Biology subjects

Kobayashi, T.

Publications and source records attributed to Kobayashi, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Characterization of a maintainable human myeloid model of VEXAS syndrome with enhanced TNF-induced cell death and DAMP release

Background: VEXAS syndrome is an adult-onset autoinflammatory disorder caused by somatic UBA1 mutations. UBA1-mutated myeloid cells have been reported to exhibit increased susceptibility to inflammatory cell death; however, the mechanisms underlying this phenotype and the extracellular consequences of enhanced cell death remain incompletely understood. Methods: Disease-associated UBA1 M41 mutations were introduced into U937 cells by CRISPR/Cas9-mediated genome editing. The resulting cells were characterized by genomic, molecular, and functional analyses, with particular focus on UBA1-associated cellular phenotypes, cell death signaling, and extracellular release of damage-associated molecular patterns (DAMPs). Results: Genome editing yielded UBA1-mutated cells that could be maintained under standard culture conditions while recapitulating multiple molecular and cellular features associated with VEXAS syndrome. Long-read sequencing revealed the intended UBA1 M41 mutation on one allele and a CRISPR/Cas9-induced on-target genomic deletion on the other. The mutant cells exhibited reduced UBA1b and increased UBA1c expression, prominent cytoplasmic vacuolization, reduced proliferative capacity, and increased basal cell death. They also showed enhanced susceptibility to TNF-induced cell death under conditions favoring either apoptosis or necroptosis. Mechanistic analyses demonstrated enhanced apoptotic and necroptotic signaling, together with increased basal abundance of RIPK1, RIPK3, and MLKL. Pharmacological inhibition of RIPK1, RIPK3, or MLKL attenuated membrane permeabilization under caspase-inhibited conditions. Enhanced cell death was accompanied by increased extracellular release of ATP, HMGB1, and S100A8/A9. Conclusions: We characterized a maintainable UBA1-mutated human myeloid model that recapitulates multiple molecular and cellular features associated with VEXAS syndrome. Using this model, we showed that UBA1 dysfunction is associated with heightened susceptibility to TNF-dependent apoptotic and necroptotic cell death and increased extracellular release of multiple DAMPs. These findings provide insight into the cellular consequences of UBA1 dysfunction and establish a tractable experimental platform for further mechanistic studies of VEXAS syndrome.

immunology↗

bioRxiv2026

Sensory neuron dysfunction and hyperexcitability in dorsal root ganglia at disease onset in the SOD1G93A mouse model of ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder traditionally characterized by motor neuron degeneration, but emerging evidence indicates sensory system involvement. Despite reports of sensory abnormalities in some patients, the molecular and functional alterations in dorsal root ganglion (DRG) neurons remain insufficiently characterized. We investigated DRG pathology at disease onset in 12-week-old SOD1G93A mice using an integrated transcriptomic, morphological, and electrophysiological approach. RNA sequencing of lumbar DRG identified 35 differentially expressed genes, predominantly upregulated, enriched in oxidative stress-related and phagosome pathways. Comparative analysis with motor neuron transcriptomes revealed distinct gene expression profiles, indicating sensory neuron-specific molecular responses. Immunohistochemistry demonstrated reduced soma diameter in both A- and C-fiber DRG neurons. Nav channel colocalization increased for Nav1.7 in A fibers and Nav1.8 in both fiber types, whereas Nav1.6 was unchanged. Whole-cell patch-clamp recordings showed depolarized resting membrane potential, increased spike amplitude, and enhanced repetitive firing in A-fiber neurons, consistent with hyperexcitability, while C fibers showed no significant functional changes. These findings demonstrate early molecular, structural, and functional alterations in primary sensory neurons in ALS, supporting pathology beyond motor neurons and identifying sensory neuron excitability as a potential therapeutic target.

neuroscience↗

bioRxiv2026

Early detection of pancreatic ductal adenocarcinoma by single-molecule profiling of pancreatic enzyme activities

Pancreatic ductal adenocarcinoma (PDAC) remains a leading cause of cancer-related mortality, largely due to diagnosis at advanced stages. Early detection through minimally invasive liquid biopsy holds promise for improving patient outcomes. Here, we report a blood-based liquid biopsy platform based on single-molecule enzyme activity profiling (SEAP), which detects proteoform-level alterations in circulating pancreatic enzymes at single-molecule resolution. Using a tissue-centric biomarker discovery strategy, we identified activity signatures of pancreas-specific digestive enzymes associated with PDAC. A combinatorial classifier detected PDAC (stage I-IV) with 95.5% specificity and 75.0% sensitivity (74.2% for stage I-II) across 690 blood samples collected from multiple hospitals and biobanks. Performance was further validated in an independent cohort enriched for early-stage disease, where 54.2% (13/24) of stage IA, 64.3% (9/14) of stage IB, and 21.4% (3/14) of stage 0 lesions were classified as positive. These findings support the clinical potential of SEAP for early detection of PDAC.

biochemistry↗

bioRxiv2026

Fast corrective responses in redundant motor control are shaped by intrinsic constraints of movement patterns

In real-world motor tasks, body movements unfold in a high-dimensional space, whereas task errors are defined in a lower-dimensional space. How such low-dimensional errors propagate across redundant motor degrees of freedom to generate rapid corrective responses remains poorly understood. To address this question, we developed a redundant bimanual task in which participants manipulated a virtual stick with both hands to move its tip to a visual target. Visual perturbations either displaced the stick tip (end-effector relevant errors) or altered the tilt angle of the stick without affecting the tip position (end-effector irrelevant errors), allowing us to dissociate errors defined in task space from those arising in redundant dimensions. Participants rapidly corrected both types of perturbations using highly stereotyped movement patterns. Corrections to end-effector relevant errors consistently involved coordinated changes across redundant dimensions, and even perturbations that did not affect task success elicited systematic corrective responses. Together, these results demonstrate that fast corrective responses in redundant motor control are not generated by flexible re-optimization, but are instead shaped by intrinsic coordination constraints that govern how visual errors propagate through the motor system.

neuroscience↗

bioRxiv2026

Visuomotor learning is determined by the tonic component of feedback responses

When a movement error occurs, the motor system updates its commands to improve performance on subsequent trials. A prominent feedback error learning hypothesis proposes that the feedback response that corrects movement within a trial serves as a teaching signal for the learning response, observed as changes in motor commands on the next trial. However, how the temporal pattern of the feedback response influences the learning response remains unclear. Here, we introduce an experimental paradigm that directly compares the temporal patterns of feedback and learning responses across different patterns of visual error. We show that although the feedback response closely tracks the temporal pattern of the visual error, this temporal pattern is not transferred to the learning response. Instead, the amplitude of the feedback response during the holding period, which reflects the temporal history of the visual error, strongly predicts the magnitude of the learning response. These findings suggest that information reflected in the tonic component of feedback responses is closely linked to the scaling of subsequent motor-command updates.

neuroscience↗

bioRxiv2025

Caveolin-1 controls lineage fidelity of adult neural stem cells via lysosomal degradation of PDGFRα

Caveolae are cell-surface signaling hubs involved in stem cell proliferation and differentiation. However, the molecular details of how they influence stem cell quiescence remain poorly understood. In this study, we show that most caveolae components, particularly caveolin-1 (Cav-1), are specifically upregulated in quiescent neural stem cells (NSCs). Furthermore, we demonstrate that Cav-1 restricts platelet-derived growth factor receptor (PDGFR) signaling, a key regulator for NSC proliferation and oligodendrocyte differentiation in the adult brain, through lysosomal degradation. Genetic ablation of Cav-1 leads to aberrant NSC activation, reduced neurogenesis, and enhanced ectopic oligodendrocyte differentiation on the ventricular surface in the ventral side of the ventricular-subventricular zone (V-SVZ), a key NSC niche responsible for neuroblast differentiation. Mechanistically, we show that Cav-1 deficiency suppresses the endo-lysosomal degradation of PDGFR, leading to PDGFR accumulation and excessive signaling for oligodendrocyte differentiation. Taken together, our results uncover a crucial homeostatic mechanism wherein caveolae function as a safeguard to maintain adult NSC quiescence and lineage fidelity by controlling lysosomal turnover of PDGFR in a niche-specific manner.

neuroscience↗

bioRxiv2025

Environmental exosomes: Evidence of extracellular RNA release by aquatic organisms

Aquatic organisms continuously interact with surrounding water, yet whether they release extracellular vesicles remains unknown. We hypothesized that pearl oysters (Pinctada fucata) release exosomes/small extracellular vesicles (sEVs) into the aquatic environment. To this end, we collected exosomes/sEV-sized components by ultrafiltration from tank water and open-sea culture areas. Microscopy revealed abundant vesicles consistent with exosome/sEV size, and RNA sequencing identified oyster-specific piRNAs that matched sequences previously detected in hemolymph exosomes. These findings demonstrated that pearl oysters actively released exosomes containing species-specific nucleic acids into surrounding water. We propose referring to these vesicles as environmental exosomes/environmental sEVs (eExosomes/esEVs). This finding suggests that aquatic exosomes serve as carriers of RNA and may contribute to inter-organismal communication networks. Beyond their functional role, eExosomes/esEVs also hold promise as novel targets for environmental DNA/RNA (eDNA/eRNA) analysis, offering new opportunities for ecological monitoring and biodiversity research.

ecology↗

bioRxiv2025

Cellular fitness is determined by ribosomal protein S12-mediated release of a truncated Xrp1

Multicellular tissues require continuous optimization to maintain their structure and function by actively eliminating viable, yet unfit cells via a mechanism known as cell competition. During this process, unfit loser cells commonly upregulate the C/EBP family transcription factor Xrp1, which causes their elimination, indicating that intracellular Xrp1 levels determine cellular fitness. However, despite its central role, the mechanism by which Xrp1 is upregulated in unfit cells remains unknown. Here, we show that Xrp1 is upregulated through a previously unrecognized post-transcriptional regulatory mechanism mediated by the ribosomal protein S12 (RpS12). Surprisingly, Xrp1 mRNA is abundantly expressed even in wild-type cells, yet it is not translated. This is due to an upstream open reading frame (uORF) located in the 5UTR of the initial coding exon of Xrp1 mRNA, which inhibits the translation of the main Xrp1 ORF. Intriguingly, in unfit cells, RpS12 causes splicing-mediated skipping of this initial exon, leading to the use of an alternate start codon that generates a short isoform of Xrp1 protein, causing cell death. Notably, protein structural analysis reveals that RpS12 is highly homologous to that of a spliceosomal component SNU13, suggesting the role of RpS12 in directly regulating alternative splicing of Xrp1 mRNA. Our findings thus provide not only a mechanistic insights into how cellular fitness is determined but also how tissue optimization is achieved.

cell biology↗

bioRxiv2025

Development of a sandwich ELISA for the detection of bovine A1 beta-casein

The genetic variant A2 beta-casein is associated with fewer digestive and absorption issues compared to A1 beta-casein, leading to increased global demand for A2 milk. However, contamination with the A1 variant during collection, transportation, or sterilization of A2 milk poses a risk, necessitating a verification test to ensure A2 milk does not contain A1 beta-casein. We developed an A1-specific monoclonal antibody (mAb) and a general mAb that reacts with both A1 and A2 variants using the iliac lymph node method. A sandwich ELISA was created using the general mAb as the capture antibody and the A1-specific mAb as the detection antibody to identify A1 beta-casein in milk. This ELISA successfully detected A1 beta-casein in raw and pasteurized A2 milk, including ultra-high temperature treated milk. The test identified A1 beta-casein when the A1 spike in A2 milk exceeded 1% in volume, indicating its capability to detect contamination from one A1A1 cow in a herd of one hundred A2A2 cows. The developed A1 beta-casein ELISA is suitable for high-throughput analysis and can be valuable for monitoring A1 beta-casein contamination in commercially produced A2 milk.

zoology↗

bioRxiv2025

A lipid-driven, microbe-independent mechanism of acne via Lrig1+ follicular progenitor cells

Long-chain saturated fatty acids (lcSFAs) are abundant in the skin, but their pathogenic roles in acne remain unclear. In human sebum profiling, C16:0, the most abundant lcSFA, was the only fatty acid significantly elevated in acne and correlated with inflammatory comedone counts. We then established a mouse model that faithfully recapitulates human acne phenotypes, in which topical C16:0 penetrated the epidermis and induced sebocyte hyperplasia, comedogenesis, and follicular inflammation. Mechanistically, C16:0 activated keratinization and inflammatory pathways and drove sebocyte-lineage differentiation from Lrig1+progenitor cells, which function as cutaneous lipid sensors. These effects persisted in germ-free mice and were unaffected by fatty acid transporter modulation, while MyD88 signaling was partially required for inflammatory cell recruitment. Together, our findings identified C16:0 as a human acne-associated lipid that recapitulates disease hallmarks through a microbe-independent, lipid-driven pathway, highlighting Lrig1+cells as central hubs in remodeling of the pilosebaceous unit.

cell biology↗

bioRxiv2025

The caninized anti-canine PD-1 monoclonal antibody in canine oral malignant melanoma: Efficacy and exploratory biomarker analysis

BackgroundCanine oral malignant melanoma (OMM) is a highly aggressive tumor with few treatment options. Because of its biological similarity to human mucosal melanoma, canine OMM represents a valuable spontaneous model for translational immunotherapy studies. Anti-PD-1 antibody therapy has shown promise in canine OMM; however, predictive biomarkers for treatment response and survival have not been identified. MethodsWe conducted a multicenter, prospective, investigator-initiated clinical trial to evaluate the safety and efficacy of a caninized anti-canine PD-1 monoclonal antibody (ca-4F12-E6) in 150 dogs with advanced OMM. The dogs were administered 3 mg/kg ca-4F12-E6 intravenously every two weeks. Treatment response was assessed using cRECIST v1.0. Biomarker analyses included peripheral blood parameters, cytokine/chemokines, peripheral lymphocyte subsets, microsatellite instability (MSI), immunohistochemistry for immune cell and mismatch repair protein markers, and RNA sequencing of the tumor tissue. Associations with clinical outcomes were determined by logistic regression and Cox proportional hazard models. ResultsThe best overall response rate was 16.7%. Treatment-related adverse events occurred in 40.0% of the dogs, which were primarily grade 1-3. Increased baseline white blood cell, neutrophil count, and C-reactive protein (CRP) levels were significantly associated with poor response, shorter progression-free survival (PFS), and reduced overall survival (OS). MSI-high tumors were associated with significantly prolonged OS compared with MSI-low/microsatellite stable tumors. Transcriptome analysis revealed differentially expressed genes and enriched immune-related pathways in responders, though limited by sample size. Conclusionca-4F12-E6 exhibited durable antitumor activity and an acceptable safety profile in dogs with OMM. Baseline systemic inflammatory markers and MSI status may serve as predictive biomarkers for clinical outcomes. The results support the use of canine OMM as a comparative model for human immuno-oncology and biomarker discovery.

cancer biology↗

bioRxiv2025

Emergence of cell polarity by reciprocal interactions between Wnt and core PCP components

Planar cell polarity (PCP) is established by asymmetric localization of core PCP components in each cell. Since some Wnt proteins can induce PCP in vertebrates, it has been proposed that Wnt concentration gradients provide spatial cues for polarization. Here, however, we present evidence that Wnt11 can regulate PCP in a gradient-independent manner. In the neural plate of Xenopus embryos, endogenous Wnt11 does not form an obvious gradient in the direction of PCP, but is polarized at cell boundaries together with core PCP components. Wnt11 polarization is dependent on core PCP components, while polarization of core PCP components can also be induced by Wnt11, indicating a mutual amplification loop between Wnt11 and core PCP components in PCP formation. Furthermore, Wnt11 and core PCP components compose an intercellular loop to coordinate the direction of polarity. Thus, we propose that local and reciprocal interactions between Wnt11 and core PCP components can generate PCP.

developmental biology↗

bioRxiv2025

Dynamic polarization of heparan sulfate proteoglycans is involved in planar cell polarity and localization of endogenous Wnt11 during vertebrate neural tube formation.

Planar cell polarity (PCP) on a tissue plane is established by asymmetric polarization of core PCP components in individual cells. Recently, we found that Wnt11 organizes PCP in Xenopus embryos by accumulating locally with core PCP components to be polarized, but not by acting as a directional cue forming a global gradient. Since heparan sulfate proteoglycans (HSPGs) associate with Wnt on cell membranes, how HSPGs are involved in Wnt polarization and PCP formation is a central issue. Here, we show that HSPGs are dynamically organized in a polarized manner, as with core PCP components and Wnt11, by associating with them. While HSPGs with HS chains enriched by N-sulfation and N-deacetylation are polarized, depending on Wnt and core PCP components, NDST1, which catalyzes these modifications, is required for Wnt11 polarization and PCP formation. Thus, mutual regulation among HS chains, Wnt11, and PCP components appears to be essential for PCP formation. HighlightsO_LIndst1 is required for Wnt11 localization and proper PCP formation. C_LIO_LINDST1-modified HS chains are polarized in the neural plate during development. C_LIO_LIPolarization of NDST1-modified HS chains depends on PCP formation. C_LIO_LIPositive feedback loop results in polarized distribution of HSPGs, Wnt11, and PCP. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC="FIGDIR/small/669988v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1db75adorg.highwire.dtl.DTLVardef@10aec47org.highwire.dtl.DTLVardef@1e50d5org.highwire.dtl.DTLVardef@4cd41c_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2025

Genome sequence data and integrative RNA-Seq data of Black Soldier Fly (Hermetia illucens) (A Japanese strain)

The black soldier fly, Hermetia illucens, is a dipteran insect with a distinctive black body in the adult stage. The larvae of this species possess biological features which can be used for industrial applications. We established an H. illucens field-collected strain to develop an industrial system for recycling organic waste. We constructed whole-genome sequences of the strain of H. illucens and prepared a gene set and its functional annotation data to promote research on the social implementation of H. illucens and genetically characterise this strain. In addition, time-course transcriptome data of the larvae and transcriptome expression data of multiple tissues from 6th instar larvae and male and female adults were prepared. The genome size and its N50 value were approximately 1.05 Gbp and 191 bp, respectively. The results of the validation analyses of the genome and transcriptome expression data showed that these data were reliable as reference data, suggesting that they can be used in research on the industrial usage of evolutionary H. illucens or evolutionary or comparative biology.

genomics↗

bioRxiv2025

Local, but not circulating, complement C3 governs immune checkpoint blockade efficacy

The roles of circulating complement C3, produced in hepatocytes, in immune defense are well defined in the literature. Interestingly, the C3 gene evolved before the circulatory system, suggesting an essential but yet unknown role of locally produced C3. Here, we investigated the effect of C3 on cancer immunotherapy and found that cancer-associated fibroblast (CAF)-derived C3, not hepatocyte-derived C3, determines the efficacy of immune checkpoint blockade (ICB). Tumors developed in CAF-specific C3 knockout mice exhibited resistance to anti-PD-1 therapy, with increased immunosuppressive M2-like macrophage infiltration. Mechanistically, the C3 degradation product iC3b suppressed myeloid cell infiltration via complement receptor 3 signaling, and the therapeutic targeting of this pathway restored ICB sensitivity of immunotherapy-resistant tumors. In human cancers, stromal C3 expression correlates with reduced M2-like macrophage infiltration and improved immunotherapeutic outcomes. These data demonstrate the role of locally produced C3 in the innate immune response, which may have been conserved across many organisms.

cancer biology↗

bioRxiv2025

Inhibiting B-cell-mediated Immunosuppression to Enhance the Immunotherapy Efficacy in Hepatocellular Carcinoma

BackgroundImmunotherapy is efficacious in hepatocellular carcinoma (HCC), but the benefits are limited to a minority of patients. Most HCC patients show resistance to immune checkpoint blockade (ICB). Agonists of the stimulator of interferon genes (STING), potent immune stimulators, showed limited effectiveness. Using preclinical models, we studied the mechanisms of resistance to ICB and STING agonism. MethodsMurine HCA-1 and RIL-175 HCCs were orthotopically grown in mice with underlying liver fibrosis, to mimic the presentation of human HCC. Established tumors were treated with a STING agonist (BMS-986301) or anti-PD1 ICB, and mice were followed to evaluate safety and efficacy, as well as the mechanisms of treatment resistance by RNA sequencing, flow cytometry, and immunofluorescence, B-cell depletion and T-cell immunoglobulin and mucin domain 1 (TIM-1) ICB. ResultsUnbiased analyses of transcriptomic data from murine HCC tissues from ICB-treated mice showed an increased abundance of intratumoral CD8+ T cells and B cells. STING agonism alone showed efficacy in the ICB-responsive RIL-175 HCC model but more limited efficacy in the ICB-resistant HCA-1 model. STING agonism increased circulating IL-10 and intratumoral infiltration by B-cells, including TIM-1+ B cells, and promoted the formation of tertiary lymphoid structure (TLS)-like structures, especially in the peritumoral areas. Strikingly, adding B cell depletion to ICB or STING agonism treatment significantly increased survival. Interestingly, unlike ICB, STING agonism also had a pronounced anti-metastatic activity. In addition, the combination of STING agonism and TIM-1 blockade augmented B cell differentiation and antigen presentation in vitro and improved the anti-tumor effects in murine HCC in vivo. This approach decreased the number of TIM-1+ B cells in the tumor and shifted B cells to higher expression of CD86 and MHC class II, enhancing the antigen presentation capability and further boosting the antitumor efficacy of CD8+ cytotoxic T cells. ConclusionOur findings demonstrate that B cells are associated with ICB- and STING-mediated therapy resistance, and that depleting B-cells or targeting TIM-1 enhances both innate and acquired therapeutic efficacy in HCC.

cancer biology↗

bioRxiv2024

Dantrolene inhibition of ryanodine receptor 1 carrying the severe malignant hyperthermia mutation Y522S visualized by cryo-EM

Mutations in the skeletal isoform of the ryanodine receptor (RyR1) pose grave risks during anesthesia or treatment with succinylcholine muscle relaxants. These can trigger a potentially lethal malignant hyperthermia (MH) episode via intracellular calcium increase mainly from RyR1 channel leakage. Dantrolene is the primary antidote to prevent death. The main target of dantrolene is RyR1, however little is known about the mechanism of inhibition. Cryo-EM of dantrolene bound to the severe MH Y522S RyR1 mutant in the closed and open states at 2.5-3.3 [A] resolution revealed that the drug binds to the channels cytoplasmic assembly, far from the ion gate, interacting with residues W882, W996 and R1000 in the P1 domain. The finding was validated by Ca2+ imaging and [3H]ryanodine binding in WT and alanine mutants. Dantrolene reduced channel opening probability by restricting the central activation module, "cooling down" the primed conformation caused by the mutation. These findings shed light on dantrolenes mechanism against MH and suggest avenues for developing more effective therapeutics.

biochemistry↗

bioRxiv2024

Low-invasive, wide-field, and cellular resolution two-photon imaging of neural population activity in brainstem and nucleus tractus solitarii

Brain-viscera communication plays a crucial role in regulating mental health, with the vagus nerve being a key structure mediating this interaction. Clinically, artificial vagus nerve stimulation (VNS) is used to treat various neuropsychiatric disorders, highlighting the importance of vagal afferent fibers in regulating emotion. The nucleus tractus solitarii (NTS) is a brainstem structure proposed to receive signals from vagal afferents and relay them to brain networks for emotion regulation. However, due to the anatomical complexity and difficulty in accessing the deep-brain NTS region in living animals, the mechanisms remain unclear. Here, we developed a wide-field and deep-brain two-photon imaging method using a double-prism based optical interface. This approach enables the identification of cellular-resolution neural activities in the NTS while preserving the cerebellum, which covers the NTS and is important for emotion regulation, intact. We systematically evaluated how NTS neurons respond to VNS and a gastrointestinal hormone, suggesting the usefulness of this method for investigating the role of the vagus-NTS pathway in vivo.

neuroscience↗