Biology subjects

Ding, X.

Publications and source records attributed to Ding, X..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

A Time-Sequenced Three-Drug-Loaded Microneedle Patch for the Prevention of Vein Graft Restenosis After Coronary Artery Bypass Grafting

Coronary artery bypass grafting (CABG) is the recommended treatment for severe coronary heart disease; however, restenosis of great saphenous vein grafts continues to limit long-term outcomes because of early thrombosis, mid-term intimal hyperplasia, and late atherosclerosis. To address these sequential pathological processes, this study develops a perivascular drug-loaded microneedle (MN) patch capable of controlled chronological release of tirofiban, rapamycin, and rosuvastatin. The MN patch exhibits uniform conical microneedles (height: 700-800 {micro}m), nanoparticle size of 125 nm, sufficient mechanical strength (1.07 N/needle) for venous intimal penetration, excellent biocompatibility, and no detectable cytotoxicity or hemolysis. In a mouse model of venous graft transplantation, the device significantly reduces early platelet thrombus formation (23.3% to 10%), alleviates mid-term intimal hyperplasia (intimal thickness: 0.84 mm to 0.45 mm at 4 weeks), and improves long-term graft patency with blood flow increasing from 13.1 to 65.8 mL/min at 32 weeks while reducing long-term mortality. Mechanistically, RNA sequencing and functional analyses demonstrate that excessive glycolysis-driven smooth muscle cell proliferation, coupled with abnormal endothelial cell apoptosis, constitutes the core mechanism underlying mid- to long-term restenosis, which the MN device effectively suppresses. This integrated MN patch provides a localised therapeutic strategy for preventing restenosis and improving long-term CABG outcomes. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/726718v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1de0e6borg.highwire.dtl.DTLVardef@184a846org.highwire.dtl.DTLVardef@43a50forg.highwire.dtl.DTLVardef@109bb85_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2026

A CAMKK2-UBR4-19S Proteasome Axis Regulates Chondrocyte Proteostasis and SOX9 Stability

ObjectiveInvestigate how Ca2+/calmodulin dependent protein kinase kinase 2 (CaMKK2) orchestrates a catabolic shift in chondrocytes during early osteoarthritis (OA). MethodsCartilage, osteochondral plugs and chondrocytes were collected from patients undergoing total hip arthroplasty or non-OA donors. SOX9 levels were assessed via immunoblotting or immunohistochemistry (IHC). Sox9 levels were also assessed by IHC in knee joints from wild-type (WT) and Camkk2-/- mice that underwent sham or destabilization of medial meniscus (DMM), with or without STO-609 (0.033 mg/kg) treatment. Co-immunoprecipitation followed by mass spectrometry was performed to identify CaMKK2 interacting proteins in chondrocytes. Kinase assays were analyzed by immunoblotting and phosphosites identified by mass spectrometry. Proteasome function was assessed in murine and human chondrocytes lacking or expressing kinase-active or kinase-inactive CaMKK2. ResultsInhibition or loss of CaMKK2 increased SOX9, whereas the expression of kinase-active, not inactive, CaMKK2 reduced Sox9 in human and mouse OA cartilage. Proteomic analysis of CaMKK2 immunoprecipitates revealed the presence of ubiquitin E3 ligase Ubr4 and the 19S proteasome regulatory particle (RP). CaMKK2 kinase activity was dispensable for its interactions with Ubr4, 19S RP, and Sox9-ubiquitin conjugates, and kinase-inactive CaMKK2 attenuated Sox9 degradation in chondrocytes. Further, CaMKK2 phosphorylated the 19S RP ATPase Psmc5 on Ser136, and an intact kinase increased proteasome activity in chondrocytes. ConclusionsOur findings identify CaMKK2 as a dual-function regulator of chondrocyte UPS with a scaffolding role to assemble UPSUbr4-19S RP around polyubiquitinated proteins such as Sox9, and a catalytic role to enhance proteasome function, potentially through Psmc5 phosphorylation, thereby linking chondrocyte inflammatory signaling to Sox9 degradation and cartilage degeneration.

cell biology↗

bioRxiv2026

G4 Eurasian avian-like H1N1 swine influenza viruses exhibit enhanced pathogenicity potential in mice and pigs

Currently circulating swine influenza viruses (SIVs) mainly include H1N1, H1N2, and H3N2 subtypes. In this study, two G4 genotype Eurasian avian-like (EA) H1N1 SIVs were isolated from 556 samples collected between 2023 and 2026. A systematic analysis was conducted on the two EA H1N1 isolates (FYD30 and YZF69) to assess their pandemic potential. The hemagglutinin (HA) proteins of both H1N1 viruses possessed residues 225E and 228S, indicating enhanced affinity for human-like -2,6-linked sialic acid receptors, which was confirmed by receptor-binding assays. Polymerase activity tests demonstrated that the two SIVs exhibited significantly higher activity in mammalian cells, relative to avian cells, which is consistent with the efficient replication in mammalian cells. Challenge experiments revealed that both H1N1 caused significant pathogenicity in mice and pigs, with YZF69 exhibited higher virulence than FYD30. The higher virulence of YZF69 may be attributed to its molecular features, including the NP Q357K mutation, and an additional glycosylation site in HA. In conclusion, currently circulating EA H1N1 SIVs have acquired key molecular signatures of mammalian adaptation, exhibit enhanced virulence in mammals, and continue to undergo extensive reassortment driven by international swine trade. These findings highlight the potential pandemic risk of SIVs and underscore the urgent need for strengthened surveillance.

microbiology↗

bioRxiv2026

A High-Fidelity 3D Fluid-Structure Interaction Framework for Predictive Microfluidic Design

The commercial maturation of microfluidics remains bottlenecked by empirical prototyping and an absence of predictive digital design capabilities. Because optimizing advanced technologies such as passive particle separation fundamentally hinges on the precise coupling of fluid dynamics and particle mechanics, conventional two-dimensional or decoupled fluid simulations inherently fail to capture authentic multiscale behaviors. To bridge this gap, we establish a high-fidelity three-dimensional fluid-structure interaction framework combining a high-order Arbitrary Lagrangian-Eulerian mapping-based finite element method with a localized hierarchical dynamic mesh strategy. Engineered to accurately resolve complex multiscale hydrodynamics, this architecture utilizes deterministic lateral displacement structures as a stringent test case. Validated against experimental data for rigid microspheres and tumor cells, the framework predicts transport trajectories and critical separation diameters with sub-micron precision. Crucially, the simulation explicitly resolves the M-shaped spatial fluctuation of local size thresholds alongside the dynamic vertical migration of particles. Unveiling these hidden physical mechanisms provides a deterministic explanation for highly debated phenomena such as mixed-mode transport. By enabling the rigorous in silico evaluation of complex non-periodic architectures, this framework serves as a powerful instrument for predictive structural optimization. Such capabilities establish the essential infrastructure for microfluidic digital design, accelerating the transition from empirical trial-and-error to precision simulation-driven engineering.

bioengineering↗

bioRxiv2026

Controlled linking of AAV capsids enables coordinated multi-vector delivery

Adeno-associated virus (AAV)-based delivery of large genetic systems splits cargo across vectors, but functional reconstitution depends on stochastic co-delivery and often requires high viral doses. Here, we present a multi-capsid linking strategy that enables coordinated delivery of AAV vectors carrying distinct or split genetic cargos. Using a substrate-confined linking process, we suppress uncontrolled higher-order aggregation while enabling programmable nanoscale capsid coupling via oligonucleotide linkers. These linkers further enable efficient downstream purification of linked AAVs and recycling of unlinked vectors, yielding [~]70% AAV dimers and trimers in <24 hours. By enforcing coordinated delivery, linked AAVs achieve co-transduction at reduced viral doses and with greater expression uniformity than conventional dual-AAV approaches. This improved coordination enhances reconstitution of split genetic systems and increases targeted prime editing efficiency in vitro and in the mouse brain. This capsid coupling strategy expands the functional scope of AAVs toward dose-efficient and uniform delivery of large genetic systems.

bioengineering↗

bioRxiv2026

Rapid Podocyte ablation Causes Acute Renal Tubule Cell Necrosis and Interstitial Fibrosis

It remains unclear whether podocyte loss directly causes acute renal tubular cell (RTC) damage and interstitial fibrosis, thereby leading to renal failure. Here, we applied intermedilysin (ILY)-mediated human CD59 (hCD59) cell ablation to generate an acute, specific podocyte-ablation mouse model. Cre-induced hCD59 transgenics (ihCD59) were crossed with Nphs2Cre to generate ihCD59+/-/Nphs2Cre+/- mice. The specific and rapid podocyte-ablation mediated by ILY injection directly caused RTC necrosis, leading to renal failure and even death within 2-3 days in a dose-dependent manner. Treating mice that received an ILY lethal dose with peritoneal dialysis or administering a non-lethal dose, we extended their survival beyond six weeks and found that mice developed interstitial fibrosis and glomerulosclerosis with persistent proteinuria and tubule damage. Podocyte-ablation caused massive disruption of glomerular function at week 1, and then partial recovery by week 2. Genes and pathways of TLRs and apoptosis, and mitochondrial functions were respectively upregulated and downregulated in both ablated-podocyte mouse and biopsied-glomerulonephritis patient kidney samples. Together, this rapid podocyte-ablation causes acute RTC necrosis that progresses to interstitial fibrosis in this mouse model, which is applicable for dissecting mechanisms underlying podocyte injury-mediated tubular damage and glomerular repair, with the potential to reveal novel therapeutic targets for kidney diseases.

pathology↗

bioRxiv2026

Telomere-to-telomere assembly and haplotype analysis of tetraploid Dendrobium officinale illuminate Orchidaceae polyploid evolution and mycorrhizal symbiosis genes

Dendrobium officinale is a typical epiphytic orchid. We report the telomere-to-telomere (T2T) genome assembly for D. officinale, representing the first T2T reference genome within the Orchidaceae family. The assembly is anchored to 19 chromosomes and contains 38 complete telomeres and 15 characterized centromeres. We further generated haplotype-resolved assemblies of the autotetraploid genome, identifying 12,761 sets of tetra-allelic genes. Based on synonymous substitution analysis, we inferred that the autotetraploidization event occurred approximately 0.86 million years ago. A systematic analysis of the SWEET gene family across the genus Dendrobium revealed that the gene family size is shaped primarily by epiphytic types and environmental factors. In D. officinale from Langshan, eight SWEET genes were specifically expressed in roots, suggesting they may play specialized roles in the root mycorrhizal system, potentially contributing to the D. officinales ability to recruit and maintain fungal partners. Together, these resources provide valuable foundations for studies of orchid evolution, functional genomics, and molecular breeding.

bioinformatics↗

bioRxiv2026

Betrayal is worse than loss during cooperation

Cooperative behavior is a cornerstone of human interaction. Although both "betrayal aversion" (the affective cost of being betrayed) and "loss aversion" (the financial detriment incurred from betrayal) are established determinants of cooperative behavior, their relative potency remains undetermined. Here, we investigated these effects by integrating computational modeling and event-related potential (ERP) techniques. In two tasks involving risk and cooperation, participants decided whether to take financial risks or to cooperate under possible betrayal. Our results showed that betrayal aversion had a stronger effect on reducing cooperation compared to loss aversion. Furthermore, ERP data demonstrated sequential processing: betrayal was encoded early in decision-making, reflected by increased P3 with weaker betrayal aversion, whereas loss aversion manifested later, marked by increased LPP. By dissociating the contributions of betrayal and loss, our finding provides novel insights into the cognitive and neural mechanisms underlying cooperative behavior.

neuroscience↗

bioRxiv2026

A universal platform for simultaneous TCRα/β removal enables safer and more potent TCR therapies and autoimmune modeling

Adoptive T-cell therapies using tumour-specific T-cell receptors (TCRs) are limited by competition with endogenous receptors, which impairs efficacy and poses risks of off-target autoreactivity. Here we present a CRISPR-based platform that completely and selectively eliminates both endogenous TCR- and -{beta} chains without affecting introduced transgenic TCRs, irrespective of codon optimization. This approach achieves >90% deletion efficiency in Jurkat and primary human T cells, markedly enhancing the expression, pairing fidelity, and functional potency of transgenic receptors. Using a clinically relevant HLA-A*02:01-restricted DMF5 TCR, we show that dual TCR ablation boosts antigen-specific activation and cytotoxicity in vitro and significantly enhances tumor clearance in vivo in human immune system (HIS) mice, while preventing graft-versus-host disease (GVHD). Targeted locus amplification revealed that CRISPR-induced double-strand breaks did not alter lentiviral integration profiles, confirming genomic safety. Extending this approach to four insulin-reactive TCRs demonstrated that removal of endogenous receptors increased transduction efficiency and functional activity, with one (1E6) showing selective activation and infiltration of stem cell-derived islet grafts (SC-islets) in vivo. This study establishes a universal, safe, and scalable genome-editing platform for generating functionally precise human T cells. By integrating cancer immunotherapy and autoimmune disease modelling within a single framework, it provides a strong preclinical rationale for dual endogenous TCR removal as a route to improved specificity, safety, and therapeutic efficacy in TCR-based cell therapies.

immunology↗

bioRxiv2025

Missense Mutation ADNP p.C687R Disrupts Chromatin Regulation and GABAergic Differentiation in HVDAS

Activity-dependent neuroprotective protein (ADNP) is a key regulator of neurodevelopment and a high-risk gene for autism spectrum disorder (ASD). Most pathogenic variants cause loss-of-function in Helsmoortel-Van der Aa syndrome (HVDAS), yet the impact of missense mutations remains unclear. Here, we characterized a ADNP missense mutation (p.C687R) identified from a HVDAS patient, predicted to disrupt its ninth zinc finger domain. Overexpression of p.C687R altered subnuclear localization and affected wild-type ADNP distribution. In utero electroporation revealed impaired neuronal migration and cortical branching. CUT&Tag profiling showed mutant- specific chromatin binding, preferentially targeting histone modification genes through both direct and distal interactions. Patient-derived iPSCs carrying p.C687R exhibited upregulated bivalent histone marks (H3K4me3 and H3K27me3) on neurodevelopmental genes, including regulators of GABAergic differentiation, which became transcriptionally activated in neural progenitors. These findings reveal a gain-of-function mechanism by which ADNP p.C687R perturbs chromatin regulation and disrupts GABAergic lineage specification, expanding the molecular framework of ADNP pathogenesis. The Paper Explained ProblemMutations in the gene ADNP cause Helsmoortel-Van der Aa syndrome (HVDAS), a severe neurodevelopmental disorder, and also increase the risk for autism spectrum disorder (ASD). While most known ADNP mutations lead to loss of protein function, many missense mutations that alter a single amino acid remain poorly understood regarding their effects on brain development and disease contribution. ResultsWe characterized a specific missense mutation, ADNP p.C687R, from a patient diagnosed with HVDAS, and found that it does not simply inactivate the protein but instead confers a disruptive new function. Across multiple models, from cells to mice, the p.C687R mutant exhibits abnormal chromatin binding, altered subnuclear localization, and impaired neuronal migration and differentiation. Importantly, in patient-derived iPSCs, it marks distinct neurodevelopmental genes with bivalent histone modifications, which become aberrantly activated during differentiation into GABAergic neural progenitor cells. ImpactThese findings confirm the pathogenicity of the missense mutation ADNP p.C687R in HVDAS, reveal a gain-of-function mechanism whereby the mutant disrupts chromatin regulation and GABAergic lineage specification, and advance our understanding of ADNP-related neurodevelopmental disorders.

neuroscience↗

bioRxiv2025

Persistent Activation of Monocytes/Macrophages and Cell Senescence in SIV-Infected Macaques on ART

Despite effective viral suppression with antiretroviral therapy (ART), people living with HIV (PLWH) experience persistent inflammation, immune dysfunction, and premature onset of cardiovascular and aging-related comorbidities. To define the underlying mechanisms, we performed longitudinal transcriptomic profiling in peripheral blood mononuclear cells (PBMCs) from a cohort of simian immunodeficiency virus (SIV)-infected rhesus macaques spanning four key stages: pre-infection, acute infection, short-term ART, and long-term ART. Bulk RNA sequencing revealed dynamic immune remodeling across infection and treatment. Acute SIV infection induced robust antiviral and inflammatory programs, with upregulation of interferon-stimulated genes (ISGs), IL-27, JAK/STAT, and NF-{kappa}B signaling, coupled with suppression of T- and B-cell activation pathways. Short-term ART effectively reversed these transcriptional perturbations, restoring adaptive immune gene expression and reducing innate antiviral responses to near-baseline levels. In contrast, chronic SIV infection on long-term ART maintained viral suppression but was characterized by reactivation of innate immune pathways, including TLR2/TLR4/MYD88, NF-{kappa}B, and inflammasome (NLRP3/or NLRP12, caspase-1) signaling, along with sustained macrophage activation, platelet/coagulation signaling, and senescence-associated secretory phenotype. Protein analyses confirmed persistent CASPASE-1 and NF-{kappa}B activation in spleen tissue. Pathologic evaluation of a carotid artery from an SIV-infected, long-term ART- treated macaque revealed macrophage-rich plaques with p21 senescent cells with intraluminal thrombus formation, recapitulating key features of HIV-associated atherogenesis. Together, these findings demonstrate that while ART normalizes acute infection-induced immune dysregulation, chronic SIV infection sustains a chronic, macrophage- and TLR-driven inflammatory state linked to vascular injury and aging process regardless of long-term suppression of viremia. Targeting inflammasome, NF-{kappa}B, and senescence pathways may mitigate non-AIDS comorbidities in PLWH.

immunology↗

bioRxiv2025

Deciphering the Antigenic Evolution of Seasonal Influenza A Viruses with PREDAC-Transformer: From Antigenic Clustering to Key Site Identification

Seasonal influenza viruses undergo continuous antigenic drift due to mutations in the hemagglutinin (HA) protein, rendering vaccines ineffective and posing a significant global public health challenge. Existing computational models can predict antigenic relationships but generally lack interpretability, making it difficult to reveal the molecular determinants driving antigenic changes. To address this, we developed PREDAC-Transformer, an end-to-end deep learning framework that integrates sequence, physicochemical, and evolutionary features, and employs a self-attention mechanism to capture long-range dependencies relevant to antigenicity. More importantly, we introduce the integrated antigenicity score, which combines attention-based attribution with information-theoretic metrics to provide continuous quantification and ranking of the antigenic contributions of individual amino acid site. Our results demonstrate that PREDAC-Transformer not only significantly improves the accuracy of antigenic relationship prediction but also successfully recapitulates major historical antigenic cluster transitions. Using integrated antigenicity score, we systematically identified two classes of key sites: global key sites with sustained impact on antigenic evolution, and cluster-transition determining sites that drive cluster transitions. These sites include most canonical epitopes and reveal additional functional residues previously overlooked, which may influence immune escape via cooperative effects or glycosylation. Collectively, these findings advance our understanding of influenza antigenic evolution and provide novel insights for refining computational models. PREDAC-Transformer achieves high-precision prediction while attributing antigenic differences to individual residues, thereby linking viral genomic variation, antigenic change, and public health decision-making. This framework has the potential to reduce experimental burdens in influenza surveillance and assist in vaccine strain recommendation, thereby supporting global influenza control efforts.

bioinformatics↗

bioRxiv2025

OASIS: in vivo AAV-mediated transduction and genome editing of adult oligodendrocytes

New viral approaches have revolutionized neuroscience by precisely delivering genes in neurons; for example, to control or monitor activity in specific neuronal cell types. In contrast, the manipulation of oligodendrocytes requires the Cre-LoxP system and gene-by-gene engineering, breeding, and genotyping. Here we introduce OASIS (Oligodendrocyte AAV-CRISPR mediated Specific In vivo editing System), a versatile platform that combines SELECTIV, an AAV-receptor-based transduction strategy, with HiUGE, an NHEJ-mediated CRISPR/Cas9 knock-in approach. We show efficient and specific oligodendrocyte transduction across the brain and tagging of endogenous cytoskeletal, myelin, cell adhesion, scaffolding, and junctional proteins. OASIS enables sparse yet reliable labeling, allowing direct visualization of a proteins subcellular localization with single-cell resolution. We successfully fused the biotin-ligase TurboID with endogenous oligodendroglial Neurofascin-155, thereby achieving targeted biotinylation of the axoglial junction. OASIS is rapidly customizable for any gene-of-interest. Together, OASIS overcomes longstanding barriers in oligodendrocyte biology, providing a powerful system for precise, customizable genome editing and subcellular visualization in the adult brain.

neuroscience↗

bioRxiv2025

Muscular dystrophy-associated lamin variants disrupt cellular organization through a nucleolar-ribosomal axis controlling cytoplasmic macromolecular crowding

Emery-Dreifuss muscular dystrophy (EDMD) arises from mutations in nuclear lamins or emerin. Current pathological models emphasize defects in nuclear mechanics and transcription regulation. Yet these models do not fully explain the complexity of EDMD or other laminopathies. Here, we uncover an emerging pathway linking nuclear lamina defects to the reorganization of cytoplasmic biophysics, revealing how nuclear dysfunction cascades throughout the cell. Using Caenorhabditis elegans EDMD models, we demonstrate that lamin mutations dramatically alter cytoplasmic organization, reducing macromolecular crowding and increasing diffusivity of 40 nm Genetically Encoded Multimeric (GEM) nanoparticles. These striking biophysical changes coincide with nuclear positioning defects and collapsed endoplasmic reticulum architecture, mirroring phenotypes associated with ribosome depletion. We propose a mechanism where mutations in the C. elegans lamin lmn-1 disrupt nucleolar density and ribosome biogenesis, creating a nucleolar-ribosomal axis that propagates defects from the nucleus to the cytoplasm. Genetic interactions between lmn-1 and ribosomes support this regulatory relationship. While individual depletion of other nuclear envelope proteins produces minimal effects, combined loss of the functionally redundant emerin ortholog emr-1 and LEM-domain protein lem-2 phenocopied lmn-1 mutants, demonstrating that cytoplasmic biophysical disruption lies at EDMDs pathogenic core. Our findings establish a paradigm where nuclear lamina defects fundamentally rewire cellular biophysics through nucleolar-ribosomal dysfunction, opening transformative therapeutic avenues for treating laminopathies.

cell biology↗

bioRxiv2025

Single-cell proteomics: a powerful new tool to study kidney cell heterogeneity

BackgroundTechnological advancements in protein mass spectrometry have significantly enhanced analytical sensitivity and throughput, enabling single-cell proteomics by mass spectrometry (SCP-MS) to become reality. SCP-MS allows high-resolution analysis of cellular heterogeneity and function, bypassing bulk analysis limitations. Here, we used SCP-MS to document at the protein level the cellular diversity of mouse kidney cells. We further focused SCP-MS on low abundance distal convoluted tubule (DCT) cells that are essential for body electrolyte homeostasis and blood pressure control. MethodsMouse kidney cells were isolated via enzymic digestion and single-cells isolated using fluorescence- activated cell sorting (FACS). DCT cells were isolated from mice with GFP expression specifically in the DCT (parvalbumin-GFP) using a similar workflow. SCP-MS analyses was performed using an Orbitrap Ascend Mass Spectrometer with a FAIMS Pro Duo interface coupled with Wide ISolation window High-resolution MS1 Data Independent Acquisition (WISH-DIA). Spectronaut was used for protein identification and quantification, and cell-type annotation and clustering were performed in Python by scanpy implementation. ResultsWorkflow benchmarking using Hela cells confirmed a successful SCP-MS setup. From 768 single mouse kidney cells, SCP-MS identified 2626 unique proteins. Computational approaches resolved various nephron segments, including distinct DCT populations. From enriched DCT cells, 1912 proteins were identified, enabling classification of three populations - DCT1, DCT2, and a proliferative subset (ProLIF) that represented a transient state between DCT1 and DCT2. ProLIF cells had elevated abundance of the sodium-chloride cotransporter NCC and represented approximately 33% of DCT cells, substantially exceeding previous transcriptomic estimates of ProLIF cell frequency (~0.1%). High proliferation (~39%) of DCT cells was confirmed using immunohistochemistry. ConclusionsThis SCP-MS analysis of mouse kidney uncovered significant cellular heterogeneity not captured effectively using transcriptomics. Despite imitations in proteome depth and throughput, SCP-MS provides a powerful approach for investigating kidney cellular dynamics at the protein level. Key pointsO_LIWe developed a single-cell proteomics by mass spectrometry (SCP-MS) workflow identifying up to ~2,000 proteins in a single mouse kidney cell C_LIO_LISCP-MS identified a substantial proliferative subset of cells in the DCT underestimated by transcriptomics C_LIO_LISCP-MS could inform on clinical strategies for kidney disease monitoring, offering robust indicators of tubular injury or regeneration. C_LI

biochemistry↗

bioRxiv2025

The tumor suppressor CYLD acts as a deubiquitinase for mTOR to constrain its activity

Proper control of mTOR (mechanistic/mammalian target of rapamycin) signaling is relevant for health, disease and ageing. Information from intra- and extra-cellular signaling cues is transmitted to mTOR through an intricate signaling network that impinges on the Rag and Rheb GTPases to regulate its localization and activity. Interestingly, although mTOR is a heavily ubiquitinated protein, the role of this post- translational modification (PTM) in regulating its activation status remains poorly understood. Here, through an unbiased RNAi screen, we identified the tumor suppressor CYLD deubiquitinase (DUB) as a direct negative regulator of both mTORC1 and mTORC2 activities. Mechanistically, CYLD interacts with mTOR and removes non-degradative, K63-linked ubiquitin (Ub) chains from multiple of its residues. Consequently, CYLD loss-of-function cells are characterized by mTORC1/2 hyperactivation, elevated rates of protein synthesis, increased cell size, and resistance to serum-starvation-induced activation of cell death pathways. Moreover, silencing of cyld-1, the C. elegans CYLD ortholog, fully reverses the extended lifespan of low- TORC1-activity mutant worms. Finally, we find that inactivation of CYLD is associated with hyperactivation of mTORC1 also in skin biopsies from CYLD cutaneous syndrome (CCS) patients. In sum, our findings highlight CYLD as a sentinel of mTOR hyperactivation via direct control of its ubiquitination, and suggest that dysregulated mTOR activity may contribute to the development and progression of CCS tumors.

cell biology↗

bioRxiv2025

Post-inhibitory rebound firing drives hypothalamic activation for male mating

Many behaviorally relevant limbic circuits are dominated by inhibitory connections, raising the question of how neuronal activation arises in such contexts. In male mating, both activation of mPOA and its inhibitory inputs are essential--a paradox previously ascribed to disinhibition. Here, we show that the mPOA largely lacks local inhibitory circuits, arguing against disinhibition. Instead, in vivo recordings reveal that mount-active mPOA neurons are suppressed prior to behavioral onset, and this inhibition negatively correlates with subsequent activation, consistent with post- inhibitory rebound firing (PIR). A biologically plausible conductance-based model demonstrates that synaptic inhibition interacts with T-type Ca{superscript 2} currents -- the ionic basis of PIR -- to promote mPOA neuron firing. Experimentally, bidirectional manipulations of mPOA T-type Ca{superscript 2} channels confirm this mechanism: knockdown reduces PIR and impairs male mating, while overexpression enhances mating. Together, these results identify PIR as a critical mechanism by which inhibition promotes activation in limbic circuits to drive male mating.

neuroscience↗

bioRxiv2025

High-throughput 3D super-resolution ultrasound imaging

Capturing fast hemodynamics in deep organs is essential for understanding microvascular regulation of organ responses to physiological demands and pathological stress. However, non-invasive three-dimensional (3D) imaging of these microscale processes remains challenging due to trade-offs between spatial resolution, imaging speed, and penetration depth. Here, we present fluctuation-based high-order super-resolution acoustic microscope (FLAME), a tracking-free 3D ultrasound imaging technology capable of fast microvascular angiography and flow measurement. Using as few as 30 volumes, FLAME improves 3D ultrasound resolution by 8-fold ([~]50 m) and shortens data acquisition time by over two orders of magnitude, from tens of seconds to tens of milliseconds, compared with conventional tracking-based super-resolution approaches1-6. This high-throughput capability supports high-fidelity imaging of tissue functions under flexible experimental settings. FLAME achieves an unprecedented 3D super-resolution frame rate of [~]40 Hz, capturing transient hemodynamic responses to various physiological and pathological stimuli in different mouse organs, from the microvascular level to the whole-body scale. With open-sourced implementation, FLAME provides an accessible platform for real-time, in-depth hemodynamic monitoring across diverse biomedical applications and clinical translation.

bioengineering↗