Biology subjects

Kim, H.

Publications and source records attributed to Kim, H..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Terminal Schwann Cells Regulate Presynaptic Vesicle Homeostasis but Not Neuromuscular Junction Integrity in Mice

Terminal Schwann cells (tSCs) are specialized glial cells persistently residing at the neuromuscular junction (NMJ), where they are widely postulated to participate in synaptic transmission and maintenance. However, elucidating their precise in vivo roles has been hindered by a lack of tSC-specific genetic models, as conventional tools simultaneously targeted axonal Schwann cells. Here, we identify Col20a1 as a highly specific marker for tSCs through single-cell transcriptomic analysis and establish a novel Col20a1-CreERT2 knock-in mouse model. Utilizing this model, we achieved highly specific systemic labeling and ablation of tSCs during the postnatal maturation window. Surprisingly, systemic tSC loss disrupted neither gross NMJ architecture nor overall motor behavior. Instead, electrophysiological and ultrastructural analyses revealed profound pre-synaptic defects, characterized by increased spontaneous miniature endplate potentials (mEPPs), accelerated synaptic depression during high-frequency stimulation, and significant physical depletion of pre-synaptic vesicles. Furthermore, long-term analysis revealed a robust glial repopulation that sustained prolonged neuromuscular integrity. Together, our findings establish Col20a1 as a definitive genetic handle for tSC research, delineating that while tSCs are dispensable for macroscopic synaptic structure, they serve a precise and critical role in regulating pre-synaptic vesicle homeostasis.

neuroscience↗

bioRxiv2026

The ENIGMA-PD-WML Pipeline: A Containerized, User-Friendly Approach for Accurate, Standardized Segmentation of White Matter Lesions in Multi-Site MRI Data

Understanding vascular contributions to disease is a major unmet need. White matter lesions (WML) are an accepted imaging marker of cerebral small vessel disease, giving insights into its related pathologies. A unified approach for WML analyses in large multi-site data is lacking despite the need for pooling of data to overcome the limitations of often small heterogenous MRI studies which make subtyping and identifying patterns within disease groups difficult. Our ENIGMA-PD-WML pipeline is an open-source containerized pipeline containing all the code and packages required for pre-processing, processing and post-processing of T1-weighted and FLAIR data, outputting accurate and reproducible binary WML maps using a UNet approach. The pipeline provides a standardized image analysis approach for WML and outputs data in both native and MNI space to allow for sharing and pooling of data from multiple sites for large-data analysis. In addition to a reliable standardized approach for WML segmentation, key priorities when developing the pipeline included: usability, i.e., requiring minimal manual input and technical expertise to use, and suitability to run on various MRI scanners and acquisition parameters as is common in multi-site data. This paper describes the pipeline in detail, with rationale for each step, providing transparency and facilitating its usage to overcome reproducibility issues in large-scale WML analyses.

neuroscience↗

bioRxiv2026

Mtf2 Safeguards Naïve Pluripotency by Restricting Trophectoderm Competence

The first fate decision in mammalian embryos segregates embryonic and extraembryonic lineages, establishing a largely irreversible transition from totipotency to pluripotency. The mechanisms that enforce this unidirectionality remain incompletely understood. Here, we identify the PRC2.1 subunit Mtf2 as a key epigenetic barrier that safeguards lineage fidelity in naive embryonic stem cells (ESCs). Despite globally elevated H3K27me3 levels in naive ESCs, loss of Mtf2 derepresses trophectoderm (TE) regulators, enhances cellular plasticity, generating both ICM- and TE-like populations. Mtf2-deficient cells readily acquire expanded potential with extraembryonic competence and form blastoids without trophoblast stem cell supplementation, whereas loss of the PRC2.2 subunit Jarid2 has minimal effects. Integrated CUT&Tag and transcriptomic analyses reveal that Mtf2 directly represses TE-associated genes in naive ESCs. Thus, reduced Mtf2 occupancy permits their activation in expanded potential states. Collectively, our findings establish Mtf2 as a central epigenetic barrier that stabilizes naive pluripotency and restricts inappropriate acquisition of extraembryonic fate.

Developmental Biology↗

bioRxiv2026

Machine Learning-Guided Discovery of Bacterial-Selective Membrane-Active Compounds Reveals Mechanistic Bias in Antibiotic Training Datasets

The rise of antibiotic resistance necessitates the discovery of antibacterial compounds with novel mechanisms of action (MoAs). Recent machine learning approaches have shown promise in antibacterial compound discovery, but often identify derivatives of known antibiotic classes rather than mechanistically novel compounds. Previous approaches applied Tanimoto similarity filters at the end of screening pipelines, but this method has substantial drawbacks: Tanimoto similarity can be misleading in chemical space, and post-hoc filtering does not influence what activity models learn to prioritize. Here, we present a machine learning pipeline that addresses chemical novelty upfront by employing an XGBoost-based MoA classifier to explicitly prioritize compounds predicted to have mechanisms distinct from known antibiotic classes, combined with graph neural networks for antibacterial activity and toxicity prediction. Applied to the Zinc20 database, our approach successfully identified non-toxic antibacterial compounds structurally distinct from known antibiotics. Notably, the majority of these hits exhibited membrane-targeting activity with selectivity for bacterial cells over mammalian cells, suggesting potential for next-generation membrane-active antibiotics. However, we did not identify compounds with novel protein targets. Systematic analysis revealed that this limitation stems from mechanistic bias in training data rather than model architecture. Specifically, our activity model learned to preferentially score compounds similar to specific groups in the training data, thus overrepresenting certain MoA classes including membrane-active compounds. Even substantial model architecture and training data enhancements did not overcome this constraint. Our findings demonstrate that the primary bottleneck for discovering mechanistically novel antibiotics is the scarcity of diverse, mechanistically-annotated training data. This work provides both a methodological framework for mechanism-aware screening and critical insights into data requirements for genuinely novel antibiotic discovery.

bioinformatics↗

bioRxiv2026

PETase Kubu enables near-complete enzymatic depolymerization of commercial PLA/PBAT blend mulch film

Agricultural mulch films improve crop productivity, but post-use recovery and recycling remain difficult because the films are thin, fragmented, dispersed across fields, and contaminated. Commercial PLA/PBAT blends are increasingly used as biodegradable mulch film materials, yet these films exhibit slow or incomplete degradation under environmental conditions. Here, we show that Kubu, a thermostable PETase from Kutzneria buriramensis, rapidly depolymerizes commercial PLA/PBAT mulch film without pretreatment at 60 {degrees}C, achieving 95% mass loss within 96 h and releasing terephthalate, adipate, and lactate, detected by LC-MS/MS and HPLC, as the major monomeric products of both PBAT and PLA components. GPC and SEM revealed extensive degradation at the polymer-water interface. DiffDock docking against Kubu, IsPETase, and TfCut indicates that the canonical W/F(Y) cleft of the PETase/Cutinase fold accommodates aliphatic and aromatic ester bonds with comparable geometry, suggesting that substrate promiscuity is an inherent property of the cleft architecture. Kubus distinctive contribution combines this permissive cleft with catalytic activity sufficient for near-complete blend depolymerization within 24 h. Integration with a socioeconomic analysis shows that complete enzymatic depolymerization could avoid social costs up to $4,087 per ton of mulch film. These findings establish a single-enzyme approach to end-of-life management of heterogeneous polyester blends.

bioengineering↗

bioRxiv2026

Structural Basis for Inhibition of the HKU5-CoV Main Protease by Clinical SARS-CoV-2 Protease Inhibitors

The identification of Pipistrellus bat coronavirus HKU5 lineage 2 (HKU5-CoV-2) as a human ACE2-adapted virus highlights the need for antiviral strategies to control emerging HKU5-related merbecoviruses. However, despite the importance of the main protease (Mpro) as a key antiviral target, structural and biochemical characterization of HKU5-CoV Mpro in the context of clinical inhibitors has remained limited. Here, we obtained high-resolution crystal structures of HKU5-CoV-1 Mpro in its apo state (1.75 [A]) and in complex with the approved covalent inhibitor nirmatrelvir (1.91 [A]) and the non-covalent inhibitor ensitrelvir (1.55 [A]). These structures reveal an induced-fit mechanism, in which the S2 loop containing Met49 adopts an open conformation in the apo state and undergoes inhibitor-specific conformational changes upon nirmatrelvir and ensitrelvir binding. These structures served as a foundation for the characterization of HKU5-CoV-2 Mpro via modeling and molecular dynamics simulations. Biochemical assays revealed that HKU5-CoV-1 and HKU5-CoV-2 Mpro exhibited nearly identical kinetic profiles, with turnover rates approximately two-fold higher than SARS-CoV-2 Mpro. Structural analysis revealed a highly conserved S1 subsite but distinct local environments in the S1', S2, and S4 substrate-binding sites relevant to inhibitor recognition. Despite these variations, nirmatrelvir and ensitrelvir showed potent inhibitory activity, with comparable double-digit nanomolar IC50 values across all three Mpro proteins. Computational analyses indicated that HKU5-CoV-2 Mpro engages nirmatrelvir and ensitrelvir in binding modes comparable to those observed for HKU5-CoV-1, supporting a conserved mechanism of inhibitor recognition and providing a structural basis for developing pan-coronavirus antivirals targeting emerging merbecoviruses.

biochemistry↗

bioRxiv2026

Carbamylome analysis reveals regulatory roles for lysine carbamylation in β-cell glycolytic and insulin-processing enzymes

In type 1 diabetes (T1D), insulin-producing {beta} cells are destroyed by an autoimmune response driven by pro-inflammatory cytokines, including interferons. {beta}-cell cytokine signaling is mediated in part by post-translational modifications, such as phosphorylation and acetylation. However, the role of other post-translational modifications in {beta}-cell cytokine signaling represents an important knowledge gap. In the context of autoimmune diseases, lysine carbamylation has gained attention for its role in pathogenesis. Here, we investigate the role of carbamylation in T1D. We found that pancreatic islet cells from the T1D model, non-obese diabetic (NOD) mice, exhibit 11% carbamylation-positive cells, whereas non-diabetic CD1 mice have only 5%. Proteomics analysis of the MIN6 insulin-producing cell line treated with a cocktail of three pro-inflammatory cytokines IFN{gamma} + IL-1{beta} + TNF identified 284 carbamylated peptides from 222 proteins impacted by the cytokine treatment. Integration of carbamylation and acetylation provided a deep view of the cytokine-regulated PTMs and potential points of interplay. A functional-enrichment analysis revealed that carbamylation was enriched in pathways related to autoimmune diseases, metabolism, DNA replication, and protein translation. Moreover, functional testing demonstrated that carbamylation inhibits the glycolytic enzyme aldolase A and the insulin-processing enzyme carboxypeptidase E, identifying a possible role for cytokine-induced {beta}-cell dysfunction. In summary, protein carbamylation is elevated in islets from NOD mice, and pro-inflammatory cytokine treatment regulates protein carbamylation in MIN6 cells. These data identify carbamylation as a potential regulatory mechanism for {beta}-cell metabolism and insulin production in the context of islet inflammation.

biochemistry↗

bioRxiv2026

Genotype and methylation interact to reconfigure transcriptional regulation in colorectal cancer

BackgroundTranscriptional regulation is shaped by both genomic variants and the environment. Yet, how the regulatory effects of genomic variants are reconfigured by dynamic epigenomic changes during tumorigenesis remains incompletely understood. MethodsWe investigated methylation context-dependent links between genotype and gene expression in colorectal cancer (CRC) using paired tumor and normal-adjacent tissue (NAT) from 80 patients, thereby controlling for germline genomic background. By integrating promoter-targeted bisulfite sequencing with RNA-seq, we systematically compared expression quantitative trait loci (eQTLs) and methylation quantitative trait loci (mQTLs). To capture regulatory complexity beyond simple mediation, we implemented a memo-eQTL framework that explicitly models genotype x DNA methylation (GxM) interactions. ResultsWe observed extensive tissue specificity in both eQTL and mQTL landscapes; tumor-specific eGenes were significantly enriched for hallmark oncogenic pathways, including WNT and MAPK signaling. Standard mediation models explained only a minority of genotype-expression relationships, whereas our explicit interaction framework revealed widespread reconfiguration of methylation-dependent genetic effects in tumors. Memo-eQTL mapping (FDR < 0.05) identified 18 NAT and 73 tumor eGenes with significant GxM interactions, and results were consistent at a more permissive threshold (FDR < 0.2). We further developed a patient-level memo-eQTL score and found that interaction-based regulatory disruption in NAT, but not in tumor, significantly correlated with clinical stage (P = 0.035). ConclusionsGenetic regulation in cancer is reorganized through context-dependent GxM interactions. Importantly, GxM signatures in NAT are specifically linked to disease progression, offering new insights into field cancerization and the clinical consequences of regulatory reprogramming in CRC.

bioinformatics↗

bioRxiv2026

The genomic basis of evolutionary stasis in the 500-million-year-old red seaweed genus Ahnfeltia

The red seaweed genus Ahnfeltia is an ancient lineage that has persisted for over 500 million years with remarkably limited diversification despite a global distribution in cold-temperate intertidal habitats. Compared to the highly diverse sister lineage, Rhodymeniophycidae, Ahnfeltia provides a unique system for investigating long-term evolutionary persistence in marine macroalgae. Here, we generated chromosome-scale genomes from five populations across three species and combined population genomics with paleogeographic niche modelling. Our results reveal remarkable genomic conservation, strong geographic isolation with limited gene flow, high homozygosity, and evidence of purifying selection. Niche projections indicate long-term stability and spatial connectivity of suitable cold-temperate habitats. These findings suggest that Ahnfeltias persistence and limited diversification are linked to genomic constraints and stable ecological niches over geological timescales. This study provides new insights into the genomic basis of evolutionary stasis in ancient marine lineages and highlights potential vulnerability to ongoing climate change affecting cold-water coastal ecosystems.

evolutionary biology↗

bioRxiv2026

Dual lineages of Langerhans cells cooperate to restore the immune barrier after skin injury

Langerhans cells (LCs) are key immune sentinels of the epidermis. How this network reorganizes to safeguard epidermal immunity after injury has remained unclear. Here, we uncover a previously unrecognized two-lineage program of LC repopulation during wound repair. Classically, tissue-resident embryonically derived LCs (eLCs) migrate to lymph nodes in response to antigens. In contrast, we find that injury triggers nearby eLCs to migrate into wounds, providing immediate coverage. In parallel, circulating monocytes infiltrate the skin and differentiate into long-lived monocyte-derived LCs (mLCs) that integrate stably into the network. We identify the chemokine receptor CXCR2 as a novel regulator of eLC migration into wounds, distinct from the CXCR4/CCR7 pathways mediating LC egress to lymph nodes. Pharmacological inhibition of CXCR2 impairs directional eLC migration and is accompanied by increased mLC infiltration, preserving immune barrier density. These findings reveal a coordinated and flexible two-lineage repair program that ensures robust restoration of epidermal immunity.

cell biology↗

bioRxiv2026

The combination of EWSR1-FLI1 and loss of one EWSR1 allele leads to the induction of trisomy 8

Ewing sarcoma is a pediatric cancer that develops in skeletal elements. The majority of Ewing sarcoma patients carry the aberrant EWSR1-FLI1 fusion gene. Despite trisomy 8 being an additional common aberration associated with a poor prognosis for patients, its induction mechanism remains unknown. When the EWSR1-FLI1 gene is formed, the cell loses one wildtype EWSR1 allele. To elucidate the induction mechanism of trisomy 8, we generated a cell line that allows for the conditional induction of EWSR1-FLI1 expression and EWSR1 knockdown (derived from a single EWSR1 allele. Specifically, the conditional cell line was generated by integrating the Tet-on EWSR1-FLI1 construct into the AAVS locus and adding a miniAID tag at the 5 end of the EWSR1 locus using auxin-degron system. A combination of the EWSR1-FLI1 expression and degradation of one allele-derived EWSR1 induced a high incidence of trisomy 8 within eight days, enhancing colony formation. Mechanistically, trisomy 8 is induced by the haploinsufficiency of EWSR1, and the remaining EWSR1 proteins are likely inhibited by interaction with EWSR1-FLI1. Our data showed that the knockout of EWSR1 alone was sufficient to increase the incidence of trisomy 8. Expression of wild-type EWSR1 in EWSR1 knockout cells rescued the high incidence of trisomy 8. In contrast, the EWSR1:R565A mutant, which lacks the ability to interact with Aurora B kinase, failed to rescue this phenotype. We propose that the combination of EWSR1-FLI1 expression and loss of EWSR1 contributes to the induction of trisomy 8 through the compromised EWSR1-Aurora B pathway. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/726567v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@999891org.highwire.dtl.DTLVardef@1ef6748org.highwire.dtl.DTLVardef@65e475org.highwire.dtl.DTLVardef@179da40_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2026

Decoy diversification underpins the regulation of an NLR-mediated autoimmunity

Appropriate activation of innate immune receptors is vital for the plant immune system. Immune activation must be strong and robust upon invasion by rapidly evolving pathogen species, while staying inactive in the absence of a pathogen to avoid constitutive defense responses that inhibit plant growth. Nucleotide-binding leucine-rich repeat receptors (NLRs) are intracellular proteins that surveil for pathogen invasion, often by direct or indirect perception of pathogen effector proteins. Indirect recognition of pathogen effector proteins, through monitoring the integrity of guardee or decoy proteins, can allow for a single NLR to detect a wide range of pathogen effectors. In this work, we investigated how the Solanaceae NLR NbPtr1 can recognize six different effector proteins from the bacterial pathogens Pseudomonas syringae, Xanthomonas perforans, and Ralstonia pseudosolanacearum. We identified several NOI-domain containing proteins that are guarded by NbPtr1. These NOI proteins share homology with, but are distinct from, RPM1 INTERACTING PROTEIN 4 (RIN4), a well-known guardee in Arabidopsis. Virus-induced gene silencing and CRISPR/Cas9-assisted deletion of NbNOI genes resulted in stunted growth dependent on NbPtr1 activity. Negative regulation of NbPtr1 requires the interaction between NbPtr1 and the suppressor NbNOIs. A conserved threonine residue of the NOI proteins is required for this interaction and fits into a putative binding pocket of NbPtr1 based on protein modeling. This threonine residue is modified by some of the recognized effector proteins. Our study uncovers the regulatory mechanism of an autoactive NLR and highlights the importance of decoy diversification in establishing compatibility with NLRs.

plant biology↗

bioRxiv2026

An Exponential Scale Mixture Model for Metatranscriptomics Data with Application to Inflammatory Bowel Disease

Metatranscriptomic (MTX) sequencing enables profiling of gene expression across microbial communities, providing a framework for linking genetic potential with functional activity. However, standard pipelines report normalized abundances rather than raw counts, limiting the use of count-based RNA-seq methods, while Gaussian-based alternatives rely on transformations and assumptions that are often poorly suited to MTX data. We propose a new modeling framework for differential expression analysis of MTX data, built on a scale mixture of exponential distributions, that incorporates DNA abundance to adjust for genomic potential, accommodates subject-specific random effects, treats zeros as left-censored, and employs a mixture prior to handle extreme sparsity. Applied to the IBDMDB multi-omics cohort, differential expression results vary substantially across models, including among Gaussian approaches with different pseudocount choices. Our approach identifies a distinct subset of candidate genes not detected by existing Gaussian methods; these may provide useful leads toward a novel understanding of transcriptomic patterns associated with dysbiosis in inflammatory bowel disease. Estimated dysbiosis effect directions are consistent between our model and Gaussian-based approaches, while effect sizes from our model tend to be larger in absolute value.

genomics↗

bioRxiv2026

MagNet: Computational Methods for Constructing High-Confidence Protein-Protein Interaction Networks in Magnaporthe oryzae

Magnaporthe oryzae, the rice blast fungus, plays a role as a model organism for molecular plant-microbe interaction research. Studies on the pathogenic mechanism of this fungus revealed many genes involved in signaling pathways. As multi-omics data are being available, genomic-level researches have been conducted to uncover the underlying biological processes during the pathogenesis of M. oryzae. Identifying the genome-wide protein-protein interaction (PPI) network is one of the omics-level approaches, which helps to understand signaling and regulatory pathways. However, existing biological network resources of M. oryzae are not sufficient to decipher pathogenesis mechanisms due to the abundance of false positives/negatives. In this study, a reliable PPI network database of M. oryzae, MagNet, was constructed with three methods, including homology-based Interolog search, co-expression network construction, and domain-domain interaction (DDI)-based prediction. With three approaches altogether, the pan-network with 5,600,976 interactions was generated, including 217,531 highly confident interactions supported by all three methods. Experimental data on M. oryzae PPIs supported that our PPI network can predict PPIs with higher accuracy compared to the previously constructed databases. MagNet would provide integrated biological network data, which can help to understand the molecular mechanisms of the rice blast fungus. The PPI data can be accessed via https:/magnet.scnu.ac.kr.

genomics↗

bioRxiv2026

Identifying Context-Specific Cell-Cell Interaction Genes Without Ligand-Receptor Databases from Spatial Transcriptomics

Current approaches to inferring cell-cell interactions (CCIs) are largely constrained by predefined ligand-receptor databases, particularly for low-resolution spatial transcriptomics (ST) platforms such as Visium. Due to the difficulties in accurately resolving interacting cells at coarse spatial resolution, other modes of interaction are often overlooked. Low-resolution ST data, however, can serve as an alternative to high-resolution ST, which suffers from low sensitivity, and to image-based ST, which is limited by restricted gene panels. Here, we present CellNeighborEX v2, a database-free framework that directly infers CCI-associated genes from ST data by detecting deviations between observed and expected gene expression at the spot-population level. These deviations are rigorously evaluated through a hybrid statistical framework involving permutation testing and are further refined by considering the abundance of interacting cell-type pairs. Compared with other conventional approaches relying on ligand-receptor databases, CellNeighborEX v2 can capture CCI genes from a broad spectrum of interactions, including both paracrine signaling and contact-dependent communication. Across datasets from hippocampus, liver cancer, colorectal cancer, ovarian cancer, and lymph node infection, CellNeighborEX v2 accurately recapitulated previously identified CCIs. Notably, it uniquely detected interactions absent from existing ligand-receptor databases, enabling detection of context-specific CCIs from Visium data. CellNeighborEX v2 is a tool that expands the analytical spectrum of Visium data and deepens our understanding of the molecular language of intercellular communication.

bioinformatics↗

bioRxiv2026

Tryptophan degradation by intestinal Bacteroides induces anti-tumor immunity and limits melanoma growth

Defining mechanisms used by gut microbiota to control anti-tumor immunity may offer novel therapeutic modalities. Here, we demonstrate that Bacteroides rodentium and closely related Bacteroides uniformis species induce anti-tumor immunity and limit melanoma development when colonized in either germ-free (GF) mice, mice with a complex microbiome, or WT mice. Enhanced CD8+ T cell infiltration seen in tumors of mice harboring B. rodentium coincided with increased expression of immune-stimulating pathways and activation of bone marrow-derived dendritic cells that were co-cultured with the B. rodentium secretome. Metabolomic analyses of cecal samples from GF mice colonized with Altered Shedlar Flora (ASF) plus B. rodentium revealed lower tryptophan levels than in ASF-colonized controls, and WT mice fed a tryptophan-deficient diet exhibited inhibition of melanoma development. In silico genomic reconstruction of metabolic pathways revealed that both B. rodentium and B. uniformis harbor tryptophanase A (TnaA) and aromatic amino transferase (ArAT) genes, both of which function in tryptophan degradation. Administration of a B. uniformis harboring TnaA mutant failed to inhibit melanoma growth in gnotobiotic mice. Notably, administration of indoles, but not kynurenines, also effectively inhibited melanoma development, increasing immune cell infiltration into the tumors. Correspondingly, levels of bacterially encoded tryptophan-degrading enzymes were higher in cohorts of melanoma patients responding to immune checkpoint blockade. These findings highlight a novel mechanism of anti-tumor immunity and tumor growth inhibition dependent on the tryptophan degradation products, indoles, produced by intestinal Bacteroides species.

cancer biology↗

bioRxiv2026

A self-limiting orexin-habenula circuit for stress resilience

Resilience requires neural systems that mobilise active coping during stress while limiting its persistence to preserve homeostasis under sustained challenge1-4. Here we identify a self-limiting orexin-habenula circuit in which lateral hypothalamic orexin neurons engage aromatic L-amino acid decarboxylase-expressing D-neurons (encoded by Ddc) in the lateral habenula via orexin receptor type 2 (OX2R)5-7. Activation of this pathway increased nucleus accumbens dopamine and promoted active coping and positive valence. Optotagging revealed rapid stress-evoked recruitment followed by post-stress suppression. In lateral habenula neurons, orexin peptides exerted dissociable effects: orexin-A engaged an OX2R-dependent inhibitory programme superimposed on a parallel inward current, whereas orexin-B did not reproduce this inhibitory profile and instead exerted a distinct membrane effect. Chronic stress disrupted this buffering system through coordinated inflammatory activation, promoter methylation and erosion of D-neuron identity and orexin responsiveness. Restoring orexin-A reversed behavioural and molecular deficits through OX2R-dependent suppression of nuclear factor kappa B signalling, preservation of Tet2 expression, and demethylation-linked maintenance of the Ddc programme. Together, these findings define a self-limiting orexin-habenula resilience circuit that enables adaptive coping while constraining stress-induced vulnerability.

neuroscience↗

bioRxiv2026

Amplification-Free Detection of Antibiotic Resistance in Enterococcus faecium using PNA-FISH

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen, with antibiotic resistance mediated by the vanA and vanB operons. Rapid and accurate detection of antibiotic resistance is critical for the timely treatment of bacteremia and sepsis. Although imaging-based approaches using fluorescence in situ hybridization (FISH) provide a potential diagnostic solution, detecting mRNAs of antibiotic resistance genes (ARGs) in individual cells remains particularly challenging due to their low copy number and transient expression. Here, we present a peptide nucleic acid (PNA)-FISH method for direct detection of vanA- and vanB-associated resistance in individual VREfm cells. A universal probe targeting the conserved region across vancomycin resistance genes and a set of probes exclusively targeting the vanB gene were designed. The universal probe showed increased fluorescence in the vanA-genotype strain upon vancomycin or teicoplanin treatment, and in the vanB-genotype strain upon vancomycin treatment. In contrast, vanB-specific probes showed increased fluorescence exclusively from the vanB-genotype strain upon vancomycin treatment, confirming their specificity to the vanB gene. Efficient cellular penetration and strong hybridization of PNA probes enabled efficient and accurate detection of antibiotic-resistant bacterial cells, even under a wide-field fluorescence microscope. No detectable signals above background were observed in other major bacterial species associated with bacteremia and sepsis. These findings demonstrate robust detection of antibiotic-resistant cells in mixed microbial populations. When integrated with microbe-capturing techniques, this method may support culture-free detection of antibiotic resistance without nucleic acid amplification or sequencing, with the potential to reduce diagnostic turnaround time.

microbiology↗