Biology subjects

Kawano, K.

Publications and source records attributed to Kawano, K..

10 recordsLinked to original sources

bioRxiv2026

Analytical methodology for unveiling the physical effect of detergents on the function of full-length membrane proteins using a single-molecule tracking system

G protein-coupled receptors (GPCRs) are important targets for drug discovery because they are the largest and most diverse family of membrane proteins in the human body. As exemplified by {beta}2-adrenergic receptor ({beta}2AR), which is a typical class-A GPCRs, they are prone to denaturation and inactivation after solubilization. Although diverse techniques have been developed, current structural and functional analyses are still limited to proteins with relatively stable and high expression. However, some mutant variants and misfolded proteins involved in diseases have extremely low expression levels and may be difficult to analyze. To overcome these limitations, we established a novel analytical platform for evaluating the ligand-binding ability of full-length {beta}2AR as a model at the single-molecule level without purification and addressing challenges such as membrane proteins with low expression levels and structural instability. This method enables the direct use of unpurified receptors immediately after solubilization and allows us to use only a small amount of sample ([~]10 ng) for observation and to distinguish between specific and nonspecific ligand binding by fitting. Furthermore, we unveiled the physical properties of detergents on the structural stability of solubilized receptors and found that the lateral pressure within detergent micelles affects ligand-binding ability. Detergents that provided a fluid microenvironment were able to maintain ligand-binding ability for several days even after solubilization; conversely, detergents that provided a rigid microenvironment caused the protein to lose its activity earlier. Our method could be a promising tool for the structural and functional analysis of membrane proteins untargeted until now. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=112 SRC="FIGDIR/small/740883v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@3d274corg.highwire.dtl.DTLVardef@defd0forg.highwire.dtl.DTLVardef@1b81eb4org.highwire.dtl.DTLVardef@e7c275_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Gingipain-containing products from Porphyromonas gingivalis promote epithelial CCL20 signaling and γδ T-cell accumulation in COPD-like airways

Chronic obstructive pulmonary disease (COPD) is a progressive inflammatory airway disease in which impaired mucosal barrier function may increase susceptibility to aspirated oral microbial products. Periodontal disease has been associated with COPD development and exacerbation, but the epithelial mechanisms linking periodontal pathogens to pulmonary immune remodeling remain unclear. Here, we investigated whether gingipain-containing Porphyromonas gingivalis culture supernatant (PCS) promotes {gamma}{delta} T-cell-associated inflammation in COPD-like airways. Repeated intratracheal administration of PCS to {beta}ENaC-transgenic mice induced airway-centered immune cell accumulation and increased {gamma}{delta} TCR-positive cell accumulation, together with elevated expression of the {gamma}{delta} T-cell-associated cytokines Ifng and Il17a. PCS also increased pulmonary Ccl20 and Ccr6 expression, whereas epithelial alarmin-related genes and M2 macrophage-associated responses were not induced in parallel. In ENaC-overexpressing human airway epithelial cells, PCS induced CCL20 and F2RL1, the gene encoding protease-activated receptor 2 (PAR-2), and reduced the N-terminal PAR-2 signal, consistent with proteolytic receptor cleavage. Direct PAR-2 activation reproduced CCL20 induction, whereas pharmacological PAR-2 inhibition suppressed PCS-induced CCL20 expression. In contrast, PAR-1 inhibition or LPS neutralization with polymyxin B did not suppress this response. These findings support a mucosal epithelial protease-sensing model in which gingipain-containing P. gingivalis products activate PAR-2-dependent CCL20 production in airway epithelial cells and are associated with CCR6-linked {gamma}{delta} T-cell accumulation in COPD-like airways. Contribution to the field statementPeriodontal disease is often associated with chronic obstructive pulmonary disease, but how oral bacterial products affect lung inflammation remains unclear. This study shows that protease-rich products from the periodontal pathogen Porphyromonas gingivalis can be sensed by airway epithelial cells under COPD-like conditions. This response induces a chemokine signal that is linked to the accumulation of {gamma}{delta} T cells, a type of immune cell involved in inflammatory responses. Our findings suggest that the airway epithelium acts as an immune sensor, converting signals from aspirated oral bacterial protease into lung inflammation. This work provides a mechanistic framework for understanding how oral dysbiosis may contribute to immune remodeling in COPD.

immunology↗

bioRxiv2026

HDAC3 inhibition stabilizes the IL-37 receptor module to enhance anti-inflammatory signaling in cystic fibrosis airway epithelium

Airway inflammation in cystic fibrosis (CF) persists despite advances in CFTR modulator therapy. IL-37b suppresses innate immune signaling through a receptor complex containing IL-18R and wild-type SIGIRR (WT-SIGIRR; IL-1R8), but this pathway is compromised in CF airway epithelial cells by the dominant-negative exon 8-skipped SIGIRR isoform ({Delta}8-SIGIRR). Here, a natural-product screen identified short-chain fatty acids as preferential enhancers of WT-SIGIRR. Pan-HDAC inhibition with panobinostat increased WT-SIGIRR, reduced {Delta}8-SIGIRR, and restored IL-37b-dependent suppression of the TLR3 ligand poly(I:C)-induced IL-8 production. Isoform-selective inhibitor screening and siRNA knockdown identified HDAC3 as a regulator of the IL-37 receptor module. Low concentrations of RGFP966 and HDAC3 silencing increased WT-SIGIRR and IL-18R protein abundance without inducing their mRNA levels. HDAC3 inhibition delayed proteasome-dependent WT-SIGIRR turnover and stabilized IL-18R, thereby enhancing IL-37b-mediated anti-inflammatory signaling in CF airway epithelial cells.

cell biology↗

bioRxiv2026

Early apelin receptor activation attenuates elastase-induced emphysema and preserves endothelial apelin receptor signaling in mice

Alveolar capillary endothelial cells are positioned adjacent to the alveolar epithelium and contribute to lung homeostasis and injury responses. Single-cell studies have identified aerocyte capillary endothelial cells (aCap), which are specialized for gas exchange, and general capillary endothelial cells (gCap), which contribute to endothelial maintenance and inflammatory signaling. Apelin and its receptor are differentially enriched across these endothelial compartments, but their roles in emphysema development remain incompletely understood. Using an elastase-induced emphysema model in male C57BL/6J mice, we combined bulk RNA sequencing, CIBERSORTx-based cell-type deconvolution, histology, inflammatory assays, pulmonary function testing, and pharmacologic activation of the apelin receptor with [Pyr1]-Apelin-13. At 24 hours after elastase exposure, the inferred fraction of gCap was reduced, and lung expression of apelin and the apelin receptor was decreased. Early [Pyr1]-Apelin-13 administration reduced lung inflammatory mediator expression, Ly6G-positive neutrophil accumulation, bronchoalveolar lavage neutrophil counts, and matrix metalloproteinase-9 activity. Early treatment also attenuated subsequent airspace enlargement, whereas treatment initiated after emphysema was established did not improve physiological or histological outcomes. In a chronic {beta}ENaC-transgenic mouse model, the inferred gCap fraction was maintained, the aCap fraction was reduced, and apelin receptor activation did not improve disease phenotypes. These findings suggest that early activation of the apelin receptor modifies acute inflammatory and endothelium-associated responses following elastase injury and limits emphysematous remodeling in mice. Together, these results support a time-sensitive role for apelin-APJ signaling during the early phase of emphysema development.

molecular biology↗

bioRxiv2026

Dietary lipids attenuate IGF-1-Akt and injure epithelial-endothelial injury program that accelerates obstructive lung disease

Background Altered lipid metabolism is increasingly implicated in chronic obstructive pulmonary disease (COPD), but it remains unclear how a pre-existing obstructive lung state modifies the response to systemic lipid excess. We investigated whether high-fat diet (HFD) amplifies COPD-relevant lung injury and examined epithelial and vascular programs associated with this response. Methods Male wild-type (WT) and beta-epithelial sodium channel-transgenic ({beta}ENaC-Tg) mice were fed control diet or HFD for 10-11 weeks. Lung structure and function, whole-lung transcriptomes, Akt-FOXO1 signaling, apoptosis-related responses, and pulmonary vascular profiles were assessed. Streptozotocin-induced insulin-deficient diabetes and pharmacological IGF-1 receptor inhibition were used as mechanistic comparators. Palmitate responses were examined in human bronchial epithelial cells, ENaC-hyperactive epithelial cells, and endothelial cells, including conditioned-medium transfer. HFD preconditioning was also evaluated in an elastase-induced emphysema model. Statistical analyses included unpaired two-tailed Student's t tests, one-way ANOVA with Tukey-Kramer or Dunnett multiple-comparison testing, Pearson correlation, and Benjamini-Hochberg correction for RNA-sequencing analyses. Results HFD produced similar increases in body weight, glycemia, and adiposity in WT and {beta}ENaC-Tg mice, while further increasing distal-airspace enlargement and reducing FEV0.1/FVC in {beta}ENaC-Tg mice. Lung transcriptomics revealed coordinated remodeling of lipid metabolic, PI3K-Akt, and vascular programs. HFD reduced Akt phosphorylation, increased FOXO1 and Fasl, and increased TUNEL-positive cells in epithelial regions. Palmitate attenuated IGF-1-induced Akt activation in bronchial epithelial cells, whereas IGF-1 receptor inhibition reproduced Akt suppression and apoptosis-related responses without fully reproducing the HFD phenotype. HFD preconditioning also increased elastase-induced airspace enlargement, accompanied by parallel upregulation of FOXO1 and TUNEL positivity. HFD reduced pulmonary CD34-positive vascular profiles, and palmitate activated endothelial cells directly and through conditioned media from ENaC-hyperactive epithelium. In men with airflow obstruction, hepatic steatosis coincided with lower percent-predicted FEV1. Conclusions Dietary lipid stress amplifies obstructive lung injury and engages complementary epithelial and vascular responses. Impaired epithelial IGF-1-Akt signaling and epithelial-endothelial crosstalk provide a mechanistic framework linking systemic metabolic stress to reduced resilience of the obstructive lung.

molecular biology↗

bioRxiv2025

Curvature-sensing peptide functions as a membrane interfactant that glues small extracellular vesicles to cell membranes and enhances vesicle cellular uptake

Small extracellular vesicles (sEVs) are lipid nanoparticles secreted from mammalian cells that are involved in the transfer of informational or therapeutically effective substances between cells. Although the scope of research on sEVs as biocompatible carriers for drug delivery to diseased tissues and cells is expanding, several challenges remain, such as low cellular uptake efficiency and difficulty in loading drugs. It is desirable to alleviate the energy barriers associated with the cellular uptake of sEVs and minimize perturbation of sEVs when loading drugs onto them. In this study, we developed a simple drug-loading system for sEVs using a dimeric curvature-sensing peptide, which enhances sEV accumulation on the cell surface by acting as an adhesive, subsequently inducing endocytic uptake of sEVs through a clathrin-mediated pathway. The dimeric curvature-sensing peptide selectively binds to the sEV surface within 10 min, even in the presence of serum proteins, and functions as a membrane interfactant to reduce the energy barriers for the cellular uptake of sEVs. The cellular uptake of sEVs and the dimeric curvature-sensing peptide under coexisting conditions increased to over fivefold and 20-fold, respectively, compared with those administered alone. Furthermore, the dimeric curvature-sensing peptide can efficiently load anticancer drugs onto the surface of sEVs, and the system effectively induces apoptosis in two types of cancer cells. Dimeric curvature-sensing peptide is a novel technique with potential applications in drug delivery.

biochemistry↗

bioRxiv2024

Wild gut microbiome suppresses the opportunistic pathogen Aeromonas in medaka under domesticated rearing conditions

BackgroundThe gut microbiome plays a crucial role in the metabolic health and pathogen resistance of various host animals. It is also well established that external environmental factors can influence the gut microbiome, leading to differences in its composition. However, the functional implications of these differences remain poorly understood. This study examined the gut microbiome of medaka (Oryzias latipes species complex) by comparing domesticated and wild populations, with the aim of gaining insights into the functional significance of their specific characteristics, particularly those of the wild-type microbiome. ResultsFor the comparative analysis of the gut microbiome, 48 domesticated and 122 wild medaka were collected from multiple laboratories, pet stores and streams across Japan. The results showed that wild medaka exhibited significantly higher gut microbiome diversity, with a broader range of bacterial members. In contrast, the gut microbiome of domesticated medaka harbored lower microbial diversity and was consistently dominated by Aeromonas, a typical opportunistic pathogen in fish. Additionally, 88.6% of Aeromonas isolates from domesticated medaka exhibited haemolytic activity. Moreover, a domesticated rearing experiment with wild populations showed no proliferation or dominance of Aeromonas in their gut, as observed in domesticated medaka. A further rearing experiment revealed that pre-exposing antibiotic-treated medaka to sediments from their natural habitats prevented Aeromonas colonisation, even when reared under domesticated conditions. ConclusionsThese findings suggest that the habitat-derived wild gut microbiome can inhibit Aeromonas proliferation in domesticated fish, highlighting its potential to mitigate opportunistic diseases in aquaculture.

microbiology↗

bioRxiv2024

Rapid screening and identification of genes involved in bacterial extracellular membrane vesicle production using a curvature-sensing peptide

Bacteria secrete extracellular membrane vesicles (EMVs). Physiology and biotechnological applications of these lipid nanoparticles have been attracting significant attention. However, the molecular basis of EMV biogenesis has not yet been fully elucidated. In the previous research, an N-terminus-substituted FAAV peptide labeled with NBD (nFAAV5-NBD) was developed, which can sense a curvature of a lipid bilayer and selectively bind to EMVs in the presence of the cells. Herein, we applied nFAAV5-NBD to a screening of hyper- and hypo-vesiculation transposon mutants of a Gram-negative bacterium, Shewanella vesiculosa HM13, to identify the genes involved in EMV production. As a result, for 16 and six genes, we found that transposon insertion within or near the gene caused hyper- and hypo-vesiculation, respectively. Targeted gene-disrupted mutants of the identified genes demonstrated that the lack of putative dipeptidyl carboxypeptidase, glutamate synthase {beta}-subunit, LapG protease, metallohydrolase, RNA polymerase sigma-54 factor, inactive transglutaminase, PepSY domain-containing protein, and Rhs-family protein caused EMV overproduction. On the other hand, disruption of the genes encoding putative phosphoenolpyruvate synthase, D-hexose-6-phosphate epimerase, NAD-specific glutamate dehydrogenase, and sensory box histidine kinase/response regulator decreased EMV production. This study shows the utility of a novel screening method using a curvature-sensing peptide and fundamental information on the genes related to EMV production. IMPORTANCEConventional methods for isolation and quantification of EMVs are generally time-consuming. nFAAV5-NBD can detect EMVs in the culture without separating EMVs from cells. In situ detection of EMVs using this peptide facilitated screening of the genes related to EMV production. We succeeded in identifying a number of genes involved in the EMV production of S. vesiculosa HM13, and the strains obtained in this study would contribute to the elucidation of bacterial EMV formation mechanisms. Additionally, the hyper-vesiculating mutants obtained in this study would be useful in the application of this bacterium, such as in the production of useful substances as cargoes of EMVs and in the production of surface-engineered vesicles.

microbiology↗

bioRxiv2021

Predicting behavior through dynamic modes in resting-state fMRI data

Dynamic properties of resting-state functional connectivity (FC) provide rich information on brainbehavior relationships. Dynamic mode decomposition (DMD) has been used as a method to characterize FC dynamics. However, it remains unclear whether dynamic modes (DMs), spatial-temporal coherent patterns computed by DMD, provide information about individual behavioral differences. This study established a methodological approach to predict individual differences in behavior using DMs. Furthermore, we investigated the contribution of DMs within each of seven specific frequency bands (0-0.1,...,0.6-0.7 Hz) for prediction. To validate our approach, we confirmed whether each of 59 behavioral measures could be predicted by performing multivariate pattern analysis on a gram matrix, which was created using subject-specific DMs computed from resting-state functional magnetic resonance imaging (rs-fMRI) data of individuals. The prediction was successful, and DMD outperformed temporal independent component analysis, a conventional data decomposition method for extracting spatial activity patterns. Most of the behavioral measures that showed significant prediction accuracies in a permutation test were cognitive-behavioral measures. Our results suggested that DMs within frequency bands <0.2 Hz primarily contributed to prediction. In addition, we found that DMs <0.2 Hz had spatial structures similar to several common resting-state networks. We demonstrated the effectiveness of DMs, indicating that DMD is a key approach for extracting spatiotemporal features from rs-fMRI data.

neuroscience↗

bioRxiv2020

Five patterns of cell signaling pathways associated with cell behavior

Cell signaling pathway is complex systems. Here, we present a concept for a new approach to analyze cell signaling pathway associated with cell behavior. In theoretically, cell behavior is recognized by energy and fluctuation. In this study, we measured phosphorylation level of signal transducers in a cell and fluctuation of the phosphorylation level in the cell population using flow cytometry. Flow cytometric data of mean fluorescence intensity (MFI) and coefficient variation (CV) were considered to the energy and the fluctuation, respectively. Topologically, the changes of MFI and CV were categorized into five patterns (we tentatively named as attractive, subsequent, passive, counter, and negative arbiter). In this study, we clarified the relationship between the cell behavior and the five patterns. Furthermore, combining the five patterns can define the signaling pathways, such as simple activated signal, oscillating signal, regulatory signal, robust signal, or homeostatic signal. These observations provide a proof of concept for general strategy to use the five patterns for connection between cell signaling pathway and cell behavior.

systems biology↗