboxysomes (-CBs) are anabolic BMCs known for their role in sequestering Rubisco, the enzyme responsible for carbon fixation in plants, algae and bacteria, along with an upstream enzyme and an assembly protein. Rubisco has low selectivity for its substrate, CO2, and has a slow enzymatic turnover rate, resulting in an inefficient metabolic pathway. Within the -CB, Rubisco has been observed at a range of concentrations and with either a liquid-like assembly or a pseudo-lattice of polymerized fibrils. The biophysical origins of the fibril ultrastructure organization are unclear; however, it is only observed inside -CBs. Quantitative knowledge of the binding constants and energies for assembly and maintenance of these fibrils is critical for understanding this organization and Rubisco regulation, but quantitative methods for in situ analysis of Rubisco polymerization have been lacking. Here, we present an approach to convert tomography-derived -CB volumes and Rubisco particle positions into polymerization binding curves. We used this procedure to determine the Rubisco polymerization constants, including the nucleus size (n) and equilibrium polymerization constant (Kpol). The adopted modeling approach is consistent with in situ constraints, such as concentration-dependent binding interactions and confinement. This approach offers a powerful tool to evaluate both in vitro and potentially in vivo biomolecular interactions, both of Rubisco and of other proteins and polymers suitable for analysis by cryo-electron tomography. Significance StatementCryogenic electron tomography (cryoET) is a powerful method to resolve structures of proteins in their native environment at subnanometer-level resolution. Because tomography data retains spatial relationships of all particles, it intrinsically contains information about component (e.g., protein) binding interactions. Here, we use Rubisco polymerization in -carboxysomes as a model system to demonstrate that quantitative, biochemical binding analysis is possible with cryoET.

biophysics↗

bioRxiv2026

A feed-forward loop between niche adenosine and Gzmk⁺ CD8 T cells propagates systemic inflammaging

The causal link between the aging microenvironment and T cell aging remains elusive. Here, we demonstrate that adenosine within aging tissues actively reprograms CD8+ T cells into a pro-aging Granzyme K+ (Gzmk+) population. Mechanistically, senescent cells create an adenosine-rich niche via p16-dependent CD39 upregulation, triggering A2aR signaling to induce Gzmk+ T cell differentiation. Once released, Gzmk promotes systemic inflammaging through PAR1 and complement activation. Crucially, targeting this axis--either via genetic Gzmk ablation or pharmacological A2aR blockade--reverses multi-organ aging phenotypes and significantly extends healthy lifespan in mice. Human analysis reveals age-dependent Gzmk+ T cell accumulation in multi organs, while coffee intake (an A2aR antagonist) inversely correlates with plasma Gzmk levels. Our findings uncover how metabolic niche changes drive T cell aging and establish the adenosine-Gzmk axis as a pivotal therapeutic target for combating age-related diseases.

immunology↗

bioRxiv2026

Brainwide dopamine dynamics across sleep-wake transitions

Dopaminergic signaling plays a critical role in regulating arousal and transitions between sleep states, yet brainwide dynamics underlying these transitions remain incompletely characterized. Here, we employed multi-site fiber photometry with the dopamine sensor GRAB-DA2m, combined with electroencephalogram (EEG)/electromyogram (EMG) recordings, to systematically assess regional dopamine (DA) dynamics across sleep-wake states in the medial prefrontal cortex (mPFC), striatal subregions, central amygdala (CeA), and midbrain nuclei in mice. We found that DA levels prominently increased in the mPFC, dorsolateral striatum (DLS), ventral tegmental area (VTA), substantia nigra pars compacta (SNc), and dorsal raphe nucleus (DRN) during transitions from non-rapid eye movement (NREM) sleep to wakefulness (WAKE), supporting their role in arousal. Conversely, DA decreased in the CeA and nucleus accumbens lateral parts (NAc-L) during these transitions. During transitions from NREM to rapid eye movement sleep (REM), DA elevations were observed in the CeA and middle accumbens subregion (NAc-M), rather than NAc-L, while other regions exhibited decreases. Cross-regional DA correlations revealed synchronized network activity across sleep-wake transitions. Optogenetic activation of VTA and DRN dopamine neurons induced robust DA release in cortical and subcortical regions, and chemogenetic activation promoted wakefulness selectively via VTA and DRN, but not SNc DA neurons. These results elucidate distinct brainwide DA dynamics across state transitions and highlight differential roles for DA signaling in modulating sleep-wake states.

neuroscience↗

bioRxiv2026

PlotGDP: an AI Agent for Bioinformatics Plotting

High-quality bioinformatics plotting is important for biology research, especially when preparing for publications. However, the long learning curve and complex coding environment configuration often appear as inevitable costs towards the creation of publication-ready plots. Here, we present PlotGDP (https://plotgdp.biogdp.com/), an AI agent-based web server for bioinformatics plotting. Built on large language models (LLMs), the intelligent plotting agent is designed to accommodate various types of bioinformatics plots, while offering easy usage with simple natural language commands from users. No coding experience or environment deployment is required, since all the user-uploaded data is processed by LLM-generated codes on our remote high-performance server. Additionally, all plotting sessions are based on curated template scripts to minimize the risk of hallucinations from the LLM. Aided by PlotGDP, we hope to contribute to the global biology research community by constructing an online platform for fast and high-quality bioinformatics visualization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/702995v3_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@73c77dorg.highwire.dtl.DTLVardef@12ddfc6org.highwire.dtl.DTLVardef@be5963org.highwire.dtl.DTLVardef@de90b6_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstract.C_FLOATNO PlotGDP: an AI Agent for Bioinformatics Plotting C_FIG

bioinformatics↗

bioRxiv2026

Characterization of Atypical Ebola Virus Disease in Ferrets

Ebola virus (EBOV) typically results in a severe--and often lethal--acute disease. However, increasing evidence suggests that EBOV can persist in certain immune-privileged tissues, which may then serve as reservoirs for the later reemergence of EBOV and disease recrudescence. Here, we report atypical EVD recrudescence in a ferret model inoculated with an otherwise lethal dose of EBOV and treated with low doses of a highly potent monoclonal antibody cocktail. Among 32 antibody-treated ferrets, 14 animals survived, while 8 succumbed to acute EVD within about 5-8 days. The remaining 10 animals succumbed to atypical EVD between 12 and 18 days post-infection (DPI) despite having shown no, or very minor, signs of illness during the acute phase of disease. While viremia disappeared by 14 DPI in most animals that succumbed to atypical EVD, it rebounded modestly just prior to death. Unlike animals that died of acute EVD, those that died of atypical EVD showed only a moderate systemic inflammatory response and few signs of organ dysfunction, in line with low levels of virus in the liver and spleen. Interestingly, however, ferrets that died of atypical EVD showed high levels of virus in the brain, consistent with increased markers of inflammation in the central nervous system and significant pathological changes, including a breakdown in the blood-brain barrier and severe meningoencephalitis. Not only does this study shed important light on the atypical and underappreciated manifestations of EVD, but it also establishes the ferret as a valuable model of EBOV persistence and recrudescence. AUTHOR SUMMARYOver the last several years, it has become increasingly apparent that Ebola virus is capable of persisting in survivors and, occasionally, reemerging to cause a recrudescent disease that is often distinct from the acute illness and characterized by neurological involvement. Since nearly all existing Ebola virus animal models are uniformly lethal, it has been difficult to understand the processes that lead to persistence and atypical manifestations of disease. In this study, we describe a late-onset, atypical Ebola virus disease in ferrets that was characterized by high levels of virus in the brain and significant markers of brain inflammation. Our comprehensive analysis of the pathogenic processes that contributed to this disease not only provides critical insight into Ebola virus persistence and recrudescence, but it also establishes the ferret as the first tractable model for investigating these atypical outcomes.

microbiology↗

bioRxiv2026

Lysosomal Aspartylglucosaminidase Couples Glycoprotein Catabolism to Cell-Surface GlycoRNA Production

GlycoRNAs, small non-coding RNAs covalently linked to N-glycans, reveal an unexpected intersection between RNA biology and glycosylation, with potential roles in immunity and cell-cell communication. The origins and biogenesis of their glycans remain unclear. Using CRISPR-Cas9 screening, metabolic glycan labeling and lysosome-targeted proteomics, we reconfirm canonical N-glycosylation enzymes (STT3A/B) and identify components of the mannose-6-phosphate (M6P) lysosomal sorting pathway (GNPTAB, M6PR) as essential contributors. Strikingly, lysosomal aspartylglucosaminidase (AGA) acts as a rate-limiting regulator, linking glycoprotein catabolism to glycoRNA formation. Loss of AGA collapses both abundance and diversity of RNA-linked N-glycans, while pulse-chase tracing shows glycoprotein-derived glycans are repurposed for RNA glycosylation. These findings position lysosomes as active participants in RNA modification and redefine glycoRNA biogenesis as a metabolically integrated process.

cell biology↗

bioRxiv2025

A Comprehensive Treatment-Induced Resistance Atlas of Glioblastoma Reveals a Fibrotic Niche Shielding the Tumor from Immunotherapy

Therapeutic resistance in IDH-wildtype glioblastoma (GBM) is driven by an intricate interplay between cellular plasticity and protective microenvironmental niches. By constructing the GRIT-Atlas--a massive transcriptomic compendium encompassing nearly one million high-quality single cells across 17 cohorts--and cross-analyzing it with 48 independent Visium spatial transcriptomics sections, we discover a recurrent "Spatial Resistance Triad" comprising mesenchymal-like (MES-like) malignant cells, myeloid-derived suppressor cells (MDSCs), and collagen-secreting cancer-associated fibroblasts (CAFs). This large-scale spatial integration reveals that the triad physically fortifies microvascular proliferation (MVP) and pseudopalisading necrosis (PAN) niches. To substantiate these findings at true single-cell resolution, we deploy high-plex spatial molecular imaging (CosMx SMI) to map over 400,000 single cells across a clinical validation cohort. This high-resolution architecture firmly validates the triads geography and, critically, provides structural gradient evidence unmasking a spatiotemporal continuum wherein PAN emerges as a direct functional consequence of MVP advancement and subsequent vascular collapse. Mechanistically, stromal CAF-derived COL6A1 engages CD44 receptors on MES-like cells to accelerate a potent neural stemness cascade within these protective domains. Utilizing a blood-brain barrier (BBB)-penetrant multi-library compound screen, we identify Lacidipine as a multi-modal stromal disruptor that successfully silences CAF activation and halts matrix secretion. Incorporating Lacidipine into the standard chemo-immunotherapy backbone (TMZ + anti-CSF1R) completely dismantles the protective desmoplastic matrix niche, forcing a profound collapse of intracranial tumor burden and dramatically extending overall survival in orthotopic models. Collectively, our study leverages unprecedented single-cell and spatial scale to provide a definitive blueprint of GBM therapeutic evasion, establishing matrix-targeted intervention as a mandatory prerequisite for successful glioblastoma eradication.

cancer biology↗

bioRxiv2025

Precise Control of Switchable Chimeric Antigen Receptor T Cells Allows Enhanced Safety and Less T Cell Exhaustion

BackgroundChimeric antigen receptor (CAR)-T cell therapies have achieved remarkable success in hematologic malignancies, yet their clinical utility remains limited by safety concerns, limited persistence, and T-cell exhaustion driven by continuous receptor signaling. While switchable CAR designs provide external control, many reported systems are irreversible, strictly binary, or compromise CAR-T potency. MethodsWe engineered an optimized chemically switchable CAR platform (CSN CAR) that pharmacologically regulates antigen engagement by controlling the surface expression of the full-length CAR. An NS3 protease module was embedded within the CAR construct to enable drug-dependent stabilization of intact CAR on T cells. Using engineered CAR-T cells, we quantified drug-controlled activation, cytotoxicity, and cytokine release against CD19+ tumor cells by flow cytometry and ELISA in vitro. We further screened clinically approved NS3/4A inhibitors in CAR-HEK and CAR-T cells to identify optimal small-molecule controllers. A chronic stimulation model was established to assess CAR-T persistence and exhaustion-associated phenotypes in vitro. ResultsCSN CAR-T cells enabled precise, dose-dependent regulation of CAR surface density, cytokine production, and cytotoxicity. In the OFF state, switchable CAR-T cells showed minimal basal activity, consistent with reduced antigen-driven activation and cytokine release in the absence of drug in the experimental conditions. Upon drug addition, intact surface CAR was detectable within 1 hour, reaching [~]80% of peak observed CAR expression by 4 hours. Reversible suppression of CAR expression enabled attenuation of cytotoxicity toward normal CD19+ B cells in vitro after target-cell reduction, supporting a potential strategy to mitigate prolonged on-target/off-tumor activity. Under chronic stimulation, switchable CAR-T cells exhibited reduced exhaustion-associated markers, more stable CAR expression, and preferential differentiation toward a central memory phenotype. ConclusionTogether, these findings establish CSN CAR as a reversible and tunable switchable CAR-T platform enabled by clinically approved NS3/4A inhibitors, supporting controllable modulation of CAR activity with potential applications for improving the precision and safety of CAR-T cell therapy.

cancer biology↗

bioRxiv2025

Target-Stabilized Base Editors Enable Robust High-Fidelity RNA Editing

RNA base editing using engineered deaminases expands target compatibility, offering a powerful tool to correct mutations at the RNA level. However, widespread off-target effects, primarily arising from dissociated free deaminases, remain a significant challenge. Here, we devised the RECODE (RNA editing with conditionally stable and enhanced ADAR1 deaminase variants) system, which employs designer degron-tagged ADAR1 deaminase (ADAR1d) with guide RNA (gRNA)-regulated stability. By promoting degradation of gRNA-unbound ADAR1d, RECODE markedly reduced transcriptome-wide edits while maintaining high on-target efficacy. Engineering gRNA for target RNA-induced conformational switching restricts ADAR1d stabilization, further enhancing editing precision. With structure-guided rational engineering of ADAR1d, RECODE achieved editing efficiencies of 90.3% and 58.8% for an Amyotrophic Lateral Sclerosis-relevant FUS mutation in vitro and in vivo, respectively. These findings establish RECODE as a highly stringent and efficient RNA editing technology and underscore a general principle for enhancing the specificity of RNA-guided protein effectors.

molecular biology↗