Biology subjects

Lee, S.

Publications and source records attributed to Lee, S..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

Cas9-enriched nanopore sequencing enables comprehensive and multiplexed detection of repeat expansions

Short tandem repeat (STR) expansion is a major genetic mechanism underlying numerous neurogenetic disorders. However, traditional PCR amplification and short-read next-generation sequencing (NGS)-based methods often fail to detect complex and large expansions including methylation information. Here, we modified an amplification-free nanopore Cas9-targeted sequencing (nCATS) platform and developed a dedicated analysis algorithm, STRiker, for the simultaneous assessment of all currently defined STR loci (56 sites) using a single test with genomic DNA from patient-derived blood cells. We ultimately identified pathogenic repeat expansions in 12 of 37 patients (32.4%) with cerebellar ataxia who remained genetically undiagnosed despite extensive prior genetic testing, in FGF14 (n=4), ATXN8OS, NOP56, RFC1 (n=2 each), and PRNP and NOTCH2NLC (n=1 each). Additionally, family-based cascade screening revealed six relatives with repeat expansions in five families. These results demonstrated a broader diversity of pathogenic repeat structures, particularly in FGF14, and illustrated that CpG methylation can mitigate the pathogenic effects of repeat expansions. This approach offers a powerful strategy for improving the diagnosis of STR-related neurogenetic diseases, for cerebellar ataxia and other STR-related diseases in the future. One Sentence SummaryImproved nanopore Cas9-targeted sequencing with STRiker enhances diagnosis of STR- related neurogenetic diseases and offers broader molecular insights.

bioengineering↗

bioRxiv2025

Neural Sensitivity to Word Frequency Modulated by Morphological Structure: Univariate and Multivariate fMRI Evidence from Korean

A central question in psycholinguistics in visual word recognition is whether morphologically complex words are obligatorily decomposed into stems and affixes during visual word recognition or whether whole- word access can occur when forms are frequent and familiar. The present study investigated how morphological complexity and lexical frequency jointly shape neural responses by leveraging Korean nominal inflection, whose transparent stem-suffix structure permits a clean dissociation between base (stem) frequency and surface (whole- word) frequency. Twenty-five native Korean speakers completed a rapid event-related fMRI lexical decision task involving simple and inflected nouns that varied parametrically in both frequency measures. Representational similarity analysis (RSA) revealed robust encoding of surface frequency--but not base frequency--in the inferior frontal gyrus (IFG) pars opercularis and supramarginal gyrus (SMG), with significantly stronger correlations for inflected than simple nouns. Univariate analyses converged with this result: surface frequency selectively increased activation for inflected nouns in inferior parietal regions, whereas base frequency showed no reliable effects in any ROI. These findings challenge models positing obligatory pre-lexical decomposition, instead supporting accounts in which morphological processing is shaped by post-lexical, usage-driven lexical statistics. Taken together, our findings shed light on a distributed perspective on morphological processing, suggesting that structural and statistical factors jointly constrain access to morphologically complex forms.

neuroscience↗

bioRxiv2025

Bacterial supplementation shapes honey bee gut microbiota, metabolism and proteome under controlled and field conditions

Gut bacteria are essential to honey bee (Apis mellifera) health, supporting digestion, immunity, and resilience against stressors. Probiotic-based strategies have been proposed to enhance core gut symbionts, yet the underlying mechanisms and influences of environmental context on these effects are not fully understood. This study examined the influence of gut-derived bacterial supplementation on the honey bee gut microbiome, proteome, and metabolome across three conditions: (i) controlled cages, (ii) semi-controlled cages allowing contact with nestmates, and (iii) field conditions. Treatment groups received a bacterial supplement containing Lactobacillus helsingborgensis, Lactobacillus apis, Bifidobacterium choladohabitans, and Bifidobacterium polysaccharolyticum, while control groups received only a sucrose solution. Gut samples from 10-day-old bees were analyzed. Supplemented bees showed strong gut colonization by Bifidobacterium and Lactobacillus, especially under controlled and semi-controlled conditions. In the field, L. helsingborgensis remained significantly enriched in the treatment group, indicating ecological resilience. Proteomic changes in supplemented bees included an increased abundance of major royal jelly protein precursors and mitochondrial proteins, alongside the decreased abundance of ribosomal and translation-related proteins involved in peptide biosynthesis and cellular protein quality control. Metabolomic analysis revealed reproducible shifts across all three conditions. Treatment groups showed higher concentrations of microbial fermentation products (acetate, succinate) and neuroactive compounds (ornithine, {gamma}-aminobutyrate), while sucrose, N-acetylglucosamine, and uridine that were constantly lower compared to controls. No significant changes were observed in body or gut weight. These findings highlight reproducible, context-dependent effects of bacterial supplementation on honey bee gut physiology and provide mechanistic insights for microbiome-based interventions in pollinator health. ImportanceHoney bees are essential pollinators whose health is influenced by their gut microbiome. Probiotic applications aimed at improving gut health have been proposed, yet outcomes remain inconsistent and vary across settings. Results from laboratory experiments often differ from those observed under field conditions, making it difficult to understand the complex dynamics of eusocial insect colonies. Here, we evaluate honey bee gut-derived bacterial supplementation across controlled, semi-controlled, and field settings using bacterial profiling, proteomic, and metabolomic analyses. We demonstrate that bacterial supplemented groups consistently reshape gut community composition and modulate host physiological processes, but in a context-dependent manner. These results provide a unified understanding of how microbial interventions function at colony and individual levels, guiding the rational design of probiotic strategies to support honey bee health under realistic conditions.

microbiology↗

bioRxiv2025

Telomere-to-Telomere Accurate and Gapless Korean Standard Reference Genome

We present KOREF1-G-TTAGGA, the first Telomere-to-Telomere Accurate and Gapless Genome Assembly, standing as the Korean standard reference genome. The paternal and maternal haplotypes spanned 2.91 and 3.03 Gb. Genome-wide, at least 99.15% of assembled sequences were reliably haplotype-resolved, and over 95% remained accurate even in the most error-prone loci, including centromeric satellite arrays and segmental duplications. Rare-k-mer copy-number concordance within satellite arrays held up 98.9% and 98.8% per haplotype. Bionano optical maps fully spanned all canonical rDNA arrays on the five acrocentric chromosomes. The assembly quality index (AQI) of 99.77 and 99.69 exceeded the reference-quality threshold of 90, and more than 99.99% of gene and cCRE sequence was free of structural error. Both haplotypes further showed high base-level accuracy, with consensus quality value (QV) of 81.19 and 79.03, corresponding to one error per 131 and 80 Mb. KOREF1-G-TTAGGA is among the highest-quality East Asian telomere-to-telomere assemblies. It moreover anchors a decade-spanning multi-ome reference dataset for defining individual molecular states. Together, these resources define the personal referenceome as a foundation for individual biology and precision medicine, with the assemblies, annotations, and all multi-omic data openly available at https://koreanreference.org.

genomics↗

bioRxiv2025

10,239 whole genomes with multiomic and clinical health information as the Korean population multiomic reference dataset

We present Korea10K, the largest genomic dataset of the Korean population, comprising 10,239 high-coverage whole genomes (mean depth 30x) with matched multiomic profiles and phenotype data. Korea10K achieves complete and near-complete discovery of very rare and ultra-rare alleles, respectively, at 9,000 Korean genomes. This dataset provides the high-quality population-specific imputation panel, enabling accurate inference of low-frequency variants. Admixture analyses confirm the genetic homogeneity of the Korean population, despite its diverse Y-chromosomal, mitochondrial, and HLA repertoires. This pattern reflects a long and continuous lineage history characterized by persistent internal admixture and genomic homogenization over thousands of years on the Korean peninsula. We also identified 16.8 million genomic variants that directly modify CG sites by creating or abolishing CG dinucleotides, providing the population-scale evidence of coordinated genomic-epigenomic regulatory mechanism in Koreans.

genomics↗

bioRxiv2025

Development of Recombinant Anti-TLR2 Antibodies and PLGA Nanoparticle-based Gene Therapy for the Treatment of Neuropathic Pain

Neuroinflammation is a key contributor to neuropathic pain, with microglial Toll-like receptor 2 (TLR2) playing a central role in initiating and sustaining proinflammatory responses. However, existing TLR2-targeting antibody therapies are limited by poor delivery to the central nervous system and short-lived efficacy. We developed a non-viral gene therapy strategy using biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) to deliver recombinant anti-TLR2 antibody genes. High-affinity nanobody and single-chain variable fragment candidates were selected through phage display screening and shown to suppress TLR2-dependent signaling both in vitro and in vivo. In mouse models of neuropathic pain, a single intrathecal administration of PLGA NP-encapsulated antibody genes produced robust and sustained analgesia, accompanied by reduced glial activation and proinflammatory cytokine expression. These findings demonstrate a modular NP-based platform for sustained antibody expression in the central nervous system and establish its potential for the treatment of chronic pain driven by innate immune activation.

neuroscience↗

bioRxiv2025

Somatostatin Receptors Shape Insulin and Glucagon Output within the Pancreatic Islet through Direct and Paracrine Effects

Aims/HypothesisPancreatic delta cells secrete somatostatin (SST), which can inhibit both alpha and beta cells of the pancreatic islet. By controlling insulin and glucagon release, delta cells play an important role in maintaining nutrient homeostasis. However, the mechanism by which a single inhibitory hormone inhibits both alpha and beta cells, which are often considered as functional antagonists in the counterregulatory control of blood glucose, has been a physiological riddle. Here, we solve this riddle through assessment of the contributions of alpha and beta cell specific somatostatin receptors to cell intrinsic behaviors and hormone release. MethodsIslets from mice constitutively expressing fluorescent sensors reporting on cyclic AMP and Ca2+ in both alpha and beta cells were imaged using stimuli to mimic the post-prandial state of a meal consisting of glucose and amino acids. This approach was coupled with cell specific somatostatin receptor antagonists to identify how somatostatin inhibits alpha and beta cell hormone output through modulation of cAMP and Ca2+ secondary messengers and paracrine interactions. ResultsOur results support and extend prior observation that somatostatin receptor 2 (SSTR2) is the only somatostatin receptor expressed by alpha cells, while somatostatin receptor 3 (SSTR3) is the only receptor expressed by mouse beta cells. Interestingly, SSTR2 and SSTR3 regulate downstream cAMP and Ca2+ signaling cascades differently within alpha and beta cells of intact islets. Stimulation of somatostatin receptors robustly inhibits cyclic AMP in alpha or beta cells. In contrast, stimulation of SSTR2 inhibits alpha cell Ca2+ with significantly greater potency compared to inhibition of beta cell Ca2+ via SSTR3. Despite the absence of SSTR2 on beta cells, blocking alpha cell SSTR2 during nutrient stimulation resulted in a significant increase in insulin release downstream of local release of glucagon. Conclusions/InterpretationOur observations address the physiological riddle of the delta cells role during the post-prandial phase where we demonstrate that somatostatin primarily inhibits alpha cell cAMP and Ca2+ via SSTR2, preventing glucagon release. Blocking SSTR2 resulted in an increase in locally released glucagon, which coupled with muted ability for SSTR3 to inhibit beta cell calcium under strong nutrient stimulation, results in potentiation of glucose stimulated insulin secretion from the beta cell. We conclude that the role of delta cells under nutrient stimulation is to modulate the volume of insulin release by tuning the strength of intra-islet paracrine potentiation of insulin secretion by glucagon, mediated via beta cell GLP1R. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LISomatostatin released by delta cells can attenuate glucagon release from the alpha cells and insulin release from the beta cells of the islet through inhibitory somatostatin receptors. C_LIO_LISomatostatin receptor 2 is a major receptor expressed on the mouse alpha cell surface, while mouse beta cells express SSTR3 on their primary cilia. C_LIO_LIParacrine signaling by alpha cell glucagon potentiates insulin release through the engagement of stimulatory glucagon-like peptide 1 receptors on beta cells. C_LI What are the key questions?O_LIHow do the cell specific somatostatin receptors of the alpha cell (SSTR2) and beta cell (SSTR3) differentially regulate cell-intrinsic Ca2+ and cAMP signaling and the indirect paracrine pathways that shape insulin secretion in the post-prandial state? C_LI What are the new findings?O_LIAlpha cells of the mouse islet express exclusively SSTR2 and not SSTR3. C_LIO_LISomatostatin is a more potent inhibitor of cytosolic calcium activity via alpha cell SSTR2 compared to beta cell SSTR3. C_LIO_LIPreventing somatostatin inhibition of the alpha cell in the presence of elevated glucose and amino acids potentiates insulin release through the engagement of glucagon-like peptide 1 receptors on the beta cell by glucagon. C_LI How might this impact on clinical practice in the foreseeable future?O_LIOur findings indicate that local feedback inhibition of beta cells is accomplished by tuning the strength of the paracrine potentiation of locally released glucagon. This is relevant in the context of developing treatments for hypoglycemia with alpha cell specific SSTR2-specific antagonists that could potentiate insulin release and potentially exacerbate hypoglycemic episodes. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/688371v1_ufig1.gif" ALT="Figure 1"> View larger version (71K): org.highwire.dtl.DTLVardef@1138ddaorg.highwire.dtl.DTLVardef@c613f9org.highwire.dtl.DTLVardef@49806eorg.highwire.dtl.DTLVardef@18ece86_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Preference-Based Fine-Tuning of Genomic Sequence Models for Personal Expression Prediction with Data Augmentation

Despite substantial progress in genomic foundation models, accurately predicting inter-individual variation in gene expression from DNA sequence alone remains a major challenge. Current sequence-based models, such as Enformer and Borzoi, trained exclusively on the reference genome, cannot capture the effects of individual-specific regulatory variants. Moreover, the acquisition of paired whole-genome and transcriptome data required for personalized modeling is hindered by privacy and data-sharing constraints. To address this limitation, we integrate genomic data synthesis with established statistical frameworks. Our approach generates thousands of synthetic training samples by simulating genetic variation from the 1000 Genomes Project and assigning pseudo-expression labels using PrediXcan, a validated eQTL-based predictor. Because simulated and real expression values differ in scale and distribution, we introduce a preference-based objective that models relative rather than absolute expression patterns. Fine-tuning Enformer through alternating cycles of real-data regression and synthetic-data preference optimization enables efficient learning from both real and synthesized data. Using the GEUVADIS dataset, our framework outperforms AlphaGenome, PrediXcan, and Enformer fine-tuned without synthesized data, demonstrating that simulation-based integration of population-level regulatory knowledge can effectively mitigate data scarcity and improve cross-individual generalization in sequence-based gene expression prediction. Availability and implementationCode and data are available at https://github.com/pacifiic/augment-finetune-genomics.

bioinformatics↗

bioRxiv2025

Neuropathological Correlates of Apathy Progression in Alzheimer's Disease and Related Dementias: A Longitudinal NACC Cohort Study

ObjectiveApathy is a prevalent and disabling symptom in Alzheimers disease and related dementias, yet its progression across neuropathological subtypes remains incompletely understood. This cohort study investigates longitudinal changes in apathy and their associations with major neuropathologies using data from the National Alzheimers Coordinating Center. MethodsWe analyzed 1,488 participants with autopsy-confirmed neuropathology and at least two caregiver-reported NPI-Q assessments. Generalized linear mixed models were used to assess associations between apathy and six neuropathologies--Alzheimers disease (AD), Lewy body disease (LBD), frontotemporal lobar degeneration (FTLD), hippocampal sclerosis (HS), cerebrovascular disease (CVD), and cerebral amyloid angiopathy (CAA). with time modeled as years to death and including interaction terms. Sex-stratified analyses were also conducted. All models were adjusted for age at death, sex, and NPI-Q total score excluding apathy. ResultsApathy prevalence increased over time across all pathology groups. FTLD (OR = 2.11, 95% CI: 1.32-3.39) and HS (OR = 2.23, 95% CI: 1.38-3.60) were consistently associated with higher odds of apathy throughout the disease course. No significant interaction effect was observed in any of the neuropathologies. In sex-stratified analyses, FTLD (OR=2.58, 95% CI 1.40-4.77), HS (OR=2.48, 95% CI 1.29-4.77), and LBD (OR=1.64, 95% CI 1.03-2.61) were significantly associated with apathy in males, while only HS (OR=2.18, 95% CI 1.06-4.47) remained significant in females. InterpretationApathy severity varied by neuropathologies but progressed similarly over time. The elevated burden in FTLD and HS, particularly among males, underscores the importance of stratified approaches to early detection and intervention targeting apathy in ADRDs.

neuroscience↗

bioRxiv2025

T-Cell Synaptosomes Orchestrate Long-Term Anti-Tumor Immunity via Proliferative and Metabolic Reprogramming

A defining feature of T-cell activation is its dependence on physical contact with antigen-presenting cells (APCs). During this interaction, activated T cells release microvesicles, known as T-cell immunological synaptosomes (TIS), onto the surface of APCs. While TIS are known to activate APCs, their broader in vivo functions remain largely unexplored. Here, we show that TIS are primarily phagocytosed by macrophages and dendritic cells, persist intracellularly for several days, and reprogram these cells in a manner distinct from LPS. TIS upregulate genes involved in metabolism, proliferation, and anti-inflammatory responses, and promote immune cell recruitment, including eosinophils. Notably, TIS induce minimal immune activation upon initial exposure but trigger a rapid and robust response upon secondary administration, indicating a priming effect reminiscent of adaptive immunity. In an MC38 colon cancer model, TIS treatment resulted in near-complete tumor suppression and durable protection upon rechallenge, highlighting their potential as a potent and long-lasting immunotherapeutic platform.

immunology↗

bioRxiv2025

A Precisely Controlled Long-Acting Immunosuppression Platform Enables Prolonged Survival of Vascularized Composite Allografts

Vascularized composite allotransplantation (VCA) restores complex tissue defects but demands lifelong systemic immunosuppression. Oral tacrolimus (TAC) is limited by a narrow therapeutic window, pharmacokinetic variability, adherence challenges, and toxicity. Local delivery could mitigate these issues, yet clinical translation has been hindered by burst release, short duration, and the inability to co-deliver agents. We developed PRECISE (Programmable, REtrievable, Controlled ImmunoSuppression Encapsulator), an injectable, in-situ-forming PLGA depot that achieves long-acting, tightly controlled TAC release via structure-guided co-formulation with drug-binding agents (DBAs). GRAS small molecules (e.g., EGCG, maltotriose) identified by in silico docking and in vitro screening suppressed burst and modulated solvent efflux. Notably, rapamycin (RAPA) served dually as an mTOR inhibitor and a TAC-binding excipient, enabling synchronized dual-agent delivery. PRECISE eliminated burst in vitro and produced coordinated TAC+RAPA release with clinically compatible injectability. In rats, monthly intragraft dosing maintained systemic TAC/RAPA within the therapeutic window ([~]5-10 ng/mL) for >300 days, prolonged hindlimb allograft survival, expanded Tregs, and induced donor-specific hyporesponsiveness. Surgical retrieval of the depot triggered rapid TAC decline, demonstrating reversibility. In a stringent, fully MHC-mismatched porcine VCA model, PRECISE maintained on-target drug levels and extended graft survival beyond 90 days with minimal rejection and preserved vascular integrity. PRECISE is, to our knowledge, the first retrievable, injectable platform to deliver long-acting, dual-agent immunosuppression with controlled kinetics, rapid attainment of therapeutic steady state, and sustained graft protection. Its modular, structure-guided design enables clinical translation across VCA and solid-organ transplantation, delivering precise, durable, and safer immunosuppression.

bioengineering↗

bioRxiv2025

Neat1 alleviates ischemic injury in skeletal muscles

Peripheral artery disease (PAD) has historically been regarded as an age-related vascular disorder; however, recent attention has shifted toward the myopathic components of the disease. Conventional interventions, such as revascularization, have had limited success in reversing muscle pathology or preventing adverse outcomes like amputation. Hypoxia-inducible factors (HIFs) are key regulators of cellular responses to ischemia, including the promotion of angiogenesis and glycolysis. While pharmacological stabilization of HIF proteins represents a promising therapeutic strategy, its efficacy is diminished in aged muscles due to their intrinsically low basal HIF expression. We hypothesized that long noncoding RNAs (lncRNAs) might enhance the hypoxic response in aged muscle through post-transcriptional regulation of HIF expression. Our study identified a long noncoding RNA Neat1, as a critical mediator of the hypoxia-induced stress response, including upregulation of Hif1. In a murine hindlimb ischemia model, Neat1 knockout mice exhibited extensive necrosis following femoral artery ligation, whereas Neat1 overexpression conferred protection against ischemic injury. Mechanistically, we found that Neat1 regulates the stability of Hif1a mRNA, as Hif1a transcript levels were significantly reduced in Neat1-deficient muscle cells. Importantly, aged muscles displayed a blunted hypoxic response due to diminished Hif1a expression--an effect that was reversed through Neat1 overexpression, resulting in improved resistance to ischemic damage. In summary, our findings highlight Neat1 as a novel regulator of muscle adaptation to hypoxia in aging. Enhancing Neat1 expression may represent a promising therapeutic strategy for improving ischemic outcomes in patients with peripheral artery disease.

cell biology↗

bioRxiv2025

Evaluating a structured expert elicitation approach for adaptive conservation: Lessons from five years in practice

Threatened species management relies on Ex ante estimates of species responses to different interventions to generate meaningful predictions. Structured expert elicitation is often used to generate these estimates, but comparisons of these expert-predicted outcomes with observed results are rare. This study aims to evaluate the utility of expert elicitation for adaptive management in the New South Wales Saving our Species (SoS) program in Australia by revisiting six species management plans that were generated from bespoke structured elicitation guidelines five years prior. Each species management plan included a defined scope, conceptual model, monitoring indicators and estimated response to management curves under different scenarios. Experts reviewed the conceptual models after five years of management and monitoring and compared the predicted response to management with observed monitoring data. In three of the six case studies, observed outcomes closely matched predictions. Where predictions diverged, factors such as unanticipated new threats and unexpected responses to interventions contributed to discrepancies. However, in all cases, the structured approach provided a clear logic for planning, enabling managers to systematically refine their understanding. The conceptual models and response curves proved valuable for collaboration, communication, and generating hypotheses for unexpected results. This work demonstrates the value of the bespoke guidelines in supporting adaptive management processes, strengthening the knowledge base for threatened species conservation while improving alignment between predictions and real-world outcomes.

ecology↗

bioRxiv2025

CRISPR-Induced NUT Suppression Promotes Differentiation and Enhances Trop2-Targeted Immunocytokine Response in NUT Carcinoma

NUT carcinoma (NC) is an aggressive malignancy driven by NUTM1 gene rearrangements with limited therapeutic options. Here, we show that direct suppression of NUTM1 using CRISPR/Cas9 induces squamous-like differentiation and upregulates TROP2 expression in NC cells. Building on this finding, we developed a TROP2-interferon beta (IFN-{beta}) mutein immunocytokine that selectively targets TROP2-expressing tumors. Combined NUTM1 suppression and TROP2-targeted immunotherapy synergistically enhanced cytotoxic and immune-mediated responses in vitro. Transcriptomic and spatial analyses of NC patient tumors revealed that differentiation status correlates with TROP2 expression, upregulated immune pathways, and favorable clinical outcomes. Our results suggest that overcoming differentiation blockade not only alters tumor phenotype but also creates a more immune-permissive microenvironment. These findings highlight the therapeutic potential of sequential tumor reprogramming followed by targeted immunotherapy in treating NC and propose a broader strategy for overcoming differentiation blockade in fusion-driven cancers.

cancer biology↗

bioRxiv2025

β-Catenin Stabilization Protects Against Pulmonary Hemorrhage Through Amphiregulin and BATF- Mediated Regulatory T Cells

Pulmonary hemorrhaging (PH) is a life-threatening condition with a high mortality rate, yet the role of immune cells in its pathogenesis remains poorly defined. Here, we investigated the protective function of {beta}-catenin stabilization in T cells and its impact on PH. Using a novel transgenic mouse model (CAT-Tg) with stabilized {beta}-catenin, we demonstrate that {beta}-catenin stabilization induces a distinct T-cell phenotype characterized by an expansion of central effector memory cells (CD44, CD122, Eomes, T-bet). Mechanistically, this effect was associated with suppression of key proinflammatory pathways, including reduced phosphorylation of STAT1, STAT3, and JAK1. PH was induced using pristane, and CAT-Tg mice were significantly protected from lung damage, showing reduced proteinuria and decreased pulmonary proinflammatory cytokine production compared with wild-type (WT) and T cell-specific {beta}-catenin knockout (cKO) mice. This protection correlated with a marked increase in FOXP3 regulatory T cells (Tregs) in CAT-Tg mice. We further identified a novel mechanism in which {beta}-catenin stabilization enhances lung expression of Amphiregulin and BATF, two molecules essential for Treg function and tissue repair. Adoptive transfer of CAT-Tg Tregs into WT mice with pristane-induced PH conferred superior protection, as evidenced by reduced lung inflammation and proteinuria. The systemic administration of a {beta}-catenin agonist to mice with PH significantly attenuated disease severity. Our bioinformatic analysis confirmed that {beta}-catenin stabilization upregulates pathways associated with tissue repair and immune homeostasis, including PI3K-Akt, angiogenesis, and STAT5 signaling. Collectively, these findings reveal that {beta}-catenin stabilization protects against pulmonary hemorrhage by inducing a specialized T-cell phenotype and establishing a protective Amphiregulin-BATF-Treg axis. This study identifies a novel immunomodulatory pathway with therapeutic potential for PH and other inflammatory lung diseases.

immunology↗

bioRxiv2025

Standardizing the mouse MCAO Stroke Model: Distinguishing Internal Carotid vs Pterygopalatine Artery Occlusion

BackgroundThe intraluminal middle cerebral artery occlusion (MCAO) model is widely used in preclinical stroke research, but outcomes remain inconsistent because of variable occlusion times and inadvertent filament placement into the pterygopalatine artery (PPA). MethodsWe applied a modified version of the Koizumi method to directly visualize the internal carotid artery (ICA)-PPA bifurcation and systematically compare targeted ICA versus PPA occlusion. We combined multiple complementary methods - survival studies, neurological scoring, 2,3,5-Triphenyltetrazolium chloride (TTC) infarct staining, Evans blue blood brain barrier (BBB) permeability assays, laser speckle cerebral blood flow imaging, small-animal magnetic resonance imaging (MRI), and evaluation of inflammatory gene expression through qPCR - to rigorously characterize injury patterns, survival, and inflammatory responses. ResultsICA occlusion approximately caused a rapid 70% reduction in cerebral blood flow, progressive infarction (15 min: smaller infarct with approximately 50% 7-day survival; 30 min: moderate infarct but >50% mortality), and cortical edema. In contrast, PPA occlusion produced only a gradual 44% decline in blood flow, small infarcts at 60 min) and preserved survival (80% at 7 days). Finally, magnetic resonance imaging in 15-minute ICA occluded mice identified the development of cortical edema which was not observed in sham, 15-minute PPA, or 60-minute PPA mice. ConclusionThese findings identify 15-minute ICA occlusion as the most practical balance between reproducible infarction and survival for long-term studies, while 30 minutes is best reserved for short-term infarct induction when survival is less critical. By resolving two critical sources of MCAO variability, occlusion duration and vascular targeting, this study provides a standardized protocol and evidence-based occlusion benchmarks that will improve reproducibility in preclinical stroke studies

physiology↗

bioRxiv2025

Elevated Temperatures Representing Heatwave Conditions Shift Active Nitrifying Communities and Their Viruses in Tidal Flats and Agricultural Soils

Global heatwave intensification associated with climate change will impact the nitrogen cycle, yet its effect on specific nitrifier groups or their interactions with viruses remains unclear. Using 13CO2-DNA-based stable isotope probing (SIP) coupled with metagenomics, we show that elevated temperatures associated with heatwave conditions restructure active nitrifying communities and their viruses in Yangtze River estuary tidal flats and adjacent agricultural soils. In tidal flats, high temperatures shifted active ammonia-oxidizing archaea and bacteria (AOA and AOB), and nitrite-oxidizing bacteria (NOB) from marine to terrestrial ecotypes. In contrast, heatwave conditions stimulated terrestrial ecotypes of AOA but suppressed AOB in agricultural soils. 13C-labeled nitrifier-infecting viruses also showed temperature-driven shifts in activity, lifestyle, and auxiliary metabolic genes in concert with hosts. Notably, AOA viruses carried the plastocyanin gene, potentially augmenting host metabolism. These findings demonstrate heatwaves drive complex shifts in nitrifier communities and their interactions with viruses, impacting global nutrient cycling under climate extremes.

microbiology↗

bioRxiv2025

Human Dicer1 hotspot mutation induces both loss and gain of miRNA function

The core miRNA biogenesis enzyme Dicer1 sustains recurrent mutations in cancer that compromise its RNase IIIb domain, which cleaves 5p arms of pre-miRNA hairpins. However, the lack of knockin models has limited fuller understanding. Here, we generated Dicer1-KO and Dicer1-S1344L (homozygous and hemizygous) human ESCs; the latter is a non-catalytic mutation in RNase IIIa that impairs RNase IIIb activity. Dicer1 knockouts lack canonical miRNAs, while S1344L induces two trends: ablation of miRNA-5p strands, and selective changes in miRNA-3p strands. Curiously, we recognized directional upregulation of miRNA-3p passenger strands, indicating a broad strand switch. We used multiple in vitro assays to show 3p arm-nicked pre-miRNAs preferentially load miRNA-3p species into Argonaute, compared to corresponding duplexes. Moreover, activity assays, RNA-seq data, and Argonaute-mRNA profiling, confirm that these confer increased repression capacity. These data expand the molecular consequences of Dicer1 hotspot mutations in cancer.

biochemistry↗