Biology subjects

Nakamura, S.

Publications and source records attributed to Nakamura, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Engineering robust cardiomyocyte differentiation from iPSCs through WNT-mediated process window expansion

Lack of robustness and high variability in cardiomyocyte (CM) differentiation from human induced pluripotent stem cells (hiPSCs) remain critical challenges for cardiac regenerative medicine and biological research. Guided by developmental principles of lineage specification, we investigated whether early WNT inhibition could expand the process window of CM differentiation and thereby improve robustness. To test this, we compared early WNT inhibition from days 1--5 with standard inhibition from days 3--5 following a 1-day CHIR99021 (CHIR) treatment for WNT activation. Focusing on CHIR concentration as a critical parameter, we found that early WNT inhibition substantially broadened the concentration window yielding [≥]80% differentiation efficiency across distinct hiPSC lines. Extending the analysis to a two-dimensional parameter space incorporating cell density revealed a markedly expanded [≥]80% process window. Temporal analysis of lineage-specific transcription factor expression showed that early WNT inhibition suppressed paraxial mesoderm (PM) specification, likely through MSGN1 suppression, in the range of high initial WNT activation that otherwise favored PM fate, thereby extending the process window. It also suppressed definitive endoderm (DE) specification within the expanded window, promoting a more favorable lateral plate mesoderm/cardiac fate. We further demonstrated substantially reduced variability under parameter perturbations and across wells and batches, accompanied by higher differentiation efficiency, without compromising CM phenotypes. These findings demonstrate early WNT inhibition as an effective strategy for expanding the process window of CM differentiation and improving robustness, while exemplifying the process engineering principle that a broader window can support more robust differentiation, with potential relevance to stem cell--based bioprocesses.

bioengineering↗

bioRxiv2026

Thermal fractuations assist high-speed rotation of the bacterial flagellar motor at low load

The bacterial flagellar motor is a proton-driven rotary nanomachine that converts ion flow into mechanical motion. Proton-coupled rotation of the MotA5-MotB2 stator complex generates torque, yet how this process drives high-speed rotation remains unclear. Here we use single-molecule nanophotometry to resolve stepwise rotation under low-load conditions. Lowering intracellular pH selectively prolongs dwell times without affecting step durations, indicating that proton dissociation triggers torque generation. Structural analysis further suggests that the rotor disengages from the stator before completing the elementary step angle (~11 degree), implying that the power stroke alone is insufficient. Notably, the rotational diffusion coefficient of stator-less motors closely matches that inferred from wild-type motors at low load. These findings support a model in which intrinsic thermal fluctuations compensate for the limited reach of the power stroke, enabling rapid rotation. Our results reveal a hybrid mechanism in which ion-driven conformational changes bias stochastic motion to achieve efficient energy transduction.

biophysics↗

bioRxiv2026

Rewired meristem regulation drives morphological evolution of the one-leaf developmental system in Monophyllaea glabra

Plants have evolved unconventional shoot systems with a "fuzzy" character that blurs organ boundaries, but the developmental programs driving this morphological diversity remain a mystery. One-leaf plants of the genus Monophyllaea are a striking example of this diversity: they develop a single indeterminate cotyledon instead of a conventional shoot system to form a unique structural unit termed a phyllomorph. Here, we integrate tissue-specific and single-nucleus transcriptomics with spatial gene expression analyses to characterize the meristems underlying this developmental system. The Monophyllaea-specific meristem of the phyllomorph retains a conserved shoot apical meristem-like transcriptional core but has a leaf lamina program superimposed. This chimeric transcriptional state integrates normally distinct developmental programs alongside altered phytohormone regulation. The one-leaf morphology emerges through the rewiring of conserved meristem regulatory networks rather than the passive suppression of meristem activity. The findings highlight the remarkable plasticity of developmental modules in plant morphological evolution.

plant biology↗

bioRxiv2026

Increased Interstitial Flow and Elastic Lamina Degradation Precede Aortic Dissection

AimsTo elucidate the relationship between interstitial flow (IF) and structural changes in the aortic media during the development of aortic dissection (AD). MethodsApolipoprotein E-deficient (ApoE[-/-]) mice infused with angiotensin II (AngII) were used as an AD model, in which AD develops exclusively in the thoracoabdominal aorta but not in the thoracic aorta. To capture characteristics present prior to AD onset, the duration of AngII infusion was shortened to generate Pre-AD group. Normal C57BL/6 mice (Normal group) and ApoE(-/-) mice without AngII infusion (Control group) were also included for comparison. Thoracic and thoracoabdominal aortas were excised from all groups, and IF was measured in vitro in accordance with our previously established methods. ResultsIF velocity was generally smaller than 4 m/s across all groups; however, velocities exceeding 4 m/s was observed predominantly in the thoracoabdominal region of the Pre-AD group. Although mean IF velocity did not differ significantly among groups, the standard deviation differed significantly and was the highest in the thoracoabdominal region of the Pre-AD group. In this region, IF velocity tended to increase with more prolonged AngII administration, indicating acceleration of IF prior to AD onset. Three-dimensional internal structural microscopy revealed fragmentation of the elastic lamina (ELs) and a reduction in elastin density only in the thoracoabdominal region of the Pre-AD group. ConclusionsOur findings suggest that increased IF and EL degradation occur in parallel and together contribute to the initiation of AD.

bioengineering↗

bioRxiv2026

Simultaneous quantification of dynamic bacterial deformation and motility by machine learning

We developed a convolutional neural network-based machine learning technique to simultaneously analyze the morphology and motility of spirochetal bacteria swimming with continuous cellular deformation. Matching probabilities between experimental images and learned models realizes quantification of cell morphology and association with motility. This method can be applied to diverse transformable cells, offering critical biophysical insights into microbial dynamics.

microbiology↗

bioRxiv2026

Acquisition of Stickland-metabolizing bacteria during infancy prevents Clostridium botulinum infection

Infant botulism is caused by intestinal colonization with Clostridium botulinum, whereas healthy adults are resistant. Although the gut microbiota has long been implicated in protection against C. botulinum, its bacterial basis has remained unknown. Here, we show that specific amino acid-metabolizing bacteria confer resistance to C. botulinum colonization through nutrient competition. Longitudinally collected human infant microbiotas exhibited a transition from susceptibility to resistance after transplantation into germ-free mice. 5-Aminovalerate marked the resistant microbiota, implicating Stickland metabolism. Resistant microbiotas were enriched in Stickland-metabolizing Clostridia, including Clostridioides difficile. Metabolomics revealed overlapping amino acid utilization, and C. difficile suppressed C. botulinum expansion through amino acid competition. These findings demonstrate that acquisition of Stickland-metabolizing Clostridia during infancy prevents C. botulinum infection through competition for shared amino-acid-dependent nutritional niches.

microbiology↗

bioRxiv2026

Trajectory inference of epithelial-centered neighborhood profiles reconstructs a pseudo-temporal continuum in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by complex lung architecture and spatially heterogeneous remodeling, which have hindered integrated analysis of cell-intrinsic activity and intercellular communication during disease progression. Here we profiled six IPF lung specimens comprising more than 630,000 cells using the Xenium 5k panel and developed an epithelial-centered neighborhood profiling framework based on the local cellular composition around each epithelial cell. This approach captured fibrosis-associated variation in epithelial niches without requiring predefined histological regions. Pseudo-temporal continuum inference of these profiles reconstructed a continuous axis that reflected the spatial progression of fibrotic remodeling from relatively preserved alveolar regions to fibrotic and airway-like remodeled regions. Within this spatial dataset, we mapped coordinated changes in epithelial states, local microenvironments, epithelial intracellular pathway activities, and directional interactions with neighboring cell types along the same axis. Our findings provide a spatial framework that generates testable hypotheses for progressive epithelial niche remodeling in IPF.

bioinformatics↗

bioRxiv2026

Transcriptomic response to histone deacetylase inhibitors in cultured feline cells

ObjectiveIn recent years, the number of cats kept as companion animals has increased, leading to a growing demand for veterinary care. Although some histone deacetylase (HDAC) inhibitors are promising for the treatment of human cancers and neurological diseases, comprehensive systematic research on HDAC inhibitors in domestic cats remains insufficient. Therefore, this study aimed to investigate the effects of HDAC inhibitors on the transcriptome of feline cells. MethodsTwo types of cells derived from domestic cats, Crandell-Rees Feline Kidney (CRFK; kidney-derived) cells and PG-4 cells (astrocyte-derived), were treated with four HDAC inhibitors (panobinostat, trichostatin A, valproic acid, and vorinostat) for 24 h. Transcriptomic changes after treatment were examined using RNA sequencing. ResultsHDAC inhibitor treatment upregulated the expression of intercellular chemical interactions and signal transduction-related genes, similar to observations in human cells. Although HDAC inhibitors did not suppress the expression of cell cycle-related genes in CRFK cells, as observed in human cells, the inhibitors downregulated the expression of organogenesis-related genes. Consistent with observations in human cells, HDAC inhibitors suppressed the expression of cell cycle- and cancer-related genes in PG-4 cells. Importantly, valproic acid, which is thought to be more effective for neurological diseases than for cancer, suppressed the expression of more cancer-related genes in PG-4 cells than the other three HDAC inhibitors. Conclusion and relevanceOur findings revealed that the responses of cells derived from feline organs to various HDAC inhibitors varied considerably depending on the organ of origin and species. Since few studies, including human studies, have comprehensively compared transcriptomic responses to multiple HDAC inhibitor classes across multiple cell types, the results of this study provide a foundation for future research on the treatment and prevention of cancer and neurological diseases in domestic cats and other mammals.

cell biology↗

bioRxiv2026

Machine learning-based decoding of emotional valence from electrophysiological signals in the monkey brain

Understanding how the brain operates in naturalistic settings requires methods that go beyond conventional repeated-measurement approaches, necessitating the development of single-trial neural activity analysis. Recent advances in machine learning offer new opportunities for analyzing brain electrophysiological signals. Here, we recorded surface electrocorticography (ECoG) and intracranial local field potentials (LFPs) from emotion-related brain regions in a monkey performing a Pavlovian conditioning task, in which sensory cues predicting reward or punishment were presented randomly, followed by the actual unconditioned outcome. We evaluated the performance of two machine learning algorithms, a Convolutional neural network (CNN) model and a Transformer-based model (EEG-Conformer), in classifying raw ECoG/LFP traces. Both models successfully classified valence type during conditioned and unconditioned stimulus presentation. Furthermore, the Transformer achieved significantly superior classification performance compared to the CNN, particularly in multi-state classification including baseline periods. By optimizing the training dataset for the Transformer model, we could detect dynamic fluctuations in emotional valence consistent with task type from continuously evolving ECoG/LFP patterns recorded throughout the task. These results demonstrate the utility of Transformer-based models for decoding emotional valence from neurophysiological signals in non-human primates.

neuroscience↗

bioRxiv2026

A novel periplasmic layer formed by an outer membrane lipo-protein governs the cell-envelope integrity and stiffness of Leptospira interrogans

The structural integrity and mechanical properties of the cell envelope are crucial for bacterial physiology and dynamics; however, how specific molecular components govern these properties remains poorly understood. Here, we report the novel structural and mechanical functions of LipL32, the most abundant outer-membrane (OM) lipoprotein in the pathogenic spirochete Leptospira interrogans. LipL32 accounts for approximately 75% of the total OM proteins, yet its functional role has remained enigmatic. Cryo-electron microscopy revealed that the highly dense LipL32 forms a hitherto unappreciated thin layer in the periplasmic space, positioned adjacent to the peptidoglycan (PG) layer. The structural analysis also demonstrates that the disruption of lipL32 by transposon insertion results in irregular deformation of OM and spatial variability in the width of the periplasmic space. Quantitative measurements by optical tweezers confirmed that the lipL32::Tn mutant exhibited a significant reduction in whole-cell stiffness. Consistent with this mechanical impairment, the lipL32::Tn mutant displayed a diminished ability to penetrate a heterogeneous agar mesh model that mimics the hosts extracellular matrix, suggesting an important role for cell rigidity in initial infection via damaged skin. These findings define LipL32 as a critical mechanical stabilizer that maintains the structural integrity of the cell envelope. This work provides a new paradigm for understanding how specialized lipoproteins contribute to bacterial cell mechanics, offering insights into pathogenic invasiveness.

microbiology↗

bioRxiv2026

Anterior cingulate cortex projections to the amygdala in primates: topographic and layer-specific organization underlying emotion and mood regulation

Emotion and mood regulation critically depends on interactions between the anterior cingulate cortex (ACC) and the amygdala. However, the detailed architecture of ACC projections to their major targets, the basal (BA) and accessory (AcBA) basal nuclei of the amygdala, remains unclear. To address this issue, a combined retrograde and anterograde tracing with viral vectors were performed in macaques to map the projection patterns from pregenual (pgACC), subgenual (sgACC), and dorsal (dACC) subareas. Data revealed that ACC neurons projecting to the BA arose predominantly from the superficial layers (II/III) of all subareas and the deep layers (V/VI) of the sgACC, whereas ACC neurons projecting to the AcBA originated mainly in the deep layers of the sgACC and dACC. The present study defines the topographic and layer-specific organization of ACC-amygdala connectivity in primates and subserves to provide an anatomical basis for future causal and translational approaches, such as targeted interventions against ACC-related mood disorders. TeaserPrimate anterior cingulate cortex has topographic and layer-specific projections to amygdala that are involved in emotion and mood regulation.

neuroscience↗

bioRxiv2025

Sensitive colorimetric ELISA measuring salivary SBP-specific secretory IgA, its application to pollinosis SLIT

BackgroundSublingual immunotherapy is safe and one of the most effective etiological treatments for pollinosis. Salivary allergen-specific secretory immunoglobulin A (SIgA) is a useful marker to objectively evaluate treatment effectiveness. However, saliva samples usually exhibit high background noise because of nonspecific binding in enzyme-linked immunosorbent assays (ELISAs). ObjectiveWe aimed to establish an amplified ELISA to measure allergen-specific salivary SIgA levels to assess the efficacy of sublingual immunotherapy (SLIT). MethodsBiotinyl tyramide (BT-Tyr) was used to amplify the signal in a colorimetric ELISA. The allergen sugi basic protein (SBP) was biotinylated and used as a reporter to obtain sensitive and specific results with low background. This ELISA was used to measure anti-SBP SIgA levels in saliva samples from SLIT-treated patients with Japanese cedar pollinosis. ResultsThe ELISA was highly sensitive and achieved more than 200-fold signal amplification compared to non-amplified conditions. It was also specific for antigens without background noise. The levels of SBP-specific SIgA in saliva increased in patients 3 months after SLIT initiation. ConclusionWe established a sensitive colorimetric ELISA to measure SBP-specific SIgA in saliva using BT-Tyr-based amplification and biotinylated antigen as a reporter. We used this sensitive ELISA to monitor the levels of SBP-specific SIgA in saliva samples from patients with pollinosis receiving SLIT. Increased salivary SBP-specific SigA levels were observed in these patients.

immunology↗

bioRxiv2025

Anti-allergic Effect of Soluble Beta-glucan on Japanese Cedar Pollinosis and Mechanism Underlying Its Allergy-ameliorating Action

Japanese cedar (sugi) pollinosis is triggered by sensitization to sugi pollen, which contains sugi antigens, sugi basic proteins (SBP: Cryj1 and Cryj2), and adjuvant polysaccharides, insoluble {beta}-glucan particles (IBG: a dectin-1 agonist). The presence of IBG in sugi pollen enhances the production of SBP-specific IgE, which participates in inflammatory response via activation of mast cells to lead to their degranulation, resulting in sneezing. Another {beta}-glucan derived from black yeast, soluble {beta}-glucan (SBG: a dectin-1 antagonist), is known to have an anti-allergic function. Herein, we demonstrated that oral administration of SBG suppressed both SBP-specific IgE production and SBP-induced sneeze in sugi pollinosis mice model. DNA microarray analyses revealed previously unknown mechanisms underlying SBG-mediated anti-allergy action. SBG ameliorated the dysregulated expression of 16 immune-inflammatory genes that were unusually either up- or down-regulated in sugi pollinosis mice. Based on the DNA microarray informatics, it was suggested that SBG mitigated the IBG-mediated hyper-response of IgE production and inflammatory reaction in immune-inflammatory cells, B and T cells, antigen-presenting cells, and granulocytes (neutrophils and eosinophils) within iliac lymph nodes. The SBG-mediated inhibition of SBP-induced sneezing in pollinosis mice results from a decline in SBP-specific IgE level. The oral administration of SBG influenced gut microbiota, notably enhancing beneficial bacteria such as bifidobacteria. Consequently, it is plausible that short-chain fatty acids, produced by these beneficial bacteria, inhibited mast cell degranulation, leading to a reduction in sneezing. Collectively, SBG demonstrated anti-allergic effects against sugi pollinosis by inhibiting the immune response, specifically SBP-induced IgE production, as well as reducing the inflammatory reaction characterized by SBP-induced sneezing.

immunology↗

bioRxiv2025

Hydrodynamic coupling drives bacterial flagellar synchrony

Synchronization is a fundamental phenomenon observed across a wide range of physical, chemical, and biological systems. Even under the low-Reynolds-number conditions that govern microbial motility, synchronization of rhythmic processes is considered essential for diverse activities ranging from single cells to populations, yet experimental evidence remains scarce. Here, we performed rotation measurements of microbeads attached to two truncated bacterial flagella and observed the emergence of phase synchronization between these nanoscale active motors. The resulting intermittent in-phase synchronization was analyzed using a hydrodynamic model incorporating elastic deformation of the flagella, showing that stronger hydrodynamic coupling promotes more stable phase-locking. This study experimentally demonstrates fluid-mediated biological synchronization using bacterial rotary machinery, deepening our understanding of the physical principles underlying rhythmic phenomena and self-organization in living systems.

biophysics↗

bioRxiv2025

Comparative Analysis of Rumen Microbial Communities in Japanese Black Cattle: Site and Fertility Influences on Microbiome Composition and Function

IntroductionThe rumen microbiome plays a crucial role in nutrition, and productivity of cattle, influencing processes such as digestion, metabolism, and overall health. Understanding the composition and diversity of microbial communities within the rumen is essential for improving cattle management and enhancing the overall health outcomes. This study aims to investigate the rumen microbiome composition across two farms and fertility levels in Japanese Black beef cattle, using 16S rRNA amplicon sequencing. Differences in functional pathways between low- and normal-fertility cattle were also investigated. ResultCore ruminal microbes were identified with Bacteroidota being predominant across sites and fertility levels. Alpha and beta diversity metrics showed that the site explained a substantial variation in microbiome composition, while fertility had a minimal impact. Differential abundance analysis using LEfSe identified distinct microbial profiles for each site. Notably, taxa such as Fibrobacterota and Negativicutes were more abundant on one site, whereas Acholeplasmataceae and Lentisphaeria were more prevalent on the other site ConclusionUsing the sparse Partial Least Squares Discriminant Analysis (sPLS-DA) key taxa associated with fertility status, including Bradymonadales and Elusimicrobiaceae for low fertility and Anaerovoracaceae and Desulfovibrionaceae for normal fertility were identified. In addition, enhanced pathways in the normal fertility group include nicotinate degradation and complex sugar catabolism, while the low-fertility group showed increased activity in glycine betaine and nitrate reduction pathways, suggesting metabolic shifts and may impact reproductive efficiency. These findings underscore the complex interplay between geographic and biological factors in shaping the rumen microbiome.

microbiology↗

bioRxiv2025

Structural basis for color tuning and passive ion conductance in red-shifted pump-fold channelrhodopsin ChR024

ChR024 is a cation-conducting channelrhodopsin with a red-shifted absorption spectrum, recently discovered from a previously unidentified clade through machine-learning-guided gene mining. Due to its unique position in the phylogenetic tree and distinctive spectral properties, it serves as an important model for elucidating rhodopsin evolution and for engineering optogenetic tools with enhanced performance, but its structure and function remain poorly understood. Here, we present cryo-electron microscopy structures of ChR024 in detergent micelles and lipid nanodiscs at resolutions of 3.22 and 2.45 [A], respectively. The structures reveal an architecture strikingly similar to that of ion-pumping rhodopsins, even more so than other pump-like channelrhodopsins such as ChRmine. Structural, theoretical, and spectroscopic analyses uncover a unique color-tuning mechanism, in which charged residues close and distant from the retinal chromophore cooperatively modulate the pKa of the Schiff base counterion and thereby determine the absorption maximum wavelength. Finally, comparative structural analysis of channel- and pump-type rhodopsins, combined with electrophysiology, provides insights into the molecular boundary between these two functional classes and demonstrates the conversion of an outward proton pump into a light-gated channel. Together, these findings illuminate diverse mechanisms of color tuning and functional specification within the rhodopsin family, paving the way for the rational engineering of next-generation optogenetic tools.

biophysics↗

bioRxiv2025

Post-Domestication selection of MKK3 Shaped Seed Dormancy and End-Use Traits in Barley

Anthropogenic selection of grain traits such as dormancy has shaped the developmental trajectories of crop plants (1). In cereals, shortening dormancy provides rapid and even post-harvest germination, but increases the risk of weather-induced pre-harvest sprouting (PHS) with harvest losses estimated at beyond 1 billion USD a year (2, 3, 4). Our understanding of how, why, when and where diversification of cereal dormancy arose is fragmentary. Here, we show in the founder cereal crop barley (Hordeum vulgare) that the Mitogen-Activated Protein Kinase (MAPK) pathway regulates dormancy primarily through a mosaic of locus haplotypes comprising copy-number variation and inherent kinase activity of Mitogen-activated protein kinase kinase 3 (MKK3). We provide evidence supporting the historical selection of specific MKK3 haplotypes that shape dormancy levels according to changing climatic pressures. Understanding the regulatory landscape of MKK3 provides a genetic framework to balance short grain dormancy and PHS-avoidance during a period of rapid climate change.

plant biology↗

bioRxiv2025

A simple and versatile plasma membrane staining method for visualizing living cell morphology in reproductive tissues across diverse plant species

Plant reproduction involves dynamic spatiotemporal changes that occur deep within maternal tissues. In ovules of Arabidopsis thaliana (A. thaliana), one of the two synergid cells degenerates at fertilization, while the fertilized egg cell (zygote) undergoes directional elongation followed by asymmetric division to initiate embryonic patterning. However, morphological analysis of these events has been hampered by the limitations of conventional cell wall staining, which fails to label cells lacking complete walls, and by the requirement for transgenic fluorescent reporters to visualize cell outlines. Here, we report that the membrane-specific fluorescent dye FM4-64 readily permeates ovules, allowing clear visualization of reproductive cell morphology both before and after fertilization. This staining method supports high-resolution time-lapse imaging and quantitative analysis of early embryogenesis in living tissues. Importantly, it is applicable not only to the angiosperm A. thaliana but also to the liverwort Marchantia polymorpha (M. polymorpha) and the fern Ceratopteris richardii (C. richardii), enabling the visualization of live reproductive cell structures within maternal tissues and revealing fertilization-associated morphological changes. This simple and robust method thus provides a valuable tool for spatiotemporal and quantitative analyses of reproductive processes across a broad range of plant species, without the need to generate transgenic lines.

plant biology↗