Biology subjects

Liang, Y.

Publications and source records attributed to Liang, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

TEDDY: An integrative workflow for TE-chimeric isoform reconstruction and systematic characterization of TE-dependent transcriptional regulation

Transposable elements (TEs) substantially expand transcriptional regulation and complexity. Despite advances in sequencing technologies, the systematic interrogation of TE-dependent transcripts across diverse samples and platforms remains limited. Here we introduce TEDDY, a computational workflow for large-scale reconstruction, quantification and regulatory inference of TE-dependent isoforms. Using TEDDY, we resolved the landscape of TE-dependent isoforms in mammalian preimplantation development, revealing a conserved pattern of stage-specific isoform generation via species-specific TE exonization. We reconstruct a TE-dependent regulatory network underlying pluripotent-to-totipotent transition wherein key transcription factors are driven by TE-derived promoters and validated experimentally. Within this network, Arid3a, a novel regulator nominated by TEDDY, was functionally validated as essential for totipotency establishment and early development. Further application of TEDDY to hepatocellular carcinoma identified recurrence-associated prognostic isoforms, underscoring their clinical relevance. Benchmarking establishes TEDDY's accuracy and efficiency with unique capabilities in full-length isoform recovery, cross-sample and -platform analysis, visualization, and TF-TE-gene network reconstruction, making TEDDY applicable across diverse biological contexts.

genomics↗

bioRxiv2026

Structural-functional calibration corrects single-neuron identity errors in volumetric calcium imaging

Volumetric calcium imaging is increasingly used to capture larger neuronal populations at higher throughput, but high-speed axial sampling can compromise single-neuron identity. Here we identify cross-plane identity duplication as a structured error in volumetric imaging: anisotropic axial blurring and plane-wise functional segmentation can repeatedly detect the same neuron across adjacent planes, creating duplicate functional nodes that inflate neuronal counts and distort network phenotypes. We developed Comprehensive Label-Guided (CLG) volumetric imaging, a structural-functional calibration framework that uses nuclear labels as stable three-dimensional identity anchors for calcium signals. CLG combines nuclear labeling, deep-learning-based 3D segmentation, anatomical registration and identity-guided trace reassignment. In larval zebrafish whole-brain recordings, CLG resolved ~30,000 redundant detections and reduced estimated neuronal counts by 37-46%. In mouse visual cortex, CLG consolidated ~40% of putative duplicates and recovered over 2,000 active neurons missed by calcium-only analysis. Across baseline and perturbed conditions, calibration stabilized graph-derived measurements of hub organization, long-range correlations and network resilience. CLG therefore defines an anatomy-constrained identity-calibration layer for reliable single-neuron-resolved volumetric imaging.

neuroscience↗

bioRxiv2026

A leakage-controlled benchmark shows apparent codon-language-model advantages in synonymous-variant prediction are evaluation artifacts

Synonymous codon choices shape mRNA stability, translation, and folding, and codon language models (cLMs) are increasingly reported to read this biology from sequence. However, when a true signal is thin relative to a confounding one, standard evaluation protocols can manufacture the reported gain rather than measure it--and we show this is what has happened for cLMs on synonymous-variant prediction. Under random splits, the codon advantage is large: tokenization gaps of +2.3-14.3 percentage points (pp) and pretrained codon leads of +2.9 pp over the strongest protein model (ESM-1b) and up to +4.9 pp over ESM-2. We find these numbers are properties of the measurement, not the models. A memorization baseline outscores every neural model; the advantage collapses under gene-held-out evaluation; the sole surviving residual dissolves into six defensible probe defaults; and the synonym-randomization drop that appeared to confirm true signal is itself variance under pooled analysis (0.3 pp, p = 0.49). No advantage survives leakage-controlled evaluation with pooled statistics. We release CodonBench, a leakage-controlled benchmark with an emergent audit cascade, and characterize how artifacts accumulate at every pipeline step. A thin signal (I({sigma}; Y |A) {approx} 0.04 bits) may exist but is not reliably detectable at current sample sizes; we specify what detecting it would require.

bioinformatics↗

bioRxiv2026

Introns encode a vast class of Kink-loop RNAs that autoregulate pre-mRNA splicing

Introns occupy nearly one-third of the human genome, yet whether they encode widespread regulatory functions remains unclear. Here, we identify a vast new class of intron-derived Kink-loop RNAs (klRNAs) that autoregulate host pre-mRNA splicing. We developed orthogonal sequencing methods to systematically uncover approximately 15,200 and 3,500 previously unannotated klRNAs in humans and mice, respectively. Bound by the conserved RNA-binding protein 15.5K, klRNAs are compact orphan RNAs characterized by stereotypically positioned terminal C/D motifs that form K-loop structures. Depletion of 15.5K broadly disrupts klRNA biogenesis. Functional and genetic perturbations establish that klRNAs suppress host intron excision through a K-loop-dependent mechanism. Together, our findings establish klRNA-mediated autoregulation as a widespread principle governing intron fate and reveal a previously unrecognized regulatory layer encoded within mammalian introns.

molecular biology↗

bioRxiv2026

MetaboXcan: A multiomic framework linking genetically predicted metabolites, gene expression, and complex traits

Genetically regulated molecular phenotypes, such as gene expression and metabolites, are widely thought to mediate the effect of disease-relevant genetic loci identified by genome-wide association studies (GWAS); however, molecular mechanisms connecting genetic variation to disease remain poorly understood. While transcriptome-wide association studies (TWAS) have identified disease-associated genes, strategies that integrate the metabolome remain underexplored. Here, we introduce MetaboXcan, a framework for predicting plasma metabolite levels from genetic data and associating them with complex traits using GWAS summary statistics. We trained lasso regression models on plasma metabolite and genotype data from the Metabolic Syndrome in Men Study (METSIM); these models outperform existing genetic metabolite predictors and generalize well across independent cohorts. MetaboXcan leverages these models to perform four complementary association analyses: (i) gene-to-trait (TWAS), (ii) gene-to-metabolite (M-TWAS), (iii) metabolite-to-trait (MWAS), and (iv) gene-expression-based metabolite-to-trait associations (g-MWAS). The framework further organizes results into metabolic pathways and gene-metabolite interaction networks to facilitate biological interpretation. Applied to chronic kidney disease (CKD), MetaboXcan identified known disease risk genes (PDILT/UMOD, SPATA5L1/GATM), as well as multiple CKD-relevant metabolites (e.g., glycine, homoarginine). Our integrated multiomic analysis enables biological interpretation by revealing glycine availability-centered biochemical axes spanning oxidative stress, cellular energetics, and vascular signaling, while nominating candidate mechanisms for downstream investigation. These results validate known disease-related biology with genetic evidence and generate new hypotheses for further investigation. MetaboXcan is publicly available and broadly applicable to any GWAS phenotype.

genetics↗

bioRxiv2026

Targeting cellular senescence alleviates bone marrow aging

Aging of the hematopoietic system impairs hematopoietic stem cell (HSC) function and alters bone marrow niche behavior, increasing susceptibility to anemia, infections, and hematologic malignancies. Here, pharmacologic clearance of senescent cells with the PROTAC compound 753b simultaneously targeting BCL-xL and BCL-2 reverses key secretory, transcriptional, and functional hallmarks of hematopoietic aging with low toxicity, restoring balanced lineage output. Single-cell RNA sequencing further demonstrates that 753b treatment attenuates aging-associated transcriptional signatures in HSCs, while selectively eliminating senescent, pro-survival niche cells without grossly perturbing niche composition. Functionally, 753b suppresses pro-inflammatory cues from both niche and hematopoietic cells including those emanating from neutrophil progenitors, rebalancing global bone marrow secretory ecosystem across stromal and hematopoietic compartments. Collectively, we identify 753b-induced senescent cell clearance as a powerful strategy to rejuvenate aged hematopoiesis and re-establish homeostatic communication between HSCs and their microenvironment, with implications for mitigating age-related hematologic dysfunction and improving hematologic health in older individuals.

cell biology↗

bioRxiv2026

Astrocyte-to-microglia purinergic signaling mediates synaptic shielding and promotes neuronal activity

How glial cells sense and regulate neuronal hypoactivity remains poorly understood. Using in vivo imaging in anesthetized, sleeping, and behaving mice, we identified a glial purinergic signaling mechanism that converts neuronal hypoactivity into local circuit regulation. Cortical hypoactivity increased spatially confined ATP release through astrocytic pannexin-1 hemichannels, which were structurally enriched near perisomatic parvalbumin-positive boutons. Local ATP release directed microglial process movement and stabilized bulbous endings through microglial P2Y12 signaling, accompanied by localized Ca{superscript 2} activity in microglial processes. Disruption of astrocytic pannexin-1 or microglial P2Y12 impaired bulbous-ending stabilization and abolished rebound increases in neuronal activity during emergence from anesthesia. Similar ATP release and bulbous-ending formation were observed during chemogenetic neuronal silencing or natural sleep in freely behaving mice. These findings identify astrocyte-to-microglia purinergic signaling as a mechanism that converts neuronal hypoactivity into synapse-selective microglial regulation of circuit activity.

neuroscience↗

bioRxiv2026

Single-cell transcriptomics reveals chondrocyte state transitions and ECM remodeling in osteoarthritic knee cartilage

BackgroundOsteoarthritis cartilage contains heterogeneous chondrocyte states, but molecular programs linked to state transitions within human cartilage remain incompletely resolved using public single-cell data. MethodsA retrospective reanalysis was conducted of a public human knee cartilage single-cell RNA sequencing dataset (GSE255460) including 8 osteoarthritis donors and 3 non-osteoarthritis donors (19 samples). Cells underwent sample-wise quality control and doublet removal, followed by batch-corrected clustering, chondrocyte subclustering with marker-based annotation, and trajectory inference using Slingshot. Regulatory chondrocytes were prioritized for osteoarthritis versus control differential expression, with downstream Gene Ontology/KEGG enrichment (Benjamini-Hochberg false discovery rate <0.05) and protein-protein interaction network hub screening. ResultsAfter quality control, 27,036 cells were retained. Chondrocytes formed multiple transcriptional states with branching-like continuous relationships, and regulatory chondrocytes localized near the main manifold and adjacent to multiple inferred branches, consistent with a transition-adjacent state. In regulatory chondrocytes, osteoarthritis versus control differential expression was enriched for collagen-containing extracellular matrix and extracellular matrix organization, endoplasmic reticulum lumen-associated secretory/proteostasis processes, cell-matrix adhesion (including focal adhesion), and transforming growth factor beta/SMAD-related signaling. Protein-protein interaction analysis of regulatory-chondrocyte differential genes identified five high-connectivity hub genes: COL5A1, COL5A2, COL6A1, COL1A2, and COL3A1. ConclusionThis public-dataset reanalysis supports a transition-adjacent regulatory chondrocyte program in osteoarthritis characterized by coordinated extracellular matrix remodeling with concurrent secretory/proteostasis and adhesion-transforming growth factor beta signatures, nominating collagen-network hubs as candidates for downstream validation.

bioinformatics↗

bioRxiv2026

Effect of CSFV on Differential Genes of Histone Lactylation at H3K18 in the PI3K-AKT Signaling Pathway

Classical swine fever (CSF) is a highly contagious disease caused by Classical swine fever virus (CSFV), posing a serious threat to the global swine industry. This study aimed to investigate the effect of CSFV on differential genes of histone lactylation at the H3K18 site in the PI3K-AKT signaling pathway. The site with the most significant change in histone lactylation antibody level was screened by Western blot. Omics analysis was performed using CUT&Tag technology to identify differential genes in the PI3K-AKT pathway between the CSFV-infected group and the mock group, followed by validation using RT-qPCR. Functional analysis of significantly differential proteins was conducted, and the protein expression level of THBS4 was detected by Western blot. The results showed that after CSFV infection of 3D4/21 cells, the H3K18la site exhibited the most significant difference in antibody level. A total of 8,859 differential genes at the H3K18la site were identified by CUT&Tag analysis, including 6,349 up-regulated genes and 2,510 down-regulated genes. Further focusing on the PI3K-AKT signaling pathway, 10 differential genes were identified, comprising 6 up-regulated genes and 4 down-regulated genes. Compared with the control group, the mRNA expression levels of CD19, LAMA1, PDGFRA, BDNF, ANGPT4, and THBS4 were up-regulated in the CSFV-infected group, while FOXO3 and NRTN were down-regulated. Western blot results showed that the protein expression level of THBS4 increased after CSFV infection. These findings lay an important foundation for understanding the molecular mechanisms regulating viral replication and immune evasion, and have significant scientific implications and potential application value.

microbiology↗

bioRxiv2026

ANCHOR: haplotype-aware allelic and isoform inference from single-cell long-read RNA sequencing with de novo variant calling

Long-read RNA sequencing enables haplotype- and isoform-resolved allelic analysis of transcriptomes, yet extending this capability to single cells and distinct cell types remains computationally challenging due to sparse coverage, sequencing errors, incomplete variant information, and reference-biased transcript assignment. Here we present ANCHOR, a haplotype-aware framework for single-cell long-read RNA sequencing that performs de novo expressed-variant discovery, molecule-level haplotype assignment and isoform-resolved allelic quantification. ANCHOR combines a signed-graph variant caller, pair hidden Markov modelling and beta-binomial UMI aggregation to infer parental allele counts for genes and splice-resolved isoforms, without requiring a pre-existing phased genotype or deep learning. In human single-cell long-read RNA benchmarks, ANCHOR improved variant-calling performance over tested long-read RNA callers at single-cell and low-to-moderate coverage, and its beta-binomial model reduced depth-driven false positives in allele-specific expression testing. Applied to newly generated single-cell long-read RNA-seq data from reciprocal mouse crosses during gastrulation, ANCHOR resolved cell-type- and isoform-specific parent-of-origin imprinting and identified an antagonistic maternally biased Sgce isoform. ANCHOR provides a general framework for allele-and isoform-resolved analysis of diploid single-cell long-read transcriptomes.

bioinformatics↗

bioRxiv2026

(2R,6R)-Hydroxynorketamine elicits rapid antidepressant effects by promoting astrocytic μ-δ opioid receptor heterodimerization

Ketamine produces rapid antidepressant effects but is constrained by psychotomimetic properties and abuse potential. The ketamine metabolite (2R,6R)-hydroxynorketamine (HNK) shows antidepressant-like efficacy without N-methyl-D-aspartate receptor (NMDAR) blockade, yet its upstream targets remain unclear. Here we show that HNK potentiates hippocampal excitatory transmission and reverses stress-induced behavioural deficits through opioid receptor signaling. Pharmacological and genetic analyses reveal a requirement for both {micro}- and {delta}-opioid receptors in astrocytes. Chronic stress reduces {micro}-{delta} receptor heterodimers in the hippocampus, and a single dose of HNK restores their abundance. PAINT-MINFLUX nanoscopy quantifies increased {micro}-{delta} heterodimerization, and molecular dynamics simulations indicate selective binding of HNK to the {micro}-receptor protomer via Asp147 and Tyr148. Mutating these residues abolishes HNK-driven heterodimer formation, downstream signaling and rapid antidepressant-like effects in vivo. Astrocytic {micro}-{delta} opioid receptor heterodimers thus represent a targetable mechanism for next-generation rapid-acting antidepressants.

neuroscience↗

bioRxiv2026

A Rare Nestin-Expressing Granule Cell Precursor Subpopulation Underlies SHH Medulloblastoma Formation

Granule cell precursors (GCPs) drive the major postnatal expansion of the cerebellum and are the cells of origin of sonic hedgehog medulloblastoma (SHH MB). Although GCPs are often treated as a uniform population, increasing evidence suggests they are heterogeneous, and whether specific subpopulations show distinct tumorigenic competence remains unclear. Here, we identified a rare Nestin-expressing GCP subpopulation in the normal early postnatal cerebellum and showed that it is spatially restricted, molecularly distinct and highly competent to form tumors. These cells are enriched in the posterior external granule layer and co-express Atoh1. Using different SHH MB mouse models, we showed that when this rare subpopulation of GCPs is targeted they can give rise to SHH MB with an efficiency comparable to targeting a larger number of Atoh1-expressing GCPs and that tumors derived from Nestin-expressing GCPs arise in the posterior-lateral cerebellum. Single cell RNA sequencing revealed that Nestin-expressing GCP have a transcriptome indicating reduced neuronal differentiation and enrichment for stem cell genes compared to bulk GCPs and more closely align with SHH MB cells. Together, our findings reveal functionally important heterogeneity within the GCP lineage and suggest that SHH MB arises preferentially from a small subpopulation of GCPs that express Nestin.

cancer biology↗

bioRxiv2026

Direct RNA Sequencing reveals epitranscriptomic regulation of brain cells and Alzheimer's Disease pathology

Alternative mRNA splicing and post-transcriptional RNA modification are key mechanisms that regulate transcript function; however, their role in neuronal activity and neurodegenerative disease remains poorly defined. In this study, we evaluated two nanopore-based long-read sequencing (LR-seq) formats: cDNA-PCR sequencing (CPS) and direct RNA sequencing (DRS). We then applied DRS to profile both full-length isoforms and RNA modifications in major brain cell types derived from induced pluripotent stem cells (iPSCs) and post-mortem Alzheimers disease (AD) brains. Relative to CPS, DRS achieved higher accuracy and sensitivity for transcript quantification, de novo transcript model construction, and open reading frame (ORF) annotation across neuropathological gene sets. Focusing on iPSC-derived neurons, we built a multi-omic atlas to connect transcriptional output with translational engagement and protein abundance, by integrating DRS-based mRNA abundance, N6-methyladenosine (m6A) status and poly(A) tail length with ribosome profiling (Ribo-seq) and mass spectrometry (MS). The combination of DRS and Ribo-seq data demonstrated synergism in predicting protein abundance. This analysis also uncovered a significant inverse relationship between m6A modification and mRNA abundance, which was dependent on the engagement of the ribosomal A-site. Lastly, we applied DRS to the epitranscriptomic analysis of AD brain samples, demonstrating that m6A profiles can be used to distinguish early-versus late-stage disease.

neuroscience↗

bioRxiv2026

An Engineered Halotolerant Chimeric T7 RNA Polymerase for High-Yield, Low-Immunogenicity Synthesis of RNA via Simple Batch Transcription

The rapid advancement of mRNA therapeutics has imposed stringent requirements on both the quality and scalability of in vitro transcription (IVT) products. However, the accumulation of double-stranded RNA (dsRNA) byproducts and 3-terminal heterogeneity during T7 RNA polymerase (T7 RNAP)-mediated transcription can robustly trigger deleterious innate immune responses and compromise translation efficiency. Existing enzyme engineering strategies frequently struggle to reconcile the trade-offs between salt tolerance, volumetric productivity, and product purity. Here, we report a novel engineering strategy for halotolerant T7 RNAP by fusing optimized mutant polymerases with diverse DNA-binding domains (e.g., Sso7d, MC1). This approach orchestrated the development of a series of chimeric T7 RNAP mutants designed to bolster catalytic activity and template selectivity under high-salt conditions while concurrently suppressing RNA-dependent RNA polymerase (RdRP) activity. Our lead chimeric mutants exhibited exceptional salt tolerance and processivity in the presence of up to 270 mM NaCl. Notably, these mutants significantly diminished dsRNA formation to less than 0.001%, while markedly improving transcript integrity and 3 homogeneity, thereby facilitating superior translation efficiency for both linear mRNA and circular RNA (circRNA). Crucially, this heightened salt tolerance does not necessitate a trade-off in RNA yield, affording broader flexibility for downstream process optimization. In an enzymatic circRNA synthesis system, these mutants enabled a non-fed-batch configuration with high initial rNTP concentrations (15 mM each), resulting in a 50% increase in yield and achieving an unprecedented titer of 15 mg/mL. This research provides a robust enzymological solution that harmonizes quality and productivity for the industrial-scale manufacturing of high-concentration, low-immunogenicity RNA.

genomics↗

bioRxiv2026

Cell vulnerability within the sublaterodorsal tegmental nucleus underlies REM sleep behaviour disorder in prodromal α-synucleinopathy

REM sleep behaviour disorder (RBD) is a prodromal manifestation of -synucleinopathies such as Parkinsons disease. Evidence suggests that degeneration of REM sleep regulating neurons in the sublaterodorsal tegmental nucleus (SLD) could give rise to RBD, yet the specific cellular populations involved and their contribution to synucleinopathy progression remain unclear. Here, we investigated the role of defined SLD cell types in RBD pathogenesis. Using viral vector and fibril-based models of -synucleinopathy, we show that -synuclein pathology in SLD neurons triggers RBD in mice. Notably, glutamatergic SLD neurons are selectively vulnerable to synucleinopathy and the loss of these cells correlates with RBD severity. Propagation of synucleinopathy from the SLD to midbrain and forebrain structures leads to the emergence of neurological deficits associated with Parkinsons disease. These findings establish that SLD neurons are critical substrates for RBD and provide insight into the cellular mechanisms at play in the early stages of synucleinopathies.

neuroscience↗

bioRxiv2026

Selective Activation of Macrophage Innate Signaling Pathways and Inflammatory Responses to Orientia tsutsugamushi Karp and Gilliam Strains

Orientia tsutsugamushi (Ot) is an obligatory intracellular bacterium that can cause scrub typhus, an emerging but severely neglected disease with high mortality rates. Ot Karp and Gilliam strains account for most reported cases in Southeast Asia. Our group has reported that Karp-infected outbred and inbred mice exhibit more severe disease outcomes than their Gilliam-infected counterparts, likely due to their excessive inflammation and tissue injury. Macrophages (M{Phi}s) serve as the main target cells for Ot replication and host defense against the infection; they also are key players in immune modulation and cytokine/chemokine production. However, it remains unclear as to how M{Phi}-Ot interactions impact disease outcomes. In this study, we focused on RNAseq from C57BL/6 mouse-derived M{Phi}s to reveal Ot infection- and strain-related immune signatures. While Ot infection modulated several common canonical pathways/genes, we identified unique pathway/gene signatures that were highly selective for Karp or Gilliam strain, respectively. Karp infection uniquely upregulated proinflammatory signaling and pattern recognition receptors, including C-type lectin receptors (CLRs), including Mincle/Clec4e and Dectin-2/Clec4n. In contrast, Gilliam infection enhanced M{Phi} proliferation and DNA replication, and Gilliam strain grew better in M0-like M{Phi}s than Karp strain. In IFN-primed (M1-like) M{Phi}s, however, Karp exhibited significantly greater resistance against host killing than Gilliam, suggesting its superior ability to evade host immune responses. Overall, Karp strain preferentially upregulated CLRs and activated type 1-skewed inflammatory responses, but it is also relatively resistant to IFN-mediated killing. This study provides new insights into potential mechanisms underlying Ot strain-associated immune responses and disease outcomes. Author SummaryOrientia tsutsugamushi (Ot), the causative agent of scrub typhus, is an understudied, life-threatening pathogen endemic to Southeast Asia. Among its various pathogenic strains, Karp and Gilliam are clinically dominant and exhibit distinct disease severity. Building on previous reports that Karp-infected mice display more severe disease than those infected with Gilliam, we explored how strain-specific interactions with macrophages (M{Phi}s), a central cell type in both Ot replication and host immunity, may contribute to these outcomes. Using RNAseq of primary M{Phi}s from C57BL/6 mice, we characterized transcriptional profiles of both strains. Karp infection was uniquely associated with increased proinflammatory signaling and the selective induction of bacterial-sensing molecules. In contrast, Gilliam infection promoted host cell proliferation and supported more efficient bacterial replication in naive M{Phi}s. Notably, Karp demonstrated increased resistance to host killing in stimulated (proinflammatory) M{Phi}s compared to Gilliam. These findings suggest that the Karp strain can drive strong inflammatory responses but is adept at evading IFN-mediated immune defenses. Our study uncovers key immunological distinctions between Ot strains and offers novel insights into the mechanisms of strain-specific pathogenesis in scrub typhus, with potential to uncover biomarkers for disease severity.

immunology↗

bioRxiv2026

Large Language Model Agent Enables Autonomous Machine Learning Model Building for Biomedicine

Machine learning accelerates biomedical discovery, but creating effective predictive models requires specialized human expertise and demanding manual effort. Researchers must iteratively design pipelines, select architectures, and debug code. This challenge is particularly severe in biomedicine because of the heterogeneous datasets, sparse annotations, and complex evaluation protocols that are common in the domain. We present AIDO.Builder, an agentic artificial intelligence system that fully automates the entire life-cycle of biomedical model development. Provided only with a natural language task description and a target metric, AIDO.Builder autonomously constructs executable training and evaluation pipelines. The system selects suitable modeling strategies, executes experiments, and uses automated feedback-loop to iteratively revise its own code, configurations, and training procedures. It flexibly adapts to new tasks by training specialized models de novo or by using pretrained foundation models to build predictive models through task-appropriate adaptation. We show that across diverse biomedical benchmarks, AIDO. Builder produces highly competitive solutions against human alternatives, while eliminating the manual iteration previously required for robust model development. By automating the translation of raw data into reliable AI models, AIDO.Builder demonstrates how AI itself can be used to accelerate AI for biomedical research.

bioinformatics↗

bioRxiv2026

Inducible nitric oxide synthase (iNOS) regulates skin eschar lesions, bacterial persistence, and inflammatory resolution in mouse models of scrub typhus

Orientia tsutsugamushi (Ot) is an obligately intracellular bacterium that causes scrub typhus, a potentially severe infectious disease characterized by systemic inflammation and multiorgan dysfunction. We recently reported a protective role for IFN-{gamma} signaling in host defense against Ot infection; however, the underlying mechanisms remain obscure. Inducible nitric oxide synthase (iNOS, encoded by Nos2) is a key antimicrobial effector induced downstream of IFN-{gamma} signaling. Here, we used transgenic mouse models to further investigate the biological functions of iNOS. We first revealed the requirement of iNOS for the restriction of Ot growth in cultured bone marrow-derived macrophages. Using an intradermal mouse model, we found that while tissues of Nos2-/- and wild-type mice exhibited comparable bacterial burdens during acute infection phases, Nos2-/- mice developed eschar-like lesions similar to those observed in Ifngr1-/- mice, indicating a critical role for the IFN-{gamma}/iNOS axis in regulating skin pathology in scrub typhus. Notably, Nos2-/- mice displayed impaired bacterial clearance during the recovery phase (day 42), with persistent bacterial burdens in multiple organs accompanied by sustained immune activation and elevated inflammatory responses. Histopathological and biochemical analyses further revealed increased tissue damage and dysregulated physiological homeostasis in Nos2-/- mice during recovery. Mechanistically, iNOS deficiency resulted in heightened myeloid cell activation and prolonged expression of proinflammatory mediators, suggesting a dual contribution of iNOS in both antimicrobial defense and inflammation resolution. Collectively, these findings provide new insight into IFN-{gamma}-mediated defense mechanisms and imply the distinct roles of iNOS during different stages of scrub typhus. Author summaryScrub typhus is a potentially severe infectious disease caused by the bacterium Orientia tsutsugamushi (Ot), which is transmitted to humans through the bite of infected mites. Despite its global impact and expanding geographic distribution, the immune mechanisms that protect against this infection remain incompletely understood. In this study, we examined the role of inducible nitric oxide synthase (iNOS), an immune effector molecule that helps the host control infection. Using mouse models, we found that iNOS plays dual and stage-specific roles during Ot infection. Mice lacking iNOS developed dysregulated immune homeostasis during acute infection and exhibited skin lesions resembling the eschars observed in some patients with scrub typhus. In addition, these mice showed delayed bacterial clearance, prolonged inflammation, and increased tissue damage during the recovery phase. Our findings indicate that iNOS contributes not only to host antimicrobial defense but also to the control of excessive inflammation following infection. These results provide new insight into host defense mechanisms in scrub typhus and may help inform future therapeutic or preventive strategies.

immunology↗