Biology subjects

Wu, H.

Publications and source records attributed to Wu, H..

At least 127 records · Page 7Linked to original sources

bioRxiv2025

Local RhoA activation induces septin recruitment

The regulation of the actin cytoskeleton is key for controlling cell shape and structure. While the Rho GTPase RhoA is well known to regulate the actomyosin cytoskeleton, its function in controlling the septin cytoskeleton remains unclear. As RhoA interactions can vary in both time and space, they can be challenging to discern from traditional bulk biochemical assays. Here we use multiple optogenetic tools to spatially and temporally increase myosin localization, stimulate contractile force, and activate RhoA, to investigate how RhoA and its downstream effector myosin impact the septin cytoskeleton. We find that neither local accumulation of myosin nor increased activity of myosin is sufficient to alter septin architecture. Local activation of RhoA, however, results in a local increase in septin accumulation. Importantly, this septin increase is independent of the scaffolding protein anillin, which can directly bind both septin and RhoA. Together these data expand the potential role of septins in mediating RhoA signaling by stimulating the remodeling of the septin cytoskeleton.

cell biology↗

bioRxiv2025

Programmable Aptamer-Embedded Circular RNAs for Targeted Antigen-Presenting Cells Immunotherapy

The targeted delivery of mRNA to specific cell types in vivo remains a major unmet need in mRNA-based therapeutics. Conventional strategies rely on nanocarriers engineered with various modifications. However, these exogenous materials could cause adverse reactions, and their complex manufacturing processes often hinder clinical application. By leveraging the innate programmability of RNA sequences, we herein report a carrier-free, scalable, and programmable aptamer-embedded circular RNAs (Apt-circRNAs) design that confers targeting capability through the strategic incorporation of different aptamers at varying densities, while retaining circular RNA stability and protein production capability. Experimental results show that antigen-loaded Apt-circRNAs enable targeted delivery to antigen-presenting cells (APCs), drive endogenous antigen expression, activate antigen-specific T cells, and elicit potent immune responses that mediate clearance of both early-and late-stage tumors in mouse models. In humanized models of colorectal cancer, the combination of Apt-circRNA and PD-1 blockade produced synergistic antitumor effects and significantly reshaped the tumor microenvironment. In a first-in-human (FIH) clinical trial, Apt-circRNA vaccine encoding KRAS G12D/G12V neoantigens exhibited a favorable safety profile and induced robust immune activation, T cell mobilization, and neoantigen-specific immune responses. With this study, we have established a new paradigm in carrier-free, APC-targeted RNA vaccine design, offering a safe and effective platform for next-generation cancer immunotherapy.

bioengineering↗

bioRxiv2025

Probing the activity of cysteine cathepsins in inflammatory bowel diseases

Cathepsin S is a cysteine protease that has been implicated in inflammatory bowel diseases (IBD) for its ability to promote visceral pain. Given its pro-inflammatory roles, we hypothesized that cathepsin S would drive other symptoms associated with IBD. Using activity-based probes, we investigated cysteine cathepsin activation in human and murine colitis. We observed a significant increase in fecal cathepsin S in patients with ulcerative colitis compared to healthy controls, while cathepsin S in mucosal biopsies was unchanged. Mice with experimental colitis exhibited a modest increase in mucosal activity of both cathepsin S and X compared to naive mice. Luminal secretion of cathepsin S was dramatically increased upon colitis induction, although differences between mouse colonies were observed. To investigate the contribution of cathepsin S and cathepsin X to colitis, we induced colitis in cathepsin-deficient mice. Cathepsin X-deficient mice exhibited no clear differences in disease indicators compared to wild-type mice. While cathepsin S-deficient mice exhibited less rectal bleeding, less splenomegaly and marginally improved histological scores, weight loss, diarrhea, colon shortening, and myeloperoxidase activity were not significantly different from wild-type mice. To determine whether pharmacologic inhibition of cathepsin S activity would ameliorate symptoms of colitis, a reversible inhibitor LY3000328 was administered to mice at the initiation of colitis. LY3000328 provoked a clear upregulation of cathepsin S and L activity in the mucosa, most likely through a compensatory mechanism. This increase in protease activity was associated with exacerbated histological scores and splenomegaly. Collectively, these results suggest that cathepsin S, but not cathepsin X, may contribute to some of the symptoms of experimental colitis. While cathepsin S has potential to be a therapeutic target in colitis, improved strategies to sustain its inhibition are required in future.

pathology↗

bioRxiv2025

Iron Metabolism and Antioxidant Defense: The Role of LI-rTMS in Cerebral Ischemia-Reperfusion

BACKGROUNDTreating cerebral ischemia/reperfusion injury (CIRI) is challenging, with iron metabolism imbalances and oxidative stress worsening the condition. While low-intensity repetitive transcranial magnetic stimulation (LI-rTMS) shows potential in brain protection, its effects on iron processing and antioxidant use are unclear. This study explored LI-rTMSs role in regulating iron homeostasis and antioxidant defense for potential therapeutic use in CIRI. METHODSA rat model of CIRI was established using a filament insertion technique, followed by neurological and infarct volume assessments. In PC12 cells, oxidative damage was induced with H2O2 or ferric ammonium citrate (FAC), then treated with LI-rTMS or deferoxamine (DFP). Parameters measured included oxidative stress markers, iron metabolism markers, and mitochondrial integrity. Gene knockdown and overexpression experiments explored mechanisms involving LI-rTMS and ACSL4. RESULTSLI-rTMS improved neurological deficits by boosting GPX4 activity and inhibiting the ACSL4-LPCAT3-LOX axis, reducing lipid peroxidation. It restored iron balance by lowering TFR and DMT1, decreasing ferritin and hepcidin, and increasing FPN for better iron efflux. LI-rTMS also prevented the Fenton reaction, reduced ROS production, and maintained mitochondrial structure and transmembrane potential. CONCLUSIONSLI-rTMS offers neuroprotection by regulating iron homeostasis through dual pathways: boosting export (via FPN) and reducing uptake/storage (via TFR/DMT1/FE). It also provides ACSL4-dependent antioxidant benefits, suggesting its potential to prevent ferroptosis in CIRI.

biophysics↗

bioRxiv2025

DELPHAI, AI Agent for Predicting Drug Response and Resistance

Patient-derived organoids preserve critical tumor features and drug sensitivity patterns that mirror patient clinical responses, enabling single-cell RNA sequencing analysis of drug responses. Analyzing these perturbation data presents significant computational challenges in predicting cellular responses while maintaining biological interpretability. We developed DELPHAI (Deep ExplainabLe Predictive Human-organoid based AI), an AI agent that integrates single-cell perturbation prediction with mechanistic analysis using large language models. We designed a comprehensive benchmarking framework evaluating methods in both computational embedding and reconstructed gene expression spaces. Applied to glioblastoma organoids treated with temozolomide, optimal transport combined with principal component analysis outperformed baseline methods in capturing population dynamics. DELPHAI correctly identified DNA alkylation as the mechanism of action without prior drug knowledge and recommended combination therapies aligning with clinical trials. These results demonstrate DELPHAPs ability to translate single-cell perturbation data into actionable therapeutic insights, representing a significant advance toward Al-driven precision medicine in cancer treatment.

bioinformatics↗

bioRxiv2025

Regulation of epithelial tissue homeostasis by active trans-epithelial transport

Epithelia are intricate tissues whose function is intimately linked to mechanics. While mechanobiology has primarily focused on factors such as cell-generated contractility and mechanical properties of extracellular matrix (ECM), a novel mechanobiological paradigm highlights the role of osmotic and mechanical pressures in shaping epithelial tissues. In our study, we developed an in vitro model of cell coated micro-sized hydrogel spheres (MHSs) which allows to decipher the interplay between cellular activities and tissue mechanics. Drastic, isotropic MHS compressions were observed once the epithelia reached confluence. Further studies revealed that the compression was a process independent of cell contractility but rather regulated by active transepithelial fluid flow. Compressive stresses of about 7 kPa are generated by such an active hydraulic mechanism. Tissue homeostasis is then maintained by a fine balance between cell proliferation and extrusion. Our findings demonstrate the critical role of fluid transport in generating mechanical forces within epithelial tissues. Supported by a theoretical mechano-hydraulic model, a mechanistic framework for understanding the intricate interplay between cellular processes and tissue mechanics was established. These results challenge traditional views of epithelial tissue mechanics, emphasizing the pivotal influence of osmotic and mechanical pressures in shaping tissues. We anticipate that this study will advance the understanding of epithelial tissue development, the maintenance of homeostasis, and the mechanisms underlying pathological conditions. Significance StatementEpithelial tissues are vital for many bodily functions, but their mechanics remain poorly understood. Our study uncovers a novel mechanism by which epithelial cells generate mechanical stress, not through traditional cell contractility, but by actively pumping ions and water across their membranes. Using a model of micro-sized hydrogel spheres (MHSs) coated with epithelial cells, we demonstrate that epithelial cells actively transport fluid across the tissue to compress the MHSs. This facilitates the establishment of tissue homeostasis, which is further maintained by a balanced cell proliferation and extrusion rate. Supported by a theoretical model, our findings highlight the overlooked role of fluid transport in tissue mechanics, offering new insights into how epithelial tissues develop, maintain stability, and respond to disease.

cell biology↗

bioRxiv2025

JointMap identified SERPINA3+ chondrocytes as a therapeutic target for osteoarthritis

BackgroundOsteoarthritis (OA) is a prevalent degenerative joint disease that affects nearly half of the population aged over 65. Recent single-cell transcriptional studies have identified heterogeneous OA-specific chondrocyte subtypes, yet a unified subtype-classification remains incomplete. MethodsIn this study, we integrated a total of 545,946 cells to develop JointMap, an interspecies atlas. The chondrocyte subtypes were analyzed using machine learning algorithms were validated via human samples, animal models, and in vitro cell culture. We performed KAS-seq and analyzed the spatial characters for JointMap. ResultsBased on clinical diagnosis and cross-omic indexes, we identified a novel OA-elevated chondrocyte subtype, SERPINA3+ chondrocytes. Notably, we uncovered their upregulated chondrogenesis function and intercellular communication patterns as potential therapeutic targets. ConclusionsIn summary, JointMap provides a unified framework for the systematic annotation of chondral tissues across species and helps in the identification of druggable networks for articular diseases.

bioinformatics↗

bioRxiv2025

The two-component nuclease-active KELShedu system confers broad antiphage activity via abortive infection

Bacteriophages and bacteria engage in a continuous evolutionary arms race, driving the development of intricate bacterial defense systems such as CRISPR-Cas, BREX, Gabija, and Shedu. Here, we characterize a two-component KELShedu system in Escherichia coli that confers resistance to phages via abortive infection. The KELShedu system comprises KELA, a dsDNA-binding protein, and KELB, a metal ion-dependent nuclease harboring the DUF4263 domain. In addition, we find that physiological levels of NTP inhibit the DNA cleavage activity of the KELShedu system, suggesting that KELShedus activation depends on reduced intracellular NTP levels during phage invasion. Our research demonstrates that the KELShedu system responds to nucleotide depletion triggered by phage replication, leading to non-specific degradation of cellular DNA and ultimately inducing abortive infection. These insights into the KELShedu system expand the repertoire of bacterial anti-phage mechanisms and lay the groundwork for novel applications in microbial engineering and therapeutic development. TeaserThe KELShedu system exerts anti-phage function by aborting infection in response to physiological changes following phage infection

microbiology↗

bioRxiv2025

Functional characterization of a Con7-related transcription factor in Coprinopsis cinerea indicates evolutionary conservation of morphogenetic roles

Fruiting bodies of mushroom-forming fungi (Agaricomycetes) exhibit the highest degree of multicellular complexity in fungi, yet the molecular underpinnings of their developmental programs remain incompletely understood. Here, we characterize gcd1, a gene encoding a transcription factor in the Con7 subfamily of C2H2-type zinc finger proteins. This subfamily has previously been implicated in pathogenic morphogenesis in Ascomycota, but their role in Agaricomycetes has not previously been addressed. In Coprinopsis cinerea, CRISPR/Cas9-mediated deletion of gcd1 resulted in strains with severely impaired fruiting body morphogenesis, with malformed cap, stipe, and gill tissues. Gcd1 deletion strains lacked universal veil, resembling species with open (gymnocarpous) development. We find that GCD1/Con7 homologs are widely distributed in most Dikarya species and are mostly encoded by a single gene in each species genome. Transcriptome analyses identified several misregulated genes in the {Delta}gcd1 mutant, which pinpoint potential mechanisms underlying its developmental defects as well as provided insights into the morphogenesis of mushroom fruiting bodies. These findings establish GCD1 as a key regulator of multicellular development in C. cinerea and broaden the known functions of Con7-like transcription factors to include fruiting body morphogenesis in Agaricomycetes. Overall, our results and the morphogenetic role of Con7-like transcription factors of Ascomycota suggest functional conservation over half a billion years of evolution.

microbiology↗

bioRxiv2025

Tafazzin-Mediated Cardiolipin Remodeling Controls Metabolic Stress Response and Effector Function of Inflammatory T Cells

Clonal expansion and effector differentiation of T cells require extensive metabolic reprogramming. This includes the restructuring of the inner mitochondrial membrane (IMM) to enhance respiration by increasing chemiosmotic coupling efficiency. Cardiolipin, a unique phospholipid that is exclusively synthesized and localized in the IMM, modulates the biophysical properties of the electron transport chain (ETC) in tissues with high energy demands, such as cardiomyocytes and skeletal muscle. However, it remains unclear whether cardiolipin is also important for metabolic remodeling during T helper (Th) cell differentiation. In this study, we show that cardiolipin transacylation, catalyzed by the enzyme Tafazzin, supports the clonal expansion and effector function of inflammatory Th1 and Th17 cells in vitro and in models of autoimmune colitis and encephalomyelitis. At the molecular level, we demonstrate that loss of Tafazzin-mediated cardiolipin maturation induces a metabolic and transcriptional stress response in Th cells to compensate for impaired coupling efficiency of the ETC complexes and disrupted cellular redox homeostasis. However, the genetic program that restores cellular homeostasis and mitigates oxidative stress concurrently impairs the effector functions of inflammatory T cells, such as cytokine production. Our findings also provide insights into the complex clinical manifestation of patients with Barth syndrome (BTHS) caused by mutations in the human TAFAZZIN gene. BTHS is characterized by cardiac and skeletal myopathy as well as neutropenia and an increased susceptibility to infections. Although the molecular basis of the immunodeficiency remains poorly understood, our findings suggest that impaired Th cell function contributes to the immunopathology observed in BTHS patients.

immunology↗

bioRxiv2025

The Pulmonary Microbial Signatures of Early Stages of Severe Infection in Children

The respiratory tract related diseases are the main causes of death in children. Child pulmonary severe infection is the most important inducement for Respiratory tract related diseases. Therefore, it is necessary to understand the composition of the microbial community and the species correlation in children with severe pulmonary infection. In this study, the bronchoalveolar lavage fluid (BALF), blood and cerebrospinal fluid (CSF) samples of 782 children with severe infection in the early stage were systematically analyzed by amplicon-sequencing technology to reveal the distribution of microbial community and its clinical correlation. Results reveal significantly higher bacterial abundance in BALF than in blood/cerebrospinal fluid, with community structures shaped by tissue microenvironments, geographical disparities, age, and clinical symptoms. Neonatal BALF harbors simplified microbiota dominated by probiotics, while diversity increases with age, showing marked differences between 1-3 and 6-12-year-olds. Sepsis samples exhibit reduced microbial diversity, with Staphylococcus and Moraxella enrichments in the CST-4 cluster. Respiratory symptom progression correlates with microbiota succession from oral colonizers to respiratory pathogens. Third-generation sequencing-derived co-occurrence networks illustrate synergistic interactions among opportunistic pathogens, providing a basis for ecological targeted therapy. This study provides a key basis for breaking through the limitations of traditional diagnosis, establishing a precise diagnosis and treatment system based on third-generation sequencing, and developing ecological targeted treatment strategies.

pathology↗

bioRxiv2025

CD8+ T cells regulate the bioenergetic reprogramming of lymphoid organs and the heart during viral infection

The activation of the immune system is a bioenergetically-costly process1. Yet, essential bodily functions require a continuous energy supply, imposing energy constraints and trade-offs between competing processes2. Our understanding of the underlying bioenergetic adaptations reconciling rapid immune activation with other vital processes remains scarce. 3-6 Here, by using experimental models of viral infections, we identified an unexpected CD8+ T cell-driven redistribution of energy substrates between lymphoid organs and the heart. Viral infection promoted systemic hypoglycaemia and ketogenesis, together with systemic reallocation of energy substrates. Across organs analysed, secondary lymphoid organs and the heart showed the most dramatic changes. The former increased glucose uptake and oxidation while the heart showed the opposite, switching to preferential fatty acid utilization. These bioenergetic adaptations were absent in infected mice lacking CD8+ T cells or with T cells lacking the glucose transporter GLUT1. Pharmacological inhibition of fatty acid oxidation forced a systemic switch to glucose oxidation. This was associated with metabolic decompensation, reduced cardiac energetics, left ventricular stress, and mortality in otherwise nonlethal viral infections. Our results reveal how the energetic cost of immune cell activation imposes bioenergetic adaptations on non-lymphoid organs, posing a major challenge for the heart by completely relying on fatty acids.

immunology↗

bioRxiv2025

Distinguishing cortical indexes of arousal and awareness in sleeping patients with unresponsive wakefulness syndrome

Consciousness is assumed to be defined by two components: awareness and arousal. However, compared with awareness, the cortical index for arousal remains lacking in clues. Since patients with unresponsive wakefulness syndrome (UWS) are supposed to have no awareness but arousal, sleep-wake cycle in patients with UWS may reflect pure arousal fluctuations, and then could highlight the cortical indexes for arousal. This study recorded nighttime polysomnography in patients with UWS, patients with minimally conscious state and healthy controls. The EEG signal showed that spectral slope could index arousal, fluctuating among sleep stages in all three groups. Both spectral entropy and Lempel-Ziv complexity indexed awareness, showing a significant difference between conscious and unconscious states. These findings provide fundamental evidence for the two-component hypothesis of consciousness.

neuroscience↗

bioRxiv2025

Intracranial Insights into the Developing Neural Basis of Moral Punishment

Third-party punishment (TPP) is a critical component of social regulation and justice, in-tegrating moral reasoning and emotional salience. However, the neural developmental basis underpinning this complex process remains largely unknown. Using rare intracranial stereo-electroencephalography (SEEG) in 14 children and 17 adults, we investigated the developing neural circuits of TPP. We found that broad-band gamma activity in the amygdala and ventromedial prefrontal cortex (vmPFC) encodes inferred intentions, with different patterns across age groups. Furthermore, the vmPFC, insula, and inferior parietal lobule (IPL) integrate punishment efficacy, also showing significant developmental differences. Combining task and resting state functional connectivity analyses, we further found age-dependent interactions among the amygdala-insula and IPL-vmPFC neural couplings during decision-making. These findings provide valuable intracranial evidence that the maturation of moral decision-making stems from the developmental refinement of subcortical-cortical circuits that integrate emotional and cognitive evaluations, explaining the shift from intuitive decisions in children to context-sensitive judgments in adults.

neuroscience↗

bioRxiv2025

A Precision Ultrasound-Localized Sonoporation-Equipment (PULSE) Enabling Intratumoral Delivery for Cancer Immunotherapy

Cancer immunotherapy offers a promising long-term treatment for solid tumors by activating the immune system, but side effects and systemic toxicity limit its broad clinical translation. While intratumoral delivery addresses these limitations, elevated stiffness within solid tumors continues to hinder both intracellular and extracellular delivery. Here, a novel therapeutic platform, the Precision Ultrasound-Localized Sonoporation-Equipment (PULSE), is presented to enable spatiotemporally controlled, ultrasound-mediated gene and drug delivery for intratumoral immunotherapy. The PULSE incorporates forward and sideward-looking miniaturized ultrasound transducers, integrated into either a 7-French catheter (cPULSE) or a 16-gauge needle (nPULSE), to minimally invasively sonicate a centimeter-sized tumor. Following the design, prototyping, and characterization of PULSE, its dual capability for intracellular and extracellular delivery is demonstrated through comprehensive in vitro cell-based sonoporation tests and phantom-based drug penetration tests. Luciferase assays (activity: ~104 RLU/g level) in the cell studies confirm significantly enhanced gene transfection with cPULSE, while increased dye diffusion (width: ~1 cm level) in the phantom tests validates improved perfusion with nPULSE. The reported PULSE shows promise for spatiotemporally precise, controlled, and localized therapeutic delivery in early-stage tumors for intratumoral immunotherapy.

bioengineering↗

bioRxiv2025

LIM Domain Proteins link molecular and global tension by recognizing strained actin in adhesions

Mechanotransduction is fundamental to cell signaling and depends on force-sensitive adhesion proteins. How these proteins differentiate and integrate their responses to tension remains an open question. We show mechanosensitive LIM domain proteins like zyxin detect global adhesion tension by recognizing strained actin within these structures. In sharp contrast, vinculin localization and intramolecular tension remain unchanged, despite vinculins well-documented role in mechanotransduction. This reveals a stark disconnect between molecular tension and global tension in adhesions. We further show tension-dependent localization is specific to LIM domain proteins that recognize strained actin and extends to LIM proteins at cell-cell junctions, suggesting a common mechanotransduction mechanism. Finally, we show zyxins tension-dependent adhesion localization stabilizes actin and recruits VASP to promote stress fiber polymerization, identical to its role in stress fiber repair. Our findings reveal a fundamental role for LIM domain protein force-sensing in adhesions and highlight the non-linear connection between molecular and global tension.

cell biology↗

bioRxiv2025

Genomic Surveillance Reveals Global Spread of Macrolide-Resistant Bordetella pertussis Linked to Vaccine Changes

The resurgence of whooping cough in regions utilizing acellular pertussis vaccines underscopes emerging public health challenges. Here, we characterized 178 Bordetella pertussis isolates collected from patients across all age groups in Shanghai (2018-2024) to assess genomic evolution and antibiotic susceptibility. Macrolide resistance to erythromycin, azithromycin, clarithromycin and clindamycin escalated from [≤]50% (pre-2020) to nearly 100% (post-2020), mechanistically linked to the 23S rRNA A2047G mutation. Genome-based analysis identified a genotype MT28-ptxP3-MRBP rapidlly dominated post-2020, exhibiting significantly higher prevalence in adults versus than age groups. Phylogenetic analysis of 178 Shanghai and 1596 global genomes revealed two major lineages corresponding to ptxP1 and ptxP3 alleles. MT28-ptxP3-MRBP cluster was identified in France, Japan and the United States in 2024, indicating potential cross-border dissemination. These findings advocate for intergrated surveillance spanning all ages and international borders to contain the global spread of macrolide-resistant Bordetella pertussis. HighlightsO_LIAfter 2020, MT28-ptxP3-MRBP lineage rapidly dominated, comprising 61.7% of isolates. C_LIO_LIMT28-ptxP3-MRBP exhibits a significant transmission advantage among older individuals. C_LIO_LIThe primary affected group shifted from [≤]36 months (pre-2020) to 37 months-18 years (post-2020). C_LIO_LIMacrolide resistance rose from [≤]50% pre-2020 to nearly 100% post-2020, with all resistant isolates carrying the A2047G mutation. C_LI

microbiology↗

bioRxiv2025

Expanding the repertoire of chemically induced covalent neoantigens

The ubiquitin-proteasome system generates peptide fragments that are displayed on MHC-I molecules. Recent studies revealed that covalent small molecules can hijack this process, leading to the presentation of hapten-modified peptide fragments, or covalent neoantigens. Here, we report a chemical proteomics platform to systematically investigate this process and harness covalent neoantigens for immune cell recruitment and activation. Using a bioorthogonal cysteine-reactive probe (IA-mTz), we demonstrate that diverse intracellular proteins can be modified, processed, and presented as covalent neoantigens across multiple cell lines. Mass spectrometry confirmed the presence of IA-mTz-modified peptides within the MHC-I immunopeptidome, and structural modeling revealed stable peptide-MHC-I interactions. We further integrated a bioorthogonal strategy that enables immune engagement: IA-mTz-modified cells conjugated with trans-cyclooctene (TCO)-IgG elicited Fc receptor activation in a reporter assay, supporting immune recruitment via antibody-dependent cellular phagocytosis. This approach was extended to additional cysteine-reactive probes and to 2,4-dinitrochlorobenzene (DNCB), a common skin sensitizer, which we show induces covalent neoantigen formation in a cell- and HLA-dependent manner. These findings reveal a previously underappreciated breadth of covalent neoantigen formation and provide a generalizable strategy for immune targeting of covalent neoantigens.

biochemistry↗