Biology subjects

Nakamura, H.

Publications and source records attributed to Nakamura, H..

At least 19 recordsLinked to original sources

bioRxiv2026

Developmental origins of thymic B-cell lymphomas

The thymus provides a specialized niche for lymphocyte differentiation, yet the developmental trajectories of its rare B-cell population and their relationship to thymic B-cell lymphomagenesis remain poorly understood. Here, we integrate single-cell multiomics and spatial profiling across human tissues to delineate a B-cell differentiation program selectively represented in the human thymus. This program links CCL17 and germinal center-like B-cell states along a trajectory distinct from canonical germinal center differentiation and can be recapitulated in vitro by signals operating in the thymic medulla. Extending this framework across human B-cell lymphomas, we identify an inherent malignant cell state in PMBCL that shares transcriptional and epigenetic features with CCL17 thymic B cells. This malignant state associates with early clonal positions in reconstructed B-cell receptor phylogenies and persists within tumor microenvironments distinct from the thymic niche. Together, these findings support a model in which thymus-specific B-cell differentiation programs are redeployed during thymic B-cell lymphomagenesis.

cancer biology↗

bioRxiv2026

An integrated Rheo-NMR platform for real-time, site-specific kinetic analysis of amyloid formation

Amyloid fibrils are protein aggregates associated with neurodegenerative diseases. Their formation proceeds through protein- and condition-dependent microscopic pathways that can generate structurally and biologically distinct aggregates, yet these pathways are inferred from ensemble-averaged measurements. Amyloid formation is often slow under quiescent conditions, prompting agitation. However, conventional agitation precludes simultaneous high-resolution solution nuclear magnetic resonance (NMR) spectroscopy, limiting site-specific kinetic analysis of microscopic aggregation pathways. Here we present an integrated Rheo-NMR platform combining controlled shear flow, time-resolved NMR spectroscopy and mechanistic kinetic modeling to quantify site-specific amyloid-formation kinetics in a single, continuously monitored reaction. Applied to -synuclein under dilute and polyethylene glycol (PEG)-induced liquid-liquid phase separation (LLPS) conditions, the method revealed environment-dependent redistribution of kinetic signatures associated with primary nucleation-elongation and secondary nucleation-elongation pathways across the sequence. The platform distinguishes changes in overall aggregation rates from differences in residue-specific kinetic profiles, providing a quantitative framework for dissecting environment-dependent amyloid formation.

biochemistry↗

bioRxiv2026

Proximity Labeling-Assisted Flow Cytometry Improves Fluorescence Contrast in Extracellular Vesicle-Enriched Fractions

Extracellular vesicles (EVs) are potential sources of circulating biomarkers, but disease-associated EVs are often scarce and their nanoscale dimensions limit analysis by conventional flow cytometry. To improve the detection of disease-associated EV signals using widely available instruments, we developed a workflow combining preparation of serum EV-enriched fractions by size-exclusion chromatography, CHL1-initiated enzyme-mediated activation of radical sources (EMARS) proximity labeling, and conventional flow cytometry. EMARS deposits fluorescein on molecules proximal to antibody-bound CHL1, thereby increasing target-initiated fluorescence. In a representative comparison, EMARS increased the signal-to-background ratio from 1.21 with conventional antibody labeling to 6.28. Because unlabeled EVs and debris interfered with analysis, we used an AI-assisted procedure to explore FITC-H cutoffs for retained P1 events. ChatGPT compared 65 candidate cutoffs across statistical and event-retention metrics and recommended 1000; the authors reviewed the complete results before adopting this value. At this data-selected cutoff, mean fluorescence intensity was higher in interstitial lung disease (ILD) than in non-ILD samples among 28 evaluable samples (P = 0.002; area under the receiver operating characteristic curve [AUC] = 0.846); neither value was adjusted for cutoff selection. Post hoc nested leave-oneout cross-validation (LOOCV), in which cutoff selection was repeated within each outer fold, yielded an AUC of 0.716 with 29 of 34 samples evaluable. The association with ILD requires validation in independent cohorts, but these exploratory findings suggest that proximity labeling may enhance measurable fluorescence contrast in serum EV-enriched fractions and support further evaluation of disease-associated EV signals using conventional flow cytometers.

biochemistry↗

bioRxiv2026

KK-LC-1 reveals evolutionary divergence in the regulation of sperm motility between humans and mice

CT83 (KK-LC-1) is a cancer-testis antigen originally identified in human lung cancer cells and has recently attracted attention as a potential target for cancer therapy. Although KK-LC-1 orthologs have been identified in up to 160 animal species, a murine homolog had not previously been identified, hindering in vivo analysis of its physiological function. In this study, we identified the mouse homolog of KK-LC-1 and performed a comparative analysis of its properties in humans and mice, together with an investigation of its biological function using gene knockout (KO) mice. The murine Kk-lc-1 gene is located on the X chromosome and, like its human counterpart, contains an N-terminal transmembrane domain. In both humans and mice, KK-LC-1 is expressed specifically in the testis and localizes to the head and tail regions of sperm. Analysis of Kk-lc-1-deficient mice revealed normal spermatogenesis, and both male and female KO mice were fertile. However, sperm from Kk-lc-1-deficient males exhibited reduced motility caused by decreased flexibility of the midpiece and failed to penetrate the oocyte zona pellucida in vitro. This defect was rescued by artificial insemination using epididymal sperm, suggesting that maternal factors in vivo may compensate for reduced sperm motility. Although impaired sperm motility during in vitro fertilization (IVF) was rescued by murine Kk-lc-1, functional rescue by human KK-LC-1 was not observed. These findings indicate that KK-LC-1 contributes to sperm motility but is not essential for fertility. Moreover, species-specific differences in KK-LC-1-mediated regulation of sperm motility suggest functional divergence during evolution. The role of KK-LC-1 in sperm motility should therefore be considered in the clinical development of cancer therapies targeting KK-LC-1.

developmental biology↗

bioRxiv2026

Patterned photocatalytic proximity labelling for spatial interactomics

Mapping protein-protein interactions and proximity within their native spatial context is essential for elucidating molecular mechanisms underlying biological processes; however, methods for spatial interactomics with subanatomical resolution are limited. Here, we introduce digital micromirror device (DMD)-PhoxID, a method that combines photocatalytic proximity labelling with patterned irradiation using a DMD to map the proximal molecular environment of proteins of interest within defined subregions of fixed brain tissue.

neuroscience↗

bioRxiv2026

Clinical isolates of Mycobacterium avium complex reveal an M. intracellulare-associated IL-17/neutrophilic pulmonary immune program in a murine disease model

Mycobacterium avium complex (MAC) is the leading cause of nontuberculous mycobacterial pulmonary disease (NTM-PD) and mainly comprises M. avium (MAV) and M. intracellulare (MI). Host-pathogen interactions may contribute to the heterogeneous clinical course of MAC pulmonary disease (MAC-PD); however, species- or isolate-associated differences in virulence and host immune responses induced by MAC strains remain poorly understood. Here, we established a panel of MAC clinical isolates exhibiting persistent pulmonary infection in mice and performed transcriptomic analyses to evaluate pulmonary immune responses. Although MAC infections induced broadly shared inflammatory responses, MI infection elicited a robust IL-17/neutrophilic inflammatory signature, whereas MAV infection showed relative enrichment of IFN-{gamma}/cytotoxicity-associated responses, indicating differences in the balance of their immune gene expression programs. The MI-associated IL-17/neutrophilic program remained evident at the isolate level and after adjustment for pulmonary growth phenotype. RT-qPCR and flow cytometric analyses using the representative isolate pair FKJ-1 (MI) and FKJ-8 (MAV) further supported the differential induction of these immune signatures in infected lungs. To define the cellular basis of these immune programs, we performed single-cell RNA sequencing on lungs infected with FKJ-1 and FKJ-8. Infection with the representative MI isolate was associated with increased Il17a-expressing CD4+ T cells and {gamma}{delta} T cells, neutrophilic inflammation, and the expansion of inflammatory macrophages. Taken together, these findings demonstrate that within a selected panel of persistent MAC clinical isolates, MI infection is associated with a robust IL-17/neutrophilic pulmonary immune program, providing a preclinical framework for dissecting species- and isolate-associated host-pathogen interactions in MAC-PD.

immunology↗

bioRxiv2025

Microbiome-host interactions drive a trade-off between sleep quality and lifespan in Drosophila

Understanding the interactions between various aging processes and the resulting heterogeneity in aging is crucial for promoting healthy aging. Here, we provide evidence that heterogeneity in microbiome and host interactions contributes to diversifying aging phenotypes in sleep, gut integrity, and longevity in Drosophila. Aged flies exhibiting sleep fragmentation preserve gut integrity, accompanied by a shift in microbiota composition, particularly an increase in Acinetobacter junii. A. junii induces sleep fragmentation via its metabolite, urocanic acid, through serotonin receptor-dependent dopamine upregulation. In parallel, A. junii exploits the host response to promote its growth, leading to lifespan extension, which is recapitulated by genetically modified Escherichia coli, suggesting a trade-off between sleep quality and lifespan. Our study demonstrates a systematic mechanism underlying aging heterogeneity, suggesting interventions through bacterial supplements.

animal behavior and cognition↗

bioRxiv2025

Sequence-encoded conformational biases shape self-assembly modes of intrinsically disordered proteins

Self-assembly of intrinsically disordered proteins (IDPs) underlies cellular functions and disease pathogenesis. This process is mediated by two intermolecular interaction modes: transient point-to-point contacts described by the sticker-and-spacer framework, and persistent surface-to-surface contacts proposed in the cross-{beta} hypothesis. We investigated the molecular basis of these modes in the context of conformational biases, defined as sequence-encoded structural preferences of local segments. Using a five-residue model, we generated lag-series IDPs from the TIA-1 prion-like domain by systematically modulating conformational biases while preserving amino acid composition. The lag-series IDPs demonstrated distinct condensate properties and varying capacities for amyloid fibril formation. Their structural analyses revealed that strongly biased regions preferentially adopt extended structures, including {beta}-strands, and the spacing between these regions influences metastable {beta}-sheet formation. Our findings demonstrate that local conformational biases shape interaction modes of IDPs, thereby linking sequence to condensate properties and amyloid fibril formation. Significance StatementProteins fold into three-dimensional structures defined by their amino acid sequences. In contrast, intrinsically disordered proteins (IDPs) lack stable structures, yet their sequences encode unique self-assembly behaviors, including phase separation and amyloid fibril formation. Can such behaviors be explained within structure-based frameworks? Using a five-residue model, we showed that IDP sequences encode local structural preferences, termed conformational biases, within short segments. These biases determine whether segments engage in transient point-to-point interactions driving phase separation or persistent surface-to-surface interactions leading to amyloid fibril formation. By bridging sequence, structure, and interaction modes, our work provides a comprehensive mechanism for self-assembly and conceptual tools for understanding IDP-related biological functions and disease mechanisms.

biophysics↗

bioRxiv2025

Strain-dependent disease progression and necrotizing granuloma formation induced by virulent Mycobacterium avium complex strains in a murine model

Mycobacterium avium complex (MAC) is the leading cause of non-tuberculous mycobacterial pulmonary disease (NTM-PD), a chronic infection with a heterogeneous clinical course. Although murine models of MAC-PD have been developed, reproducing the key features of progressive human disease, including diverse pathological features and therapeutic sensitivities, remains challenging. In this study, we evaluated five clinical MAC strains, including a newly identified highly virulent isolate, NBRC112750, in immunocompetent BALB/c mice. Among these, FKJ-1 and NBRC112750 induced progressive pulmonary infection with increasing bacterial burdens and extensive lung involvement by 25 weeks post-infection. Notably, both strains led to the formation of necrotizing granulomas resembling those observed in M. tuberculosis-infected C3HeB/FeJ mice. These lesions featured neutrophilic infiltration, foamy macrophages, and collagen encapsulation. Using FKJ-1 strain, we also established an inhalation infection model, in which low-dose exposure reproduced necrotizing granulomas. Despite in vitro drug susceptibility, FKJ-1 infection exhibited poor response to standard therapy, highlighting strain-dependent variability in treatment efficacy. These findings establish a murine model that reflects both key pathological and therapeutic aspects of MAC-PD and provides a valuable platform for investigating MAC pathogenesis and evaluating novel therapies.

microbiology↗

bioRxiv2025

Title Mafb Deficiency in Myeloid Cells Increases Susceptibility to Mycobacterium tuberculosis Infection in Mice

v-Maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) is a candidate gene associated with early tuberculosis onset identified by a genome-wide association study. Here, we investigated the role of Mafb in susceptibility to Mycobacterium tuberculosis (Mtb) infection in myeloid-specific Mafb-knockout (Mafb-cKO) mice. We infected bone marrow-derived macrophages (BMMs) from Mafb-cKO mice and Mafb-cKO mice with Mtb. The absence of Mafb promoted Mtb proliferation in BMMs. RNA sequencing (RNA-seq) revealed activation of the metabolic process and impairment of the response to type interferons (IFNs) in Mtb-infected BMMs from Mafb-cKO mice, which conforms to our previous findings in Mtb-infected human macrophages with MAFB knockdown. Mafb deficiency increased mortality and bacterial burden in the lungs and spleens during Mtb infection in mice. RNA-seq revealed weakened leukocyte or lymphocyte chemotaxis in Mtb-infected Mafb-cKO mouse lungs. Flow cytometry demonstrated an alteration in the proportion of immune cells in Mtb-infected mouse lungs due to Mafb deficiency. Together, Mafb in myeloid cells is involved not only in the functional antibacterial process of macrophages but also in immune cell recruitment in the lungs, thereby contributing to host defense against Mtb infection.

immunology↗

bioRxiv2025

Single-cell transcriptomic profiling reveals a novel signature of necrotizing granulomatous lesions in the lungs of Mycobacterium tuberculosis-infected C3HeB/FeJ mice

Tuberculosis (TB) pathology involves complex immune responses within granulomatous lesions. Using single-cell RNA sequencing, we characterized the cellular compositions of necrotizing granulomatous lesions that developed in the lungs of Mycobacterium tuberculosis-infected C3HeB/FeJ mice. We identified 11 distinct major cell types, including phagocytes such as neutrophils and macrophages, and T cells, natural killer cells, B cells, dendritic cells, and plasmacytoid dendritic cells. Among T cells, particularly, Pdcd1 {gamma}{delta} T cells were detected in necrotizing granulomatous lesions, suggesting their potential role in the pathogenicity of M. tuberculosis. Within the macrophage populations, we identified a cluster with significantly higher Plin2 expression compared to other clusters, whose transcriptomic profile was consistent with that of foamy macrophages. A subset of the Plin2-expressing macrophages was identified as a major source of Ifnb1 and Cxcl1, suggesting their involvement in type I interferon signaling and neutrophil recruitment. Furthermore, we identified Flrt2, Hyal1, and Mmp13 as novel molecular markers of Plin2-expressing macrophages, which were localized to the peripheral rim regions of necrotizing granulomas. In conclusion, our results provide the immune landscape of necrotizing granulomas and reveal novel functional states of macrophages contributing to TB pathogenesis.

microbiology↗

bioRxiv2024

Implication of S-1 biomarkers on aging and prognosis in elderly patients with lung cancer treated by adjuvant S-1 chemotherapy: Accompanying results of the Setouchi Lung Cancer Group Study 1201

BACKGROUNDSThe management of elderly patients presents several challenges due to age-related declines; however recent recommendations advocate for age not being the sole determinant for adjuvant treatment decisions in patients with non-small cell lung cancer (NSCLC). Aging may alter expression levels of 5-fluorouracil (5-FU) biomarkers. METHODSExpression changes with aging were explored using The Cancer Genome Atlas (TCGA) database. 5-FU-related biomarker expressions, including thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyltransferase, epidermal growth factor receptor (EGFR), and excision repair cross-complementation group-1 (ERCC1), were assessed by the quantitative reverse-transcription PCR assays in 89 NSCLCs elderly patients ([≥] 75 years old) receiving adjuvant S-1, an oral fluoropyrimidine agent, therapy in a SLCG1201 trial. RESULTS: TCGA database analysis (n=955) indicated decreased TS expression with aging, particularly in those 75 or older. In the SCLG1201 trial, univariate analysis revealed that high EGFR and low TS expressions correlated with favorable recurrence-free survival (RFS)(p=0.0264) and overall survival (OS)(p=0.0365), respectively. Multivariable analysis confirmed pathological stage as an independent prognostic factor for both RFS and OS (p=0.0052 and 0.0352, respectively). EGFR mutant tumors (n=27) showed significantly higher DPD (p=0.0066) and EGFR (p<0.0001) expressions, and lower TS(p=0.0125) and ERCC1(p=0.0015) expressions. CONCLUSIONDespite pathological stage being an independent prognostic factor, high EGFR and low TS expressions may predict better clinical outcomes in elderly NSCLC patients using adjuvant S-1. The age-related decrease in TS expression supports the potential benefit of 5-FU-based therapy in them compared to younger patients. Further research is warranted to validate these clinical implications.

pharmacology and toxicology↗

bioRxiv2024

Spatial confinement induces reciprocating migration of epidermal keratinocytes and forms triphasic epithelia

Epithelial cells undergo epithelial-mesenchymal transition (EMT) during migration and regain their epithelial phenotype in the post-migration phase (mesenchymal-epithelial transition; MET). We established an experimental system that reproduces a three-compartment epithelial structure comprising the original epithelium, its EMT state, and its MET state. Keratinocytes (KCs), skin epithelial cells, placed on a microporous membrane migrated through 3.0-{micro}m or larger micropores. The 3.0-{micro}m-pored membrane induced an epithelial structure with three distinct states: stratified KCs above the membrane, KCs showing EMT within the micropores, and a new stratified epithelium under the membrane. The membrane with larger micropores failed to maintain the three-compartment epithelial structure. Live imaging revealed that KCs moved in an oscillatory manner, with actin-rich filopodia-like structures extending into and out of the 3.0-{micro}m micropores, while the cells migrated unidirectionally into larger micropores. Piezo1 and keratin 6 were identified as negative modulators of KC entry into and exit from the 3.0-{micro}m micropores. These results demonstrate that non-cancerous epithelial cells migrate through confined spaces in an oscillatory manner, which might contribute to the formation of a three-compartment epithelial structure that recapitulates key aspects of wound healing.

cell biology↗

bioRxiv2024

ER sensing of lipid metabolism drives PRA family-dependent regulation of COPII vesicle transport

Newly synthesized secretory proteins and many lipids are transported from the endoplasmic reticulum (ER) to the Golgi prior to their ultimate destinations. The ER-to-Golgi transport must be tightly regulated during adaptation to environmental stress. However, the sensing mechanism and regulatory pathways governing the consecutive formation, budding and transportation of COPII vesicles from the ER remain insufficiently explored. Here, we present evidence indicating that COPII-mediated vesicle transport is transcriptionally controlled through the phosphatidic acid-dependent Opi1-Ino2/Ino4 regulatory circuit. Our analysis indicates that YIP3, a target gene of Ino2/Ino4, exerts a negative regulatory impact on COPII-mediated vesicle transport. Furthermore, we demonstrated that Ino2/Ino4 but not Yip3 modulates Sar1 activation, the initial step in COPII vesicle formation, whereas Yip3 hinders Sec16 assembly on the ER membrane, thereby implying that Ino2/Ino4 governs COPII-mediated trafficking at multiple steps. Thus, this study provides the first evidence for an ER sensing system that transcriptionally fine-tunes multiple steps of anterograde vesicular transport in response to alterations in lipid composition of the ER membrane.

cell biology↗

bioRxiv2024

Self-organization of spermatogenic wave coordinates sustained sperm production in the mouse testis

Spermatogenesis takes place in the testis, relying on the ordered turnover of differentiating cells supplied from stem cells. Classic histological analyses have revealed that this process shows hierarchical spatiotemporal patterning known as the spermatogenic cycle, wave, and descent of segmental order, indicative of currently underexplored mechanisms of tissue- and organ-scale homeostasis. Here, using mice, we conducted high-resolution, wide-field, and ultra long-term live imaging studies in vivo and ex vivo, combined with whole-organ mapping of differentiation stages. Such trans-scale measures demonstrate how stereotypic local cell turnover is coordinated into characteristic phase waves propagating along the seminiferous tubules, further organized into organ-scale patterning over the tubule loops. Minimal mathematical modeling shows that such higher-order dynamics can emerge from the local coupling of autonomous oscillators, which are rooted in delayed feedback interplay between stem and differentiating cells via retinoic acid signaling. These findings highlight a self-organization mechanism underpinning organ-scale homeostasis and constant sperm production.

developmental biology↗

bioRxiv2024

Light-guided actin polymerization drives directed motility in protocells

Motility is a hallmark of lifes dynamic processes, enabling cells to actively chase prey, repair wounds, and shape organs. Recreating these intricate behaviors using well-defined molecules remains a major challenge at the intersection of biology, physics, and molecular engineering. Although the polymerization force of the actin cytoskeleton is characterized as a primary driver of cell motility, building a minimal platform to test this process in protocellular systems has proven elusive. The difficulty lies in the daunting task of distilling key components from motile cells and integrating them into model membranes in a physiologically relevant manner. To address this, we developed a method to optically control actin polymerization with high spatiotemporal precision within cell-mimetic lipid vesicles known as giant unilamellar vesicles (GUVs). Within these active protocells, the reorganization of actin networks triggered outward membrane extensions as well as the unidirectional movement of GUVs at speeds of up to 0.43 {micro}m/min, within the range of adherent mammalian cells. Notably, our findings reveal the requirements of both branched and linear actin networks for efficient membrane protrusions. This approach offers a powerful platform for unraveling the intricacies of cell migration, designing synthetic cells with active morphodynamics, and advancing bioengineering applications, such as self-propelled delivery systems and autonomous tissue-like materials.

synthetic biology↗

bioRxiv2024

A microendovascular system can record precise neural signals from cortical and deep vessels with minimal invasiveness

Minimally invasive intravascular electroencephalography (ivEEG) signals are a promising tool for developing clinically feasible brain-computer interfaces (BCIs) that restore communication and motor functions in paralyzed patients. However, current ivEEG techniques can only record signals from the superior sagittal sinus (SSS), making it challenging to record motor responses related to hand and mouth movements from brain regions distant from the SSS, despite their critical role in BCIs. Here, using micro intravascular electrodes, ivEEGs recorded from the cortical or deep veins of eight pigs could measure cortical activities with greater signal power and better spatial resolution than those recording in the SSS, thus allowing mapping of the sensorimotor and visual functional areas. Additionally, electrical stimulation in the cortical vein between the micro intravascular electrodes induced muscle contractions contralateral to the stimulated area in five anesthetized pigs. These results demonstrate that ivEEG using micro intravascular electrodes is a promising tool for developing BCIs.

neuroscience↗

bioRxiv2024

Transcriptome Analysis Identified SPP1+ Monocytes as a Key in Extracellular Matrix Formation in Thrombi

Thrombi follow various natural courses. They are known to become harder over time and may persist long-term; some of them can also undergo early spontaneous dissolution and disappearance. Hindering thrombus stability may contribute to the treatment of thrombosis and the prevention of embolisms. However, the detailed mechanisms underlying thrombus maturation remain unknown. Using RNA sequencing, we revealed the transcriptional landscape of thrombi retrieved from the cerebral vessels and identified SPP1 as a hub gene related to extracellular matrix formation. Immunohistochemistry confirmed the expression of osteopontin in monocytes/macrophages in the thrombi, particularly in older thrombi. Single-cell RNA sequencing of thrombi from the pulmonary artery revealed increased communication between SPP1-high monocytes/macrophages and fibroblasts. These data suggest that SPP1-high monocytes/macrophages play a crucial role in extracellular matrix formation in thrombi and provide a basis for new antithrombotic therapies targeting thrombus maturation. TeaserSPP1+ monocytes play a key role in thrombus maturation, which can be a potential target for novel antithrombotic therapies.

molecular biology↗