Biology subjects

Jiang, S.

Publications and source records attributed to Jiang, S..

At least 73 records · Page 4Linked to original sources

bioRxiv2025

Insect-derived long non-coding RNAs function as epigenetic effectors to reprogram plant immunity

Cross-kingdom RNAs are emerging as critical mediators of interspecies interactions, yet the functions of long RNAs such as mRNAs and long non-coding RNAs (lncRNAs) in recipient organisms remain largely unexplored. Here, we show that the brown planthopper (Nilaparvata lugens, BPH), a major rice pest, translocates mRNAs and lncRNAs into rice plants, where they migrate systemically from feeding sites to distal tissues. Compared with BPH mRNAs, BPH Salivary gland Cross-kingdom LncRNA (BSCLs) exhibit markedly higher stability in rice. Among them, BSCL1 functions as a virulence factor that promotes BPH feeding and reproduction by suppressing host defense. Mechanistically, BSCL1 associates with the HIRA histone chaperone complex and displaces histone H3.3 from the promoters of transcription factors, including bHLH genes central to jasmonic acid signaling, thereby repressing transcriptional immunity. Our results identify BSCLs as systemic, RNA-based effectors that reprogram host defense at the epigenetic level, revealing a previously unrecognized mode of insect-mediated manipulation of plant immunity and highlighting lncRNAs as cross-kingdom regulators. Significance StatementPlant-herbivore interactions are traditionally viewed as battles over nutrients and defense signaling, mediated largely by proteins and small RNAs. Here, we demonstrate that long non-coding RNAs from brown planthopper saliva are translocated into rice, migrate systemically, and function as epigenetic effectors that suppress key transcription factors in jasmonic acid-mediated defense. Unlike insect mRNAs, these lncRNAs persist in the plant and reprogram immunity by interfering with histone deposition. This discovery uncovers a new class of mobile, RNA-based virulence factors, expands our understanding of cross-kingdom regulation, and suggests innovative strategies for pest control targeting RNA effectors.

plant biology↗

bioRxiv2025

Clonal dynamics shaped by diverse drug-tolerant persister states in melanoma resistance

Most advanced melanomas initially respond to targeted therapy but eventually relapse. Rather than acquiring new mutations, resistance is driven by drug-tolerant persister cells that enter a reversible drug-refractory state. We developed MeRLin, a high-resolution lineage tracing platform integrating cellular barcoding, single-cell transcriptomics, RNA fluorescence in situ hybridization (FISH), and computational analyses to track clonal and transcriptional dynamics in patient-derived melanoma models during prolonged therapy. Clonal dynamics revealed that persister subpopulations first responded to treatment but persisted and expanded during minimal residual disease, ultimately leading to tumor recurrence. Pre-treatment melanoma populations diversified into four conserved persister states characterized by stress-like, lipid metabolism, PI3K signaling, and extracellular matrix remodeling programs associated with adaptive resistance. Spatial transcriptomics showed the organization of these adaptive programs and a complex signaling network of autocrine and paracrine interactions among persister subpopulations. Barcoded RNA-FISH enabled spatial mapping of clonal identity and gene expression, revealing in situ co-localization of a dominant resistant clone with SLC2A1 expression. MeRLin provides a robust framework for dissecting cancer heterogeneity and identifying vulnerabilities in persister populations.

cancer biology↗

bioRxiv2025

Granularity of thalamic head direction cells

Head direction signaling is fundamental for spatial orientation and navigation. The anterodorsal nucleus of the thalamus (ADn) contains a high density of head direction (HD) cells that process sensorimotor inputs for subsequent synaptic integration in postsynaptic cortical areas. We tested the hypothesis that individual HD cells show differences in their firing patterns and connectivity by recording and juxtacellularly labeling single HD cells in subregions of the ADn in awake mice during passive rotation. We identified HD cells that exhibited different response profiles to light, sound, and movement. We also identified a mediolateral gradient of calretinin-expressing (CR+) ADn cells, with CR+ HD cells having narrower tuning widths, lower maximal firing rates, and different intrinsic properties compared to CR-cells. Axons of labeled HD cells could be followed to the retrosplenial cortex, with collaterals innervating the thalamic reticular nucleus (type I cells); others additionally innervated the dorsomedial striatum (type II cells). Most medial CR+ cells preferentially projected to ventral retrohippocampal regions. Surprisingly, we also identified a subpopulation of medial CR+ cells with twisted dendrites and descending axons that avoided the thalamic reticular nucleus, termed tortuosa HD cells (type III cells). We conclude that HD cells of the mouse ADn comprise distinct cell types, providing parallel head-direction-modulated sensorimotor messages to synaptic target neurons within the head direction network.

neuroscience↗

bioRxiv2025

Enteric ChAT-expressing neurons as new target for Lactobacillus plantarum ameliorates inflammatory bowel diseases

The gut microbiota plays a crucial role in inflammatory bowel diseases (IBD), yet how specific microbial components influence disease progression remains incompletely understood. Our study reveals that decreased abundance of Lactobacillus species correlates with ulcerative colitis severity in patients. Among tested strains, L. plantarum A736, isolated from traditional fermented foods, demonstrated remarkable efficacy in ameliorating dextran sulfate sodium (DSS)-induced colitis in a mouse model. Strikingly, multi-omics analysis revealed that L. plantarum A736 uniquely enhances choline acetyltransferase-expressing (ChAT+) neurons in the enteric nervous system and elevates acetylcholine (ACh) levels. Further analysis identified that L. plantarum A736 metabolizes tryptophan to produce indole-3-lactic acid (ILA), and supplementation with ILA significantly mimics this strains anti-inflammatory effects by activating ChAT+ neurons. Crucially, selective ablation of colonic ChAT+ neurons completely abolished the therapeutic benefits of both L. plantarum A736 and ILA, establishing their essential role in mediating these effects. These findings demonstrate for the first time that microbial metabolites can directly modulate enteric neuronal circuits to regulate gut immunity. The identified ILA-ChAT+ neuron axis represents a novel neuro-immune pathway that could be targeted for IBD treatment. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/673902v1_ufig1.gif" ALT="Figure 1"> View larger version (63K): org.highwire.dtl.DTLVardef@1e0d3c5org.highwire.dtl.DTLVardef@16f5481org.highwire.dtl.DTLVardef@152283org.highwire.dtl.DTLVardef@18cfad0_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

Single-Cell Super-Resolution Quantification of Oxidative DNA Damage via Aptamer-Assisted DNA-PAINT

Accurately quantifying oxidative DNA damage at the single-cell level remains a major challenge due to the limitations of conventional ensemble-based assays, which obscure cell-to-cell variability and lack molecular specificity. To address this, we developed a super-resolution imaging strategy that combines an 8-oxo-dG-specific DNA aptamer with DNA-PAINT, enabling quantitative visualization of 8-oxo-dG lesions with [~]22 nm spatial resolution in individual cells. Our approach reliably detects 8-oxo-dG clusters, distinguishes oxidative damage levels across cells, and exhibits superior specificity and labeling efficiency compared to antibody-based methods. Furthermore, it enables direct evaluation of the repair efficiency of hOGG1 and its catalytic mutants at the single-cell level, overcoming the confounding effects of variable transfection efficiency. This method also serves as a platform for assessing the effects of pharmacological modulators and antioxidants on DNA repair. Looking forward, this strategy can be extended to map other small molecular targets, such as epigenetically modified DNA or RNA bases, with single-cell precision.

biochemistry↗

bioRxiv2025

Red blood Cell-derived Extracellular Vesicles as biomaterials: the opportunity of freezing-induced accelerated aging

Red blood cell-derived extracellular vesicles (RBC-EVs) are emerging as promising biomaterials for next-generation drug delivery, due to their inherent biocompatibility, immune evasion capabilities, and minimal oncogenic risk. However, their clinical translation remains limited by unresolved challenges related to heterogeneity, reproducibility, and long-term storage. This study proposes a method that leverages freezing-induced ageing for obtaining highly homogeneous RBC-EV batches, an important step towards using RBC-EVs as healthcare biomaterials and advancing their clinical translation in EV-based nanomedicine. This method was made possible thanks to the analytical support of discontinuous sucrose density gradient and high-resolution interferometric nanoparticle tracking analysis, which allowed the identification of a bimodal subpopulation distribution, in terms of vesicle size, interferometric contrast, and subpopulation profiles, in freshly prepared samples, and then tracked how long-term cold storage at -80 {degrees}C channeled this heterogeneity into a monomodal population. Finally, we evaluated the functionality of homogenized RBC-EV samples by assessing surface-associated enzymatic activity and uptake in cancer cell lines, demonstrating that freeze-thaw-induced accelerated-aging provides a viable strategy for producing RBC-EV preparations that retain membrane integrity and remain readily internalized by cells. These findings offer valuable insights into the optimization and standardization of RBC-EV handling and storage protocols, providing a foundation for their reliable integration into EV-based therapeutic applications.

cell biology↗

bioRxiv2025

WNK1 kinase activity is required for maintenance of podocyte structure

The filtration-function of glomeruli requires slit diaphragms formed by interdigitating podocyte foot processes, which are actin-based membrane protrusions. Dysregulation of mechanisms that maintain these membrane extensions lead to foot process effacement, proteinuria, and progression to chronic kidney disease. Building on our previous work that showed WNK1 kinase activity is necessary for the maintenance of normal biomechanical properties of glomeruli and podocyte foot process architecture, we tested the hypothesis that WNK1 kinase activity affects the structure of podocyte foot processes through modulation of actomyosin activity and focal adhesion complexes. Using a WNK1 kinase specific inhibitor, we determined by immunofluorescence microscopy of nascent focal adhesions, podocyte membrane spreading/extensions, and NMII paralog localization and extent of activation calculated from quantification of phosphorylated myosin, that all were sensitive to WNK1 kinase activity. Moreover, biochemical evidence of WNK1 kinase activity-dependent signalosomes supports a role for WNK1 in the maintenance of podocyte foot processes, and sarcomere-like structures (SLSs) that are induced in models of podocyte injury. Using primary and immortalized podocyte cell lines developed from control and Col4a3-/-Alport Syndrome model mice, we measured WNK1 kinase activity-dependent improvement in properties of injured podocytes in vitro. Physiological relevance of WNK1 kinase activity-dependent structural maintenance of podocyte foot processes was confirmed by significant acute proteinuria measured in response to WNK1 inhibition in vivo. Collectively, the results provide evidence that WNK1 kinase signalosome activity that includes formation of nascent focal adhesions and regulation of NMII localization and activity at membrane protrusions and extensions, are necessary for physiological maintenance of slit diaphragms. SignificanceTerminally differentiated podocytes are arborized cells with interdigitating foot processes that form the renal filtration barrier. Loss of foot process structural integrity causes progressive proteinuria, which can lead to irreversible renal injury, but the mechanisms that maintain foot process structure are incompletely understood. We report evidence that WNK1 kinase activity is required for maintenance of normal glomerular filtration in vivo, and this is mediated in part through WNK1 activity-dependent modulation of non-muscle myosin II activity, and formation of nascent focal adhesions that are necessary for lamellipodial extensions. Using glomeruli and podocyte cell lines developed from an Alport Syndrome podocyte injury model, we show that aspects of abnormal podocyte structure associated with chronic kidney disease can be suppressed through increased WNK1 activation.

cell biology↗

bioRxiv2025

Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin's biochemical activity without changing thick filament structure

BackgroundInhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten is a second in class myosin inhibitor with promising clinical data showing improvements in hemodynamics and symptoms in patients with HCM. While it is known that Aficamten inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, effects on myosin structure and kinetics during loaded contraction are lacking. MethodsPermeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues from human induced pluripotent stem cell cardiomyocytes were used to evaluate effects on force and contraction kinetics. ResultsIn contrast to Mavacamten, Aficamten does not structurally sequester myosin heads along the thick filament. Aficamten inhibits ATPase activity by shifting myosin heads from higher to slower ATPase state, with the emergence of a super slow biochemical nucleotide turnover state. This results in decreased force and calcium sensitivity without altering cross-bridge cycling. These contractile mechanical changes are comparable to Mavacamten. Our myofibril mechanical assay showed inhibition of force with accelerated relaxation. In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Mavacamten reduces the activation kinetics while both result in faster relaxation. ConclusionsWe used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both. While both compounds inhibit contractility, differences in mechanisms of action and kinetics of force activation and relaxation could allow use in different patient populations.

biophysics↗

bioRxiv2025

PASTA: Versatile Tyramine-oligonucleotide Amplification for Multi-modal Spatial Biology

Spatial proteomics techniques have revolutionized our understanding of tissue architecture, but are frequently limited by detection sensitivity, bioconjugation limitations, multiplexing capacity, and multi-modal integration. Here we present Protein and nucleic Acid Serial Tyramine Amplification (PASTA), a novel signal amplification approach that significantly enhances detection sensitivity while maintaining compatibility with diverse spatial profiling methodologies. PASTA utilizes horseradish peroxidase (HRP) recruitment pathways to generate tyramine radicals that deposit oligonucleotides, enabling adaptable signal amplification across multiple biomarkers at high-plex via cyclical imaging using complementary fluorophore-labeled oligonucleotides. We demonstrate that PASTA achieves up to 100-fold signal enhancement for markers with minimal background in blank controls. The method is compatible with in situ hybridization for DNA/RNA detection, proximity ligation assays for protein-protein interactions, sequential antibody staining protocols, or any modular combination thereof. PASTA enables antibody rescue of markers with suboptimal signal-to-noise ratios and is versatile in its applications to unconjugated antibodies, and multi-round probe-based RNA detection systems beyond current capabilities. This technique addresses key limitations in spatial-omics by enhancing sensitivity for challenging targets while maintaining compatibility with established multiplexing strategies, providing a versatile, cost-efficient, and valuable tool for comprehensive spatial tissue analysis in both research and clinical applications.

molecular biology↗

bioRxiv2025

Super-Resolution Diffusivity Mapping Reveals Spatial Correlations Between Lateral Mobility and Structural Heterogeneities on Cellular Membranes

Cellular membranes orchestrate critical processes such as molecular transport and signal transduction, both regulated by the lateral mobility of lipids and proteins. However, resolving nanoscale diffusional heterogeneities and elucidating their underlying mechanisms remains a formidable challenge due to the membranes intricate architecture and compositional diversity. Here, we present point-cloud single-molecule diffusivity mapping (pc-SMdM), a cutting-edge super-resolution technique that offers a point-cloud data format with enhanced spatial resolution for diffusivity mapping. Using pc-SMdM, we visualize nanoscale diffusion slowdown clusters with [~]50 nm in diameter on plasma membranes. These clusters are predominantly governed by cholesterol content, alongside contributions from membrane protein assemblies and topographical features. Leveraging two-color pc-SMdM, we concurrently imaged multiple lipids and membrane probes, revealing distinct "fingerprint" diffusivity maps shaped by their interactions within lipid bilayers. Our findings position pc-SMdM as a transformative tool for spatially resolving molecular interactions and membrane dynamics in live cells, offering new insights into the underlying mechanisms that govern membrane mobility at the nanoscale.

biophysics↗

bioRxiv2025

Respiratory Airway Secretory Cells act as Immune Sentinels in Human Distal Airways

Pulmonary immunity in the human distal respiratory airways is essential for lung function but remains poorly explored, mainly due to limited physiologically relevant models. Here, we develop distal lung organoids containing respiratory airway secretory (RAS) cells, a recently identified epithelial population unique to the distal airways of humans and large mammals, using human pluripotent stem cells (hPSCs). Lineage tracing identified RAS cells as descendants of SOX9brightNKX2-1bright progenitors. Single-cell transcriptomics elucidated that RAS cells exhibited a distinct immune-competent phenotype with enriched genes associated with viral host entry and pattern recognition receptor signaling. Functionally, RAS cells infected by respiratory syncytial virus (RSV) exhibited enhanced antiviral activity marked by upregulation of interferon-stimulated genes (ISGs) and complement component 3 (C3). In contrast, bacterial flagellin or Pseudomonas aeruginosa (PAO1) triggered a TLR5-driven immune response in RAS cells, inducing complement system activation, a response suppressed by selective TLR5 inhibition. Notably, in chronic obstructive pulmonary disease (COPD), RAS cells displayed dysregulated immune activation, characterized by elevated expression of adaptive immune mediators. Together, our findings identify RAS cells as previously unrecognized sensors and effectors of mucosal immunity in the human distal airways and reveal their roles in viral infection, bacterial stimulation, and chronic inflammatory lung disease, providing potential therapeutic targets for respiratory diseases.

cell biology↗

bioRxiv2025

Hydrogen-Induced Calcium Influx via the TRPC4-TRPC4AP Axis

BackgroundCalcium ions (Ca{superscript 2}) serve as universal intracellular messengers regulating diverse physiological processes, while dysregulated Ca{superscript 2} homeostasis triggers cytotoxicity. Molecular hydrogen (H2) exhibits protective effects against oxidative stress-related pathologies, but its mechanism of action remains incompletely understood. Transient receptor potential canonical 4 (TRPC4) channels and their associated protein TRPC4AP are critical mediators of Ca{superscript 2} influx ( [Ca{superscript 2}]i), yet their role in H2-mediated calcium signaling is unexplored. This study investigates the molecular mechanism by which H2 modulates Ca{superscript 2} dynamics through the TRPC4-TRPC4AP axis, aiming to establish its therapeutic potential for calcium-related disorders. MethodsThe study employed heterogeneous cellular models (e.g., mesenchymal stem cells, neurons, fibroblasts) and in vivo two-photon calcium imaging in C57BL/6J mice. Techniques included CRISPR-Cas9 knockout, siRNA-mediated gene silencing, molecular docking (AlphaFold 3), and protein-protein interaction analysis. Calcium flux was quantified via fluorescence imaging, while mitochondrial integrity and cytoskeletal dynamics were assessed using JC-1 staining, ATPase activity assays, and live-cell imaging. Structural validation of TRPC4-TRPC4AP binding sites utilized mutagenesis and complementation experiments. ResultsH2 selectively enhanced extracellular Ca{superscript 2} influx via TRPC4-TRPC4AP, with no cytotoxicity or mitochondrial dysfunction observed. Key arginine residues (730Arg-731Arg) in the TRPC4 CIRB domain formed hydrogen-bond networks essential for channel activation. In vivo, H2 increased neuronal Ca{superscript 2} transient frequency and amplitude in the primary motor cortex. TRPC4AP knockout abolished H2-induced Ca{superscript 2} influx, while mutagenesis of 730Arg/731Arg disrupted channel activity. H2 also promoted cytoskeletal remodeling and cell motility, dependent on TRPC4AP-mediated Ca{superscript 2} signaling. ConclusionsThis study identifies H2 as a novel calcium agonist that activates the TRPC4-TRPC4AP axis to regulate extracellular Ca{superscript 2} influx. The 730Arg-731Arg motif in TRPC4 serves as a critical H2-sensitive site, enabling dynamic calcium homeostasis without overload. These findings provide a mechanistic basis for H2-based therapies targeting calcium dysregulation in neurodegenerative, inflammatory, and metabolic diseases, while highlighting TRPC4AP as a pivotal molecular switch for gasotransmitter signaling.

biochemistry↗

bioRxiv2025

Pathological tau activates inflammatory nuclear factor-kappa B (NF-κB) and pT181-Qβ vaccine attenuates NF-κB in PS19 tauopathy mice

Tau regulates neuronal integrity. In tauopathy, phosphorylated tau detaches from microtubules and aggregates, and is released into the extracellular space. Microglia are the first responders to the extracellular tau, a danger/damage-associated molecular pattern (DAMP), which can be cleared by proteostasis and activate innate immune response gene expression by nuclear factor-kappa B (NF-{kappa}B). However, longitudinal NF-{kappa}B activation in tauopathies and whether pathological tau (pTau) contributes to NF-{kappa}B activity is unknown. Here, we tau oligomers from human Alzheimers disease brain (AD-TO) activate NF-{kappa}B in mouse microglia and macrophages reducing the I{kappa}B via promoting its secretion in the extracellular space. NF-{kappa}B activity peaks at 9- and 11-months age in PS19Luc+ and hTauLuc+ mice, respectively. Reducing pTau via pharmacological (DOX), genetic (Mapt-/-) or antibody-mediated neutralization (immunization with pT181-Q{beta} vaccine) reduces NF-{kappa}B activity, and together suggest pTau is a driver of NF-{kappa}B and chronic neuroinflammation tauopathies. SummaryNeuronal tau activates microglial NF-{kappa}B constitutively by secreting its inhibitor I{kappa}B. NF-{kappa}B activation in PS19Luc+ and hTauLuc+ mice peaks at 9- and 11-months of age, respectively. Neutralizing pTau with pT181-Q{beta} vaccine (targeting phosphorylated threonine 181 tau) alleviates NF-{kappa}B activity in tauopathy mice.

neuroscience↗

bioRxiv2025

PRRSV evades innate immune cGAS-STING antiviral function via its Nsp5 to deter STING translocation and activation

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is an important pathogen that seriously endangers pig breeding, causing significant economic losses to the global swine industry. Our previous study found that the DNA sensing innate cGAS-STING signaling pathway plays an important role in inducing interferon (IFN) upon PRRSV infection and inhibition of PRRSV replication. However, the immune evasion and its mechanism by PRRSV is still unclear. In current study, we found that PRRSV Non-structural protein 5 (Nsp5) strongly inhibits the cGAS-STING-IFN antiviral response. Further, we found that Nsp5 interacts with STING, blocking STING transport from ER to Golgi apparatus and interfering with STING recruitment of TBK1/IKK{varepsilon}/IRF3. Finally, we demonstrated that the Nsp5 36-47 and 58-67 amino acid regions are critical regions for inhibiting STING activity and PRRSV replication. The study describes a novel mechanism by PRRSV for suppression of the host innate antiviral response and have implications for our understanding of PRRSV pathogenesis. Author summaryThe innate immune system is the first line of host defense against infections. PRRSV is an immunosuppressive pathogen that has developed different strategies to evade the host innate immune responses. Here, we demonstrate for the first time that PRRSV utilizes its non-structural protein Nsp5 to inhibit the innate immune cGAS-STING-IFN signaling pathway, promoting the viral replication. Nsp5 can inhibit the cGAS-STING pathway by interacting with STING, blocking STING transport from ER to Golgi apparatus and interfering with STING recruitment of TBK1/IKK{varepsilon}/IRF3. In particular, the 36-47 and 58-67 amino acid regions of the Nsp5 is indispensable. This information will help us understand PRRSV-host interactions and provides new insights into the prevention and control of PRRSV.

immunology↗

bioRxiv2025

Themis dominates T cell exhaustion by regulation of TCR and PD-1 signaling

T cell exhaustion is important to protect the host from immunopathology during chronic viral infection, but it also impairs T cell anti-tumor immunity1-5. A fundamental unresolved question is whether and how T cell exhaustion is determined at the onset of TCR signaling6-8. Here we report an unexpected role of Themis, a TCR-proximal signaling molecule9, in T cell exhaustion. Chronic viral infection in mice usually leads to T cell exhaustion and survival of the host. Surprisingly, Themis T-cell conditional knockout mice died from severe CD8+-dependent lung immunopathology in chronic viral infection, showing Themis importance in establishing T cell exhaustion. We found that Themis-deficient CD8+ T cells were hyperactivated at the single-cell level - producing more TNF and IFN{gamma} compared to wild-type counterparts - but defective in population-level expansion. Moreover, TCF-1 and TOX expression were inhibited in Themis-deficient CD8+ T cells, thereby impairing differentiation of exhausted T cell precursors (T-pex) and maintenance of terminally exhausted T cells (T-ex), respectively. Mechanistically, Themis initially promotes TCR signaling to induce PD-1 expression and subsequently mediates PD-1 signaling. In the latter, Themis binds to PD-1 and promotes PD-1 phosphorylation and its recruitment of SHP2, thereby acting as a negative regulator to inhibit T cell effector functions. Without Themis, the orderly regulation of TCR and PD-1 signaling, and therefore exhaustion, is disrupted. Thus, our results unequivocally demonstrate that Themis-mediated early TCR signaling plays a decisive role in T cell exhaustion and provide a novel mechanism of PD-1 signaling through Themis.

immunology↗

bioRxiv2025

A Temporal and Spatial Atlas of Adaptive Immune Responses in the Lymph Node Following Viral Infection

The spatial organization of adaptive immune cells within lymph nodes is critical for understanding immune responses during infection and disease. Here, we introduce AIR-SPACE, an integrative approach that combines high-resolution spatial transcriptomics with paired, high-fidelity long-read sequencing of T and B cell receptors. This method enables the simultaneous analysis of cellular transcriptomes and adaptive immune receptor (AIR) repertoires within their native spatial context. We applied AIR-SPACE to mouse popliteal lymph nodes at five distinct time points after Vaccinia virus footpad infection and constructed a comprehensive map of the developing adaptive immune response. Our analysis revealed heterogeneous activation niches, characterized by Interferon-gamma (IFN-{gamma}) production, during the early stages of infection. At later stages, we delineated sub-anatomical structures within the germinal center (GC) and observed evidence that antibody-producing plasma cells differentiate and exit the GC through the dark zone. Furthermore, by combining clonotype data with spatial lineage tracing, we demonstrate that B cell clones are shared among multiple GCs within the same lymph node, reinforcing the concept of a dynamic, interconnected network of GCs. Overall, our study demonstrates how AIR-SPACE can be used to gain insight into the spatial dynamics of infection responses within lymphoid organs.

immunology↗

bioRxiv2025

Early Proteasome Gene Downregulation And Impaired Proteasomes Function Underlie Proteostasis Failure In Alzheimer's Disease

Alzheimers disease (AD) is characterized by the accumulation of pathogenic proteins, notably amyloid-beta and hyperphosphorylated tau, which disrupt neuronal function and contribute to cognitive decline. Although proteotoxic stress is well-established in AD, the role of the ubiquitin-proteasome system (UPS) in maintaining neuronal proteostasis, and how it becomes compromised during disease progression remains incompletely understood. Here we integrated multiple approaches to characterize proteasome function, composition, and regulation in post-mortem human AD brain tissue compared to age-matched controls. These included proteasome kinetic assays, affinity purification of intact 26S proteasomes, in-gel activity assays and proteomics. According to Braak staging, we further interrogated bulk RNA-seq and single-nucleus RNA-seq (sn-RNA-seq) datasets spanning the progression of AD pathology. Finally, we examined Nrf1/NFE2L1 binding and subcellular localization to understand the transcriptional regulation of proteasome genes in AD. We found that proteasome activity is significantly impaired in AD brains, affecting both 26S and 20S complexes. This reduction in proteolytic capacity persisted after proteasome purification, implicating intrinsic defects within the proteasome complex. Proteomic profiling revealed diminished abundances of constitutive proteasome complexes and the co-purification of proteasomes with aggregation-prone substrates (e.g., tau, -synuclein), suggesting proteasome entrapment in pathological aggregates. Transcriptomic analyses showed progressive downregulation of constitutive proteasome subunit genes in individuals along the Braak stage axis, with downregulation apparent even at the earliest Braak stages, in tissue without overt tau aggregation. Neurons were disproportionately affected, whereas non-neuronal cells did not show substantial differences in proteasome-related gene expression, possibly through immunoproteasome induction. Despite elevated NFE2L1 expression, a key transcription factor normally driving proteasome gene transcription, AD brains exhibited impaired Nrf1 nuclear localization, preventing the expected compensatory upregulation of proteasome components. Collectively, our findings suggest that proteasome dysfunction in AD arises early and deepens over the disease course. Intrinsic alterations in proteasome complexes, coupled with early transcriptional downregulation of proteasome subunits and disrupted Nrf1-mediated regulatory pathways, contribute to a vicious cycle of proteotoxic stress and neuronal vulnerability. Restoring proteasome function and enhancing Nrf1-driven transcriptional responses may represent promising therapeutic strategies to preserve proteostasis and mitigate neurodegeneration in AD.

pathology↗

bioRxiv2025

Decoding the Molecular Language of Proteins with Evola

Proteins, natures intricate molecular machines, are the products of billions of years of evolution and play fundamental roles in sustaining life. Yet, deciphering their molecular language--understanding how sequences and structures encode biological functions--remains a cornerstone challenge. Here, we introduce Evolla, an interactive protein-language model designed to transcend static classification by interpreting protein function through natural language queries. Trained on 546 million protein-text pairs and refined via Direct Preference Optimization, Evolla couples high-dimensional molecular representations with generative semantic decoding. Benchmarking establishes Evollas superiority over general large language models in functional inference, demonstrates zero-shot performance parity with the state-of-the-art supervised model, and exposes remote functional relationships invisible to conventional alignment. We validate Evolla through two distinct applications: identifying candidate eukaryotic signature proteins in Asgard archaea, with functional Vps4 homologs validated via yeast complementation; and interactively discovering a novel deep-sea polyethylene terephthalate (PET) hydrolase, PsPETase, confirmed to degrade plastic films. These results position Evolla not merely as a predictor, but as a generative engine capable of complex hypothesis formulation, shifting the paradigm from static annotation to interactive, actionable discovery. The Evolla online service is available at http://www.chat-protein.com/.

bioinformatics↗