Biology subjects

Zhang, L.

Publications and source records attributed to Zhang, L..

At least 91 records · Page 5Linked to original sources

bioRxiv2026

IKKβ as a putative non-covalent and quinone-mediated covalent target of 4-methylcatechol in RANKL/NF-κB signaling: a combined computational and experimental analysis

Excessive osteoclast activity contributes to pathological bone loss in osteoporosis, rheumatoid arthritis, and osteolytic malignancies. The effects of small catechol derivatives on receptor activator of nuclear factor-{kappa}B ligand (RANKL)-induced osteoclastogenesis remain poorly understood. This study investigated the effects of 4-methylcatechol (4-MC) on RANKL-induced NF-{kappa}B activation and osteoclast differentiation. 4-MC reduced RANKL-induced NF-{kappa}B luciferase activity in HEK-293T/RANK cells. 4-MC also suppressed RANKL-induced TRAP activity in RAW264.7 cells in a concentration-dependent manner and reduced the number of TRAP-positive multinucleated osteoclasts, without affecting cell viability. Molecular docking predicted non-covalent binding of 4-MC within the ATP-binding hinge region of IKK{beta} (PDB: 4KIK), forming a close polar contact with Glu97, predicted hydrogen bonds with Cys99, and a hydrophobic contact with Ile165, within the pocket occupied by the co-crystallized inhibitor K252a. Covalent docking predicted that the oxidized quinone form of 4-MC engages Cys179 in the IKK{beta} activation loop. Quantum chemical calculations confirmed a markedly higher electrophilicity index for the oxidized quinone than for the parent catechol, supporting this mechanism. In silico ADMET profiling indicated favorable drug-likeness and safety. These findings identify IKK{beta} as a plausible molecular target of 4-MC through both non-covalent and covalent mechanisms. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/741661v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1d9f82eorg.highwire.dtl.DTLVardef@134a24borg.highwire.dtl.DTLVardef@8aeb91org.highwire.dtl.DTLVardef@6fb85d_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Genetic background diversifies mutational paths but converges on membrane depolarization for bleomycin resistance in yeast

Drug resistance evolution is shaped by both selective agent and genetic background in which resistant mutations arise, but most experimental evolution studies rely on a single genetic background. Here we performed parallel experimental evolution across six genetically diverged Saccharomyces cerevisiae strains, propagating 48 replicate populations for [~]225 generations under the selection of DNA-damaging chemotherapeutic bleomycin. Over 70% of the evolved populations acquired resistance with the magnitude and repeatability of adaptation varying markedly across genetic backgrounds. Whole-genome sequencing showed that bleomycin elevated single-nucleotide and insertion-deletion mutation rates, and induced background-specific aneuploidies and structural variants, including Ty-element-mediated translocations and tandem duplications. Notably, mutation rates varied up to 10-fold among backgrounds, with the most commonly-used lab strain exhibiting the highest mutagenic susceptibility, underscoring that evolutionary insights derived from single-background models may not universally generalize across a species. Despite this background-dependent genomic heterogeneity, we identified recurrent loss-of-function mutations in PMA1 and PTK2 that arose independently across nearly all genetic backgrounds and conferred strong resistance. Functional and biophysical assays revealed that these mutations impair Pma1p H+-ATPase activity and depolarize the plasma membrane, thereby limiting the cellular uptake of the positively charged drug. Our findings demonstrate that while the mutational routes to drug resistance are heavily shaped by genetic background, a convergent, background-independent mechanism centered on membrane potential regulation can dominate phenotypic adaptation. This work underscores the necessity of integrating genetic diversity into experimental evolution paradigm and nominates plasma membrane potential as an important lever for modulating chemotherapeutic efficacy.

genetics↗

bioRxiv2026

Catch-bond engineering surpasses high-affinity maturation for T cell receptor therapies against solid tumors

The sensitivity of T cell receptors (TCRs) has traditionally been attributed to their affinity, a principle that has long underpinned both T cell biology and TCR engineering. Currently, high-affinity maturation remains the predominant strategy employed to enhance TCR sensitivity; however, this approach has been associated with severe off-target toxicities in clinical settings. In this study, using the only FDA-approved TCR-T therapy as a clinical reference, we demonstrate that force-induced catch bonds, rather than static affinity, primarily determine TCR sensitivity and facilitate the development of more effective TCR engineering strategies. Among polar and charged amino acids, histidine was determined to be the most effective residue for pinpointing engineering hotspots. TCRs engineered to form catch bonds exhibited superior performance compared to the FDA-approved TCR subjected to high-affinity maturation, improving the efficacy of TCR-T cell therapies against solid tumors without eliciting off-target toxicity or alloreactivity. Mechanistically, a correlation was observed between TCR sensitivity and the strength of catch bonds, whereas no such relationship was found with affinity. The duration of T cell-tumor cell interactions, immunological synapse formation, and the intensity of subsequent signaling are governed by catch bonds rather than by affinity. Structural and computational investigations have demonstrated that force-induced reconfiguration of the ligand-receptor interface, along with the formation of a novel hydrogen bond network, augments the specificity of interactions between the TCR and antigenic peptides. Furthermore, bispecific T cell engagers utilizing the TCR scaffold also formed catch bonds with tumor antigens, indicating intrinsic mechanosensory properties of the extracellular domains of TCRs and suggesting a novel therapeutic modality based on catch-bond-engineered TCRs.

immunology↗

bioRxiv2026

Uncovering key genes related to potato tuber starch concentration from the tuber bulking, maturation stage to storage

Potato is the worlds fourth major staple crop, and tuber starch is a critical dietary energy source and a food processing raw material. In this study, hybrid progenies of the high-starch cultivar Huasheng No.7 and China main commercial varieties were used as materials, high-throughput sequencing was performed on tuber samples at tuber bulking, maturation and storage stages. Combined with bioinformatics analysis, parent-progeny resequencing, SNP screening, the regulatory mechanism of tuber starch metabolism was explored. The results showed significant stage-specific transcriptional reprogramming in potato tubers: few differentially expressed genes (DEGs) were identified at tuber bulking and maturation stages, but massive transcriptional changes occurred during storage. XET family genes regulated cell wall remodeling and carbon translocation across all stages, and WRKY transcription factors specifically controlled starch homeostasis in stored tubers. The DEGs at different stage were induced by the SNPs from their parent, and the allele from the high-starch parent, Huasheng 7 may contribute the high-starch allele to the offsprings. One key gene, Soltu.DM.02G019910, which encoded {beta}-glucosidase, have a negatively relationship with tuber starch concentration. The low-starch potato breeding tubers shown higher significantly {beta}-glucosidase activity than high-starch breeding population at maturation stage. This study clarifies the molecular regulatory network of potato tuber starch metabolism and screens core regulatory genes from the tuber bulking stage to tuber storage, thereby providing theoretical support and genetic resources for molecular breeding of high-starch and storage-resistant potato varieties.

plant biology↗

bioRxiv2026

Effects of different shading levels on growth, physiology and leaf surface micromorphology of Sorbus sibirica "Dong Hong" seedlings

BackgroundSorbus sibirica Dong Hong is a promising ornamental cultivar, but its optimal light conditions remain unclear. AimsThis study evaluated the effects of shading on seedling growth, physiology, root morphology, and leaf surface micromorphology. MethodsOne-year-old seedlings were grown under full sunlight (CK) and 30%, 50%, or 70% shade for 100 days. Growth, biomass, root traits, chlorophyll, invertase, soluble protein, stomatal characteristics, and epicuticular wax morphology were determined. ResultsShading significantly affected all measured traits. The 30% shade treatment produced the greatest seedling height, which increased by 118.18% compared with CK, and the highest chlorophyll content, which increased by 120.21%. Soluble protein content was slightly increased, whereas invertase activity decreased under moderate shading. Although total biomass decreased by 18.63%, root development remained relatively stable under 30% shade, with slight increases in total root length and average root diameter. Stomatal density was highest under this treatment, and the epicuticular wax structure remained relatively regular. In contrast, 70% shade markedly inhibited biomass accumulation and root development. ConclusionsModerate shading, particularly 30%, provided the most favorable light environment for Sorbus sibirica Dong Hong seedlings and is recommended for summer nursery cultivation in Northeast China.

plant biology↗

bioRxiv2026

CNVeil resolves haplotype-specific copy number and uncovers subclonal architecture hidden from total copy number profiling in single-cell cancergenomes

Single-cell DNA sequencing (scDNA-seq) resolves copy number variation (CNV) at single-cell resolution, revealing tumor heterogeneity and subclonal structure. Most existing methods, however, infer only total copy number. Haplotype-resolved copy number, which captures allelic imbalance and clonal evolution, remains far less developed, largely because low coverage, allelic dropout, and technical noise in scDNA-seq make phased allelic inference substantially harder than total copy number estimation. We present CNVeil, a haplotype-aware framework that infers total, allele-specific, and chromosome-scale haplotype-resolved copy number from scDNA-seq data. CNVeil first builds robust total copy number profiles through highly variable bin selection, hierarchical clustering, subclone-aware ploidy estimation, and cross-cell consensus segmentation. Using this profile as a stable scaffold, it infers allele-specific copy number with an expectation-maximization algorithm applied to heterozygous SNP allele counts, then reconstructs haplotype-specific copy number by enforcing coherent haplotype orientation across adjacent segments via dynamic programming. We benchmarked CNVeil against 12 state-of-the-art methods, including eight total copy number callers, two allele-specific callers, and two haplotype-resolved callers, across 20 simulated and real datasets spanning six experimental settings, including high-multiplexed single-nucleus sequencing, Acoustic Cell Tagmentation (ACT), and 10x Chromium. This constitutes the largest comparative evaluation of single-cell copy number inference methods to date. CNVeil consistently outperformed existing tools in segmentation accuracy, ploidy inference, subclone identification, and allele-specific copy number estimation. In a breast cancer multi-omics (wellDR-seq) cohort, CNVeil uncovered haplotype-specific subclonal diversification invisible to total copy number analysis alone and linked allele-specific copy number states to transcriptional variation. By transforming sparse single-cell allelic signals into chromosome-scale haplotype-resolved profiles, CNVeil closes a major methodological gap and provides a scalable framework for studying tumor evolution and functional genomic heterogeneity at single-cell resolution.

bioinformatics↗

bioRxiv2026

Diagnostic Performance of Fluorescence-Based Rapid On-Site Specimen Evaluation for Helicobacter pylori Antimicrobial Susceptibility Testing Before and After Algorithm Optimization: A Two-Round Comparative Study

ObjectiveThis study was designed to evaluate the diagnostic performance of fluorescence-based rapid on-site specimen evaluation (F-ROSE) for antimicrobial susceptibility testing of Helicobacter pylori (H. pylori) and to compare test performance before and after iterative optimization of fluorescence image acquisition and of the artificial intelligence (AI) recognition algorithm, using two consecutive rounds of paired testing, so as to provide trial data supporting clinical implementation of rapid susceptibility testing. MethodsForty patients with a urea breath test (UBT) or rapid urease test (RUT) positive for H. pylori within the preceding 2 weeks, together with strongly positive endoscopic findings, were prospectively enrolled. In order of enrollment they were allocated to two rounds in which F-ROSE was compared with culture-based susceptibility testing. Twenty patients were tested in round 1 on an automated fluorescence immunoassay scanner running the original algorithm; a further 20 were tested in round 2 with the optimized algorithm. With in vitro culture and E-test as the reference standard, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of F-ROSE were calculated for each round for amoxicillin, levofloxacin hydrochloride and clarithromycin, and agreement between methods was assessed with Cohens kappa. Receiver operating characteristic (ROC) analysis was used to identify the optimal cutoff of the fluorescence residual rate (FRR) and to determine how far threshold adjustment improved performance. ResultsCulture succeeded in 15 patients in round 1 and in 14 patients in round 2, leaving 29 evaluable samples. Overall diagnostic performance after algorithm optimization was clearly better than before. Pooled across the three antibiotics, sensitivity was 92.9%, specificity 71.0% and accuracy 77.8% before optimization; after optimization sensitivity rose to 100.0%, specificity increased slightly to 74.2% and accuracy to 81.0%. NPV reached 100.0% for all three agents after optimization, and no resistant isolate was missed. The gain was largest for clarithromycin, for which sensitivity rose from 85.7% to 100.0%, accuracy from 66.7% to 78.6% and kappa from 0.348 to 0.571, indicating a clear improvement in agreement between the two methods. ROC analysis showed that drug-specific optimal cutoffs derived from the Youden index improved specificity appreciably compared with the uniform 50% FRR threshold currently applied, the gain being most evident for levofloxacin hydrochloride. ConclusionsWith sharper fluorescence images and an improved AI recognition algorithm, F-ROSE performs better in H. pylori susceptibility testing, and the reduction in missed resistant strains is particularly noteworthy; the assay is therefore a plausible option for rapid susceptibility screening in clinical practice. Multicenter studies with larger samples are still required to confirm the stability of the technique.

microbiology↗

bioRxiv2026

Adipose GPD2 Couples Glycolytic Signals to Epigenetic Control of Metabolic Homeostasis

The glycerol-3-phosphate shuttle (G3PS) is a classic reducing equivalent transferring system, yet its role in adipose metabolism remains poorly understood. Here, we identify mitochondrial glycerol-3-phosphate dehydrogenase (GPD2), the core enzyme of G3PS, as a critical orchestrator of adipose thermogenesis and systemic metabolic homeostasis. Adipocyte-specific GPD2 knockout mice exhibited impaired thermogenic capacity and energy expenditure, rendering them susceptible to obesity and metabolic dysfunction. Mechanistically, GPD2 deficiency elevated cytosolic NADH, which suppressed glycolysis and decreased the levels of key metabolites, such as acetyl-CoA, thereby diminishing H3K27ac at thermogenic gene loci and silencing these genes. NADH depletion with an NADH oxidase rescued both glycolysis and thermogenic gene expression. In support of this mechanism, we defined a previously undescribed enhancer of Ucp1 whose H3K27ac was tightly controlled by GPD2. Remarkably, replenishing the acetyl-CoA pool or directly restoring H3K27ac at the Ucp1 enhancer reversed UCP1 expression in GPD2-deficient adipocytes. Importantly, we find that human adipose GPD2 expression is inversely correlated with BMI, WHR, and HOMA-IR, underscoring its clinical relevance. Collectively, our findings establish GPD2 as an essential node in the metabolic-epigenetic axis that governs thermogenic activation and energy balance.

biochemistry↗

bioRxiv2026

Pan-cancer NET-associated immunothrombosis coupling identifies a neutrophil-linked but neutrophil-irreducible tumor ecology

BackgroundNeutrophil extracellular traps (NETs) connect innate immunity with thrombosis, metastasis and immune escape, but bulk-tumor NET-associated signatures may mainly report neutrophil abundance. We tested whether an established NETs score also captures a neutrophil-irreducible tumor ecology. MethodsWe reanalysed a published 19-gene NETs score in 9,496 TCGA tumors across 32 cancer types. Tumor-stratified correlations mapped Hallmark and custom immunothrombosis modules. Within each cancer type, the score was residualized against a curated neutrophil proxy and partitioned into four median-defined ecological quadrants. Clinical survival and two independent immune-checkpoint-blockade cohorts provided outcome context. ResultsRaw NETs score was positively associated with TNF-alpha/NF-kB signaling in 32/32 cancers (median Spearman rho=0.404; FDR<0.05 in 30) and hypoxia in 32/32 (median rho=0.394; FDR<0.05 in 31). After neutrophil residualization, hypoxia (median rho=0.206; positive in 30/32) and TNF-alpha/NF-kB signaling (median rho=0.165; positive in 29/32) remained dominant. NETs-high/neutrophil-low tumors retained myeloid-inflammatory, immunothrombotic and hypoxic enrichment. Residual NETs score was associated with adverse overall survival in eight of 23 evaluable cancers after available clinical adjustment. It did not discriminate response in IMvigor210 (AUC=0.519, P=0.642) or GSE91061 (AUC=0.527, P=0.808), although it differed across IMvigor210 immune phenotypes (P=0.045). ConclusionsThe published NETs score contains a neutrophil-linked yet incompletely neutrophil-reducible component that organizes hypoxic, inflammatory and immunothrombotic tumor ecology. Its translational value is context dependent rather than that of a universal standalone immunotherapy biomarker.

cancer biology↗

bioRxiv2026

Conditional Deletion of All Neurexins Defines Diversity of Essential Synaptic Organizer Functions for Neurexins

Presynaptic neurexins are key regulators of synapse properties that arguably represent the best-studied synaptic adhesion molecules. Despite thousands of papers, however, no direct comparison of overall neurexin functions in different types of synapses is available. A decade ago, we provided such an analysis but we recently retracted this paper because four images contained microduplications that, although without discernible impact on the paper's conclusions, could not be corrected. As a result, the scientific community lost access to primary data that established the fundamental principle that neurexins perform profoundly different functions in different types of synapses. In the present study, we have therefore reanalyzed the original raw data and expanded their conclusions with new experiments to document, in a single study, the basic contributions of neurexins to different synapses. We used triple conditional knockout mice targeting all neurexins except for Neurexin-1r and applied neuron-specific manipulations combined with slice electrophysiology, two-photon Ca2+-imaging, and immunohistochemistry. With this approach, we focused on excitatory climbing-fiber synapses in the cerebellum and on inhibitory synapses formed by parvalbumin- or somatostatin-positive neurons in the cerebellum, hippocampus, and medial prefrontal cortex. Our results show that pan-neurexin deletions produce dramatically different phenotypes in synapses, ranging from modest to massive impairments in synapse assembly (climbing-fiber and parvalbumin-positive synapses) to severe but selective decreases in action potential-induced presynaptic Ca2+-transients (somatostatin-positive synapses). Thus, neurexins perform powerful but distinct context-dependent roles in different synapses that shape the brain's circuits.

neuroscience↗

bioRxiv2026

Single-nuclear RNA sequencing reveals an ATF3-independent sensory-neuron program after surgical incision

BackgroundAcute postoperative pain is common and often treated with opioids, but which sensory-neuron changes are responsible for the peripheral drive of pain is poorly defined. Skin incision induces activating transcription factor 3 (ATF3), the canonical marker of nerve injury, in dorsal root ganglion (DRG) neurons, and ATF3 marks the neurons that remain hyperexcitable. Whether ATF3 is required for postoperative pain is unknown. MethodsAdult mice of both sexes underwent hind-paw plantar incision. Postoperative nociceptive behavior was compared between sensory-neuron-specific ATF3 conditional-knockout mice (AvilCre/+; Atf3fl/fl) and littermate controls using assays that probe distinct primary-afferent modalities (von Frey; Hargreaves; dynamic light touch) together with an operator-independent index of spontaneous injury behavior. Single-nucleus RNA sequencing of wild-type and ATF3-null DRG sensory neurons (naive and postoperative day 1) characterized the initiating injury program; transcription-factor-activity inference, RNAscope, and phospho-c-Jun immunostaining examined c-Jun; and the oral dual leucine zipper kinase (DLK) inhibitor GNE-3511, given at the time of incision, was tested behaviorally. ResultsIncision transiently induced ATF3 in a subset of DRG neurons, returning to baseline by day 14, in parallel with the two-week course of nociceptive behavior. Deleting ATF3 in sensory neurons did not change the onset or resolution of nociceptive behavior on any readout, in either sex, across mechanical, thermal, and tactile modalities. Single-nucleus profiling showed a broad surgery-responsive nociceptor program in which ATF3 was the most strongly induced gene, yet only 23% of surgery-responsive neurons expressed it. Without ATF3, the program was remodeled but not abolished, and transcription-factor-activity inference nominated c-Jun as a candidate ATF3-independent factor; incision induced c-Jun in vivo. Systemic inhibition of DLK, which activates c-Jun pathway, lowered c-Jun phosphorylation and reduced evoked nociceptive hypersensitivity. ConclusionsOur results indicate that ATF3 marks part of the injured sensory neuron but does not drive acute post-surgical nociceptive behavior, and that it is not required for the postsurgical pain behaviors across afferent modalities carried by molecularly distinct nociceptor classes. The underlying injury program is broader than ATF3 and is nominated to depend on c-Jun. Because DLK inhibition, given at the time of incision, reduced evoked hypersensitivity, we propose the DLK-JNK-c-Jun axis as a candidate perioperative non-opioid target, which will require further genetic validation.

neuroscience↗

bioRxiv2026

Pluripotency and Transcriptional Pausing Disrupt Circadian Rhythms to Facilitate the Enrichment of Cancer Stem Cells

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype, enriched for cancer stem cells (CSCs), which are the cause of tumor recurrence. CSCs are responsible for tumor initiation and propagation; however, the molecular mechanisms underlying their formation remain largely unclear. We show that carboplatin treated TNBC cells lose their circadian rhythms and promote the enrichment of CSCs. Notably, genetic ablation of the circadian clock by itself, without carboplatin treatment, facilitated the formation of CSCs along with their ability to generate 3D tumorspheres and mouse tumors enriched with CSCs. Mechanistically, we identified an antagonistic interplay between the circadian clock and pluripotency, whereby the core pluripotent factor OCT4 disrupts the expression of circadian timekeeping genes to disable the circadian clock. Furthermore, we uncovered a transcriptional pausing program, controlling the circadian clock, to be perturbed in carboplatin treated TNBC cells. Concordantly, based on gene expression analysis from The Cancer Genome Atlas (TCGA), we found that the uncoupling of transcriptional pausing and the circadian clock correlated with low survivability across diverse cancer types. Moreover, cancer patients with poor prognosis exhibit low expression of the core timekeeping genes Clock, Npas2, Bmal1 and Rorc. Lastly, we restored circadian rhythms in Oct4 deficient TNBC cells and impaired their ability to generate tumorspheres and decreased the number of CSCs in mouse tumors. Overall, our findings demonstrate an unprecedented mechanism for the formation of CSCs that is dependent on the loss of circadian rhythms and thereby has eminent implications for developing new cancer therapies.

cancer biology↗

bioRxiv2026

Spatial Glyco-Codes Define Human Liver Pathology and Progression

Glycosylation is a fundamental process regulating cellular function, tissue organization, and disease progression. However, comprehensive glycan profiling at single-cell spatial resolution remains largely inaccessible, particularly in clinical archival tissues. Here we develop spatial-GPT, a multimodal platform for simultaneous profiling of glycans, proteins, and/or transcripts in archival formalin-fixed paraffin-embedded (FFPE) tissues. Using a panel of 30 DNA-encoded lectins recognizing major mammalian glycan motifs and structural classes, sequencing-based spatial-GPT (DBiT-GPT) mapped the spatial glycome, proteome, and transcriptome across 16 human liver specimens encompassing steatosis, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), leading to identification of spatial glyco-codes - combinatorial glycan states associated with distinct cellular identities, tissue features, and pathological processes. Unexpectedly, glyco-codes alone were sufficient to resolve major cell types, disease states, and HCC subtypes, revealing a previously unappreciated level of biological information encoded within the tissue glycome. Spatial glycomics uncovered tumor-like glyco-codes in premalignant regions, suggesting that glycan reprogramming may precede overt malignant transformation. Using imaging-based single-cell spatial glycan-protein profiling (CODEX-GP), we track glyco-codes across the whole-tissue architecture of 3 representative HCC samples. We further examined the glyco-codes across more than 300 patient specimens and quantified cell-type- and disease-specific glyco-codes as well as glycan-defined immune-evasion, T-cell-exhaustion, and steato-fibrotic niches. Together, these findings establish spatial glyco-codes as a previously unrecognized layer of tissue organization that encodes cellular identity, tissue function, and disease progression. The ability of glyco-codes to distinguish major liver pathologies across independent patient cohorts further highlights their potential as a new class of molecular histopathology biomarkers.

pathology↗

bioRxiv2026

Senescence-associated loss of intestinal α1,2-fucose disrupts a modifiable host-microbiome homeostasis axis in people with HIV

BackgroundPeople with HIV (PWH), despite effective antiretroviral therapy (ART), experience disrupted intestinal homeostasis characterized by microbial dysbiosis and impaired intestinal barrier integrity, which contribute to chronic inflammation and aging-associated comorbidities. However, tractable mechanisms contributing to this dysfunction remain poorly defined. ObjectiveTo determine whether acquired loss of intestinal 1,2-fucose, a host-derived intrinsic prebiotic glycan that supports colonization by short-chain fatty acid (SCFA)-producing bacteria essential for intestinal barrier integrity, contributes to microbiome disruption, impaired epithelial resilience, inflammation, and biological aging in PWH. DesignIleal and colonic biopsies, isolated crypts, stool, and blood from PWH on ART and controls underwent multi-omic analyses. Findings were mechanistically interrogated using stool anaerobic fermentation assays and 3D intestinal organoid models of stress-mediated epithelial disruption. ResultsIn intestinal tissues, PWH exhibited reduced 1,2-fucosylation and increased senescence-associated expression of the fucose-degrading enzyme -L-fucosidase. Lower 1,2-fucose tracked with depletion of SCFA-producing bacteria, increased inflammation, and premature biological aging. In anaerobic fermentations, stool from PWH produced fewer SCFAs than controls, whereas supplementation with the human-milk-oligosaccharide-derived 1,2-fucose donor 2'-fucosyllactose restored SCFA production and improved intestinal organoid resilience to stress-mediated disruption. ConclusionThese findings identify acquired loss of intestinal 1,2-fucose as a modifiable host-microbiome mechanism linking epithelial senescence, microbial metabolic dysfunction, impaired barrier resilience, inflammation, and biological aging in treated HIV infection. SUMMARY BOXO_ST_ABSWhat is already known on this topicC_ST_ABSPeople with HIV on suppressive antiretroviral therapy frequently have persistent intestinal barrier dysfunction, microbial dysbiosis, chronic inflammation, and accelerated biological aging, but the host mechanisms that maintain this disrupted mucosal state remain incompletely defined. What this study addsThis study identifies acquired loss of intestinal 1,2-fucosylation as a feature of treated HIV infection and links this defect to a host fucosidase-high, senescence-enriched mucosal niche, depletion of SCFA-producing bacteria, impaired tight junction-associated barrier signatures, inflammation, and biological aging phenotypes. How this study might affect research, practice or policyThese findings support intestinal glycan ecology as a modifiable host-microbiome axis and provide a rationale for testing 1,2-fucose-replenishing strategies, such as 2'-fucosyllactose, to restore microbial metabolic output and improve epithelial resilience in people with HIV.

microbiology↗

bioRxiv2026

Long-Timescale Molecular Dynamics Reveal a Coordination-Biased Conformational Selection Mechanism for Sorcin Activation

Sorcin is a dimeric penta-EF-hand Ca2+-binding protein that regulates intracellular Ca2+ homeostasis through Ca2+-dependent conformational activation and target recognition, and it has also been implicated in multidrug resistance in cancer. Although crystal structures have defined the apo inactive and Ca2+-bound active states of Sorcin, the transition pathways connecting these states and the conformational ensembles populated under each condition remain poorly understood. Here, we used long-timescale all-atom molecular dynamics simulations on Anton 3, totaling [~]90 s, to define the Ca2+-coupled conformational landscape of dimeric human Sorcin at atomic resolution. Starting from the Ca2+-bound structure, we directly observed the transition from the active to the inactive state following Ca2+ removal, demonstrating that loss of Ca2+ coordination is sufficient to drive inactivation on the microsecond timescale. Simulations initiated from the Ca2+-bound crystal structure with retained ions unexpectedly revealed ultrafast Ca2+ dissociation and rebinding at all EF-hand sites, indicating weak intrinsic Ca2+ affinity and highly dynamic ion exchange. In complementary simulations initiated from the apo structure, Sorcin spontaneously sampled active-like conformations even in the absence of stable Ca2+ binding, supporting a conformational selection mechanism in which Ca2+ shifts the population toward pre-existing active states rather than inducing the transition de novo. Across all conditions, we also observed pronounced and persistent structural asymmetry between the two protomers, revealing that the Sorcin homodimer is dynamically heterogeneous despite its symmetric crystal structures. Together, these results support a coordination-biased conformational selection model for Sorcin activation, in which weak and rapidly exchanging Ca2+ binding stabilizes, rather than induces, the active state. This work provides a dynamic framework for understanding Sorcin function as a fast Ca2+ sensor and offers broader mechanistic insight into activation principles of EF-hand Ca2+-binding proteins.

biophysics↗

bioRxiv2026

Culture-Free Rapid Phenotypic Antimicrobial Susceptibility Testing for Helicobacter pylori Based on Fluorescence Rapid On-Site Evaluation Technology: A Preliminary Study

BackgroundPhenotypic antibiotic susceptibility testing (AST) for Helicobacter pylori (H. pylori) has relied on bacterial culture for three decades, requiring 5-7 days to yield results. Genotypic rapid tests can only detect known resistance mutations and fail to reliably identify amoxicillin resistance. To our knowledge, no culture-free rapid phenotypic AST method for H. pylori has been previously reported. MethodsWe developed a phenotypic AST method based on fluorescence rapid on-site evaluation (ROSE) technology that completely bypasses bacterial culture. Gastric mucosal biopsy specimens from 40 H. pylori-positive patients were homogenized and co-incubated with an acridine orange/ethidium bromide (AO/EB)-based viability staining reagent and three first-line antibiotics--amoxicillin, clarithromycin, and levofloxacin--at concentrations corresponding to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints for H. pylori, at 37 {degrees}C for 1 hour. Fluorescence intensity was measured using a microplate reader. A reduction in fluorescence relative to an antibiotic-free control indicated susceptibility, whereas no significant reduction indicated resistance. Conventional culture-based AST (E-test) served as the reference method. The overall concordance rate, sensitivity, specificity, and Cohens kappa coefficient were calculated. ResultsFourteen of the 40 samples had unsuccessful culture and were excluded, leaving 26 samples for statistical analysis of each antibiotic. The overall concordance rates between the ROSE method and culture-based AST were 84.6% (22/26) for amoxicillin, 76.9% (20/26) for levofloxacin, and 69.2% (18/26) for clarithromycin. Cohens kappa coefficients indicated moderate agreement for all three antibiotics ({kappa} = 0.523, 0.539, and 0.412, respectively). Unlike genotypic methods, the ROSE method successfully assessed amoxicillin susceptibility in all 40 patients--a critical first-line antibiotic for which no reliable genetic resistance marker currently exists. The turnaround time was approximately 1 hour (55-65 minutes), compared with 5-7 days for culture-based methods; preliminary estimates indicated a cost reduction of approximately 3,000-5,000 Chinese yuan (CNY) per patient, mainly attributable to the elimination of culture media, prolonged incubation, and repeat clinic visits. ConclusionsThis study reports, for the first time, a culture-free 1-hour phenotypic AST for H. pylori. The method enables same-day, susceptibility-guided treatment decisions, addressing an unmet clinical need spanning three decades. Algorithm optimization and a prospective randomized controlled trial are currently underway to further improve diagnostic accuracy and validate clinical utility.

microbiology↗

bioRxiv2026

Linguistic Analysis of Chinese Oral Performance in Different Tasks of Chinese Second Language Learners and Native Speakers

This study investigated how different forms of task influence the oral performance of Chinese second-language learners and native speakers. By analyzing data from 40 Chinese second-language learners and 40 native speakers through picture description tasks, formal and informal questions, and questions with different emotions (happy and unhappy), it was found that different task characteristics significantly affected language performance. Short picture tasks led to higher communication efficiency and noun rates but more errors, while long story tasks showed higher verb rates, function word rates, etc. Formal questions had more characters and nouns but lower communication efficiency compared to informal ones. Also, happy emotion questions resulted in fewer characters, sentences, and errors than unhappy emotion questions. These findings contribute to the theoretical understanding of task-based language performance in Chinese as a second language and offer practical implications for teaching, textbook compilation, and student evaluation.

scientific communication and education↗

bioRxiv2026

PACAPergic BAC engages basolateral amygdalar and anterodorsal thalamic networks during looming-threat learning and memory in a labyrinth

The bed nucleus of the anterior commissure (BAC) is a small pituitary adenylate cyclase-activating polypeptide (PACAP)-rich glutamatergic cell population located at the intersection of the anterior commissure, stria terminalis, stria medullaris, and fornix. Here, we provide functional neuroanatomical characterization of the BAC using PACAP-Cre mice and Cre-dependent viral tracing. BAC axons traverse these major forebrain conduits to engage distributed limbic and diencephalic networks, with dense innervation of the posterior basolateral amygdala (pBLA) and anterior dorsal thalamic nucleus (AD), both implicated in emotional processing and spatial orientation. To determine the functional significance of the BAC, we developed a novel looming-threat memory test (LTMT) paradigm in which mice learned the location of a shelter within a dual-maze labyrinth before exposure to an overhead looming stimulus. Chemogenetic inhibition of BAC PACAP neurons impaired shelter-directed escape, increased freezing behavior, prolonged escape trajectories, and disrupted efficient safety-seeking responses. Selective deletion of PACAP from BAC neurons also blocked key aspects of the looming-induced behavioral phenotype. Fos mapping revealed robust neuronal activation by the looming stimulus, within the AD, pBLA, and dorsal periaqueductal gray (dPAG). BAC silencing blocked Fos induction. PACAPBAC depletion blocked fos induction in AD, but not in pBLA or dPAG. Preliminary ex-vivo recordings further indicated that PACAP modulates the intrinsic excitability of AD (and pBLA) neurons. Together, these findings identify the BAC as a previously unrecognized PACAPergic forebrain hub that links emotional and spatial-orientation networks to coordinate looming-threat learning, memory, and adaptive defensive behavior.

neuroscience↗