Biology subjects

Kasai, T.

Publications and source records attributed to Kasai, T..

13 recordsLinked to original sources

bioRxiv2026

Multiparametric Extracellular Vesicle Profiling Identifies a Distinct Circulating Signature of Bicuspid Aortic Valve Disease

Background: Calcific aortic valve disease (CAVD) resulting in life-threatening aortic stenosis, heart failure and death, develops earlier and progresses more rapidly in patients with bicuspid aortic valves (BAV) than tricuspid aortic valves (TAV), yet the circulating molecular drivers remain poorly understood. Extracellular vesicles (EVs) capture disease-relevant cellular activation and offer a powerful window into a BAV-specific pathophysiology. Methods: Circulating large extracellular vesicle (LEV) abundance and morphology were characterized by complementary single-vesicle nano-flow cytometry and super-resolution microscopy. Proteomic profiling further delineated LEV molecular cargoes and associated biological pathways between patients with BAV and TAV. Results: We demonstrate that patients with BAVs exhibited fundamentally altered circulating LEV profiles compared to TAVs. Super-resolution imaging revealed BAV-specific organization of platelet- and leukocyte-associated adhesion molecules into discrete EV membrane microdomains. Proteomic profiling identified distinct LEV cargoes between BAV and TAV, including a BAV-specific, previously unrecognized neutrophil-associated signature enriched for proteins involved in neutrophil degranulation and innate immunity. Consistent with this phenotype, BAV displayed alterations in platelet- and neutrophil-associated LEV populations, suggesting enhanced (thrombo-)inflammatory activity. Lastly, distinct associations between LEV populations and clinical parameters in BAV and TAV suggest that LEV profiles reflect morphology-specific aspects of disease severity. Conclusions: Comprehensive single-vesicle analysis and bulk proteomic profiling identified a distinct circulating LEV signature in BAV-associated CAVD, characterized by endothelial activation and reciprocal platelet-neutrophil interactions. These findings reveal previously unrecognized differences in circulating LEVs between BAV and TAV and highlight their potential as biomarkers of valve morphology-specific pathophysiology.

molecular biology↗

bioRxiv2026

Platelet-endothelial crosstalk drives distinct endothelial dysfunction in bicuspid aortic valve disease

Background: Bicuspid aortic valves (BAV) predispose individuals to premature development of aortic stenosis (AS). The abnormal hemodynamic environment of BAV affects both platelets and valvular endothelial cells (VECs), but whether platelet-derived signals contribute to valve morphology-specific endothelial dysfunction remains unknown. Methods: Platelets and VECs were obtained from patients with BAVs or tricuspid aortic valves (TAVs) undergoing valve replacement for AS. Platelets were activated to generate platelet releasate (PR). PR composition and its effects on VECs were investigated using complementary molecular, multi-omic, functional, and computational approaches. Results: BAV and TAV patients exhibited markedly distinct PR profiles, which translated into differential endothelial responses. Strikingly, BAV-derived PR elicited a substantially more pronounced endothelial dysfunction phenotype, characterized by widespread molecular reprogramming and functional alterations. Integrated multi-omics uncovered mitochondrial dysfunction as a central feature of BAV PR-induced endothelial injury, accompanied by activation of pathways spanning endothelial remodeling, migration, and immune-cell recruitment. These molecular signatures translated into mitochondrial depolarization, altered mitochondrial dynamics, enhanced VEC migration, and increased neutrophil adhesion and chemotaxis. Finally, computational ligand-receptor and ligand-target analyses identified candidate signaling axes linking platelet-derived mediators to endothelial reprogramming, highlighting potential therapeutic targets. Conclusions: We provide the first direct evidence that platelet activation contributes to AS initiation through the induction of endothelial dysfunction and that these effects are amplified in BAV. These findings identify platelet-VEC crosstalk as a previously unrecognized mechanism linking abnormal valve hemodynamics to disease onset. Targeting platelet-derived mediators may represent a novel therapeutic strategy for AS, specifically in BAV.

molecular biology↗

bioRxiv2025

Selective PPAR-α activation with pemafibrate attenuates macrophage-mediated progression of calcific aortic valve disease

BACKGROUNDCalcific aortic valve disease (CAVD) compromises valve compliance and cardiac hemodynamics leading to aortic stenosis (AS) and cardiovascular dysfunction. With treatment for severe AS limited to valve replacement and no effective pharmacotherapies, new interventions are urgently needed for patients. This study evaluated pemafibrate, a selective peroxisome proliferator-activated receptor alpha (PPAR) activator as a novel therapeutic for CAVD and AS. METHODS AND RESULTSIn an aortic valve wire injury (AVWI) model of AS in Ldlr-/- mice, pemafibrate administration (0.2 mg/kg/day) for 15 weeks improved aortic valve function and reduced valvular calcification by 39% (p<0.001), accompanied by reduced leaflet inflammation and CD68 macrophage infiltration. These effects were independent of changes in plasma triglyceride levels. In vitro, pemafibrate suppressed inflammation-mediated calcification of primary human valvular interstitial cells (VICs) by modulating macrophage-derived secreted factors, identifying macrophage-VIC crosstalk as a key disease mechanism. Direct treatment of macrophages with pemafibrate, or exposure to serum from pemafibrate-treated participants in the PROMINENT randomized controlled trial, shifted macrophages toward a less inflammatory and less chemotactic phenotype. Proteomic analyses of patient serum substantiated these findings by reflecting a systemic reduction in inflammatory parameters and monocyte activation. Network integration of the in vitro derived pemafibrate-responsive proteome with human calcified AV tissue proteomes identified aberrant protein translation (GNB2L1, GSPT1) and disrupted bioenergetics (MYDGF, PDIA4) as potential clinically relevant pemafibrate-responsive pathways and effector proteins relevant to AS progression. CONCLUSIONSPemafibrate slows experimental AS progression and valve calcification through modulation of macrophage-VIC crosstalk, independent of lipid lowering. These findings support further evaluation of pemafibrate as a potential pharmacological approach for CAVD and support further testing in randomized clinical trials.

pharmacology and toxicology↗

bioRxiv2025

Nanoparticle-enabled plasma proteomics of a mouse atherosclerosis model

BackgroundDyslipidemia, marked by elevated LDL-cholesterol (LDL-C), is a major risk factor for coronary heart disease. Mouse experimental models, such as Ldlr-/- mice that develop atherosclerosis and metabolic disorders when fed a high-fat diet (HFD), are indispensable for studying disease mechanisms and identifying potential biomarkers. ObjectivesWe aimed to profile the plasma proteins in experimental murine atherosclerosis with the primary goal of detecting low-abundant proteins. MethodsLdlr-/- mice were fed a chow diet or HFD for 3 (n = 27 per group) or 6 months (n = 12 per group). Plasma samples were processed using nanoparticle technology (Proteograph(R) XT Assay; Seer, Inc), and peptides were analyzed using the Orbitrap Astral (Thermo Fisher Scientific) in data-independent acquisition mode. For tissue proteomics, ten aortas were pooled for n = 4 pools per time point and diet, and n = 6 livers per time point and diet. Peptides were analyzed on the Orbitrap Exploris 480 in data-dependent mode. Proteomes were queried against the Tabula Muris mouse single-cell, STRING, and Gene Ontology databases, and queried against a genome-wide association list of 419 risk loci for coronary artery disease. ResultsWe sequenced 5,080 plasma proteins, surpassing previous reports by 10-fold. The prototypical apolipoproteins and complement factors were the most abundant proteins, whereas proteins associated with cytokine/chemokine signaling represented the previously uncharted mouse plasma proteome. We divided the proteome into quartiles (Q1-Q4) to monitor sweeping changes over time. Proteins with a sustained enrichment in HFD (n = 705) were indicative of liver cell subtypes (Tabula Muris). Whereas proteins that moved up from the lower quartiles - Q2 (n = 228), Q3 (115) and Q4 (63) - associated with leukocyte, fibroblasts, and endothelial cell markers, indicating that signatures of inflammation and endothelial activation increase with disease progression. Notably, 86 and 146 proteins were increased at 3 and 6 months, including MMP-12 and COL6A3. Classical apolipoproteins exhibited heterogeneous responses - SAA3 and APOC2 increased, while APOA1, APOE, and LCAT decreased with high-fat feeding, suggesting impaired high-density lipoprotein (HDL) functionality. Proteins shared between plasma and aorta were enriched for extracellular matrix components, while those overlapping with liver reflected metabolic processes. Finally, 120 coronary artery disease (CAD)-associated proteins from human GWAS were detected in Ldlr-/- plasma, of which 4, including lipoprotein lipase, exhibited an increase in abundance with HFD. ConclusionsNanoparticle-dependent proteome enrichment coupled to mass spectrometry may allow to identify novel plasma biomarkers in Ldlr-/- mice and facilitate monitoring of candidate proteins associated with human disease mechanisms in preclinical interventional studies, thereby opening new avenues for understanding disease pathology and uncovering novel molecular contributors.

pathology↗

bioRxiv2025

Cross-species proteomics quantification pipeline distinguishes donor versus host extracellular matrix in explanted biomaterials

Xenogenic biomaterial durability, including bioprosthetic heart valves (BPVs), is compromised by pathological extracellular matrix (ECM) remodeling, resulting in progressive structural degeneration. Mass spectrometry-based proteomics can help reveal BPV degeneration mechanisms; however, peptide sequence similarity between donor and host species complicates protein-level analysis. We present a cross-species proteomic analytical strategy for xenogenic biomaterials and cross-species proteomic datasets. In silico tryptic digestion of human and bovine protein databases identified over 400 overlapping proteins with a high protein percent identity. Explanted human BPV tissue was divided into macroscopically distinct regions of degeneration and analyzed by mass spectrometry. A peptide-level strategy quantified protein abundances in a species-delineated analysis. We highlighted degeneration region-specific depositions of key human ECM proteins and bovine ECM proteins whose abundance is time dependent. We demonstrated that single-species analysis of a cross-species proteome results in inaccurate quantification. This study highlights the importance of distinguishing between donor and host species proteomes for accurate protein quantification. While focused on clinically explanted biomaterials, our approach is broadly applicable to all forms of xenotransplantation and the use of xenogenic matrices.

bioengineering↗

bioRxiv2024

Cross-regulation of amino acid synthesis and catabolic electron transfer in bacteria

Amino acids are essential for life, serving primarily as the building blocks of proteins. Although the intracellular metabolic fate of amino acids is known, their roles in the global regulation of metabolic pathways are poorly characterized. Here, we investigated the function of amino acids as signaling molecules that modulate the catabolic activity of bacterial cells. Using the model bacterium Shewanella oneidensis MR-1, we showed that methionine activates catabolic processes, including extracellular electron transfer, at submillimolar concentrations. This regulation is mediated by MetR, a transcription factor that regulates methionine biosynthesis. In addition, the MetR-mediated regulation of catabolic electron transfer was observed in Aeromonas hydrophila. These findings reveal methionine-induced signaling cascades that cross-regulate amino acid biosynthesis and catabolism in bacteria. Our study highlights a novel physiological role for amino acids, suggesting their involvement in the coordinated regulation of anabolism and catabolism, particularly in bacteria that thrive in nutrient-limited habitats.

microbiology↗

bioRxiv2024

Sexual Dimorphism of Plasma and Tissue Proteomes in Human Calcific Aortic Valve Stenosis Pathogenesis

BACKGROUNDCalcific aortic valve stenosis (CAVS) is a global clinical burden, impacting around 2% of the population over 65 years of age. No pharmacotherapeutics exist, with surgical repair and transcatheter valve replacement being the only intervention. Females are underrepresented in studies of CAVS, leading to delay in timely intervention and increased mortality. Histopathology demonstrates female CAVS presents with decreased valvular calcification but increased fibrosis and severity of symptoms. We hypothesize that the underlying molecular mechanisms contributing to disease progression and fibrocalcific burden in AS differs between male and female patients. Our goal for this study is to use previously acquired proteomic datasets of a clinically-defined human AS cohort to examine sex disparities and underlying sex-specific disease signatures. METHODS and RESULTSAge-matched human AS tissue samples (n=4 females, n=14 males) were each segmented into non-diseased, fibrotic, and calcified disease stages and analyzed using LC-MS/MS proteomics and quantitative histopathology. CAVD plasma samples (n=20 females, n=30 males) were analyzed for circulating sex-specific biomarkers via LC-MS/MS. Unbiased principal component analysis shows sex- and stage-specific proteome clustering. AS pathogenesis drove sex-specific disparities in the valvular proteome: 338/1503 total proteins were differentially-enriched by sex across disease stages. Compared to sex-specific non-diseased controls, female fibrotic tissue resulted in 2.75-fold greater number of differentially-enriched proteins than did male fibrotic tissue (female: 42, male: 16; p<0.05 threshold). In contrast, female calcific tissue identified 2.473-fold less differentially-enriched proteins than male calcific tissue (female: 157, male 356; q<0.05 threshold). By Functional Enrichment Analysis revealed specific proteins responsible for the exacerbated valvular fibrosis signature in females, implicated adenosine phosphate metabolism as a potential male-specific driver of AS, and further reinforce the shared contribution of aberrant lipid and cholesterol activity to AS progression in both sexes. CONCLUSIONSWe reveal a sexually-dimorphic AS proteome, including the novel overabundance of ECM remodeling pathways in female calcified aortic valve tissues. This analysis allows for identification of potential sex-specific protein drug targets implicated in AS pathobiology.

pathology↗

bioRxiv2024

FtsZ of wall-less bacteria forms ring-like structures

The FtsZ protein is involved in bacterial cell division. In cell-walled bacteria, such as Bacillus subtilis, FtsZ forms a ring-like structure, called the Z ring, at the cell division site and acts as a scaffold for cell wall synthesis. The inhibition of cell wall synthesis in B. subtilis has been shown to interfere with the function of the Z ring, causing a loss in cell division control. Spiroplasma, a cell wall-less bacterium, lacks most of the genes involved in cell division; however, the ftsZ gene remains conserved. The function of Spiroplasma eriocheiris FtsZ (SeFtsZ) remains to be determined. In the present study, we analyzed the biochemical characteristics of SeFtsZ. Purified SeFtsZ demonstrated lower polymerization capacity and GTPase activity than FtsZ from E. coli and B. subtilis. We also investigated the relationship between SeFtsZ and SeSepF, which anchors FtsZ to the cell membrane, and found that SeSepF did not contribute to the stability of FtsZ filaments, unlike the B. subtilis SepF. SeFtsZ and SeSepF were produced in E. coli L-forms, where cell wall synthesis was inhibited. SeFtsZ formed ring-like structures in cell wall-less E. coli cells, suggesting that SeFtsZ forms Z rings and is involved in cell division independently of cell wall synthesis.

microbiology↗

bioRxiv2024

Autonomous adaptive optimization of NMR experimental conditions for precise inference of minor conformational states of proteins based on chemical exchange saturation transfer

In scientific experiments where measurement sensitivity is a major limiting factor, optimization of experimental conditions, such as parameters of measurement instruments, is essential to maximize the information obtained per unit time and the number of experiments performed. When optimization in advance is not possible because of limited prior knowledge of the system, autonomous, adaptive optimization must be implemented during the experiment. One approach to this involves sequential Bayesian optimal experimental design, which adopts mutual information as the utility function to be maximized. In this study, we applied this optimization method to the chemical exchange saturation transfer (CEST) experiment in nuclear magnetic resonance (NMR) spectroscopy, which is used to study minor but functionally important invisible states of certain molecules, such as proteins. Adaptive optimization was utilized because prior knowledge of minor states is limited. To this end, we developed an adaptive optimization system of 15N-CEST experimental conditions for proteins using Markov chain Monte Carlo (MCMC)to calculate the posterior distribution and utility function. To ensure the completion of MCMC computations within a reasonable period with sufficient precision, we developed a second-order approximation of the CEST forward model. Both simulations and actual measurements using the FF domain of the HYPA/FBP11 protein with the A39G mutation demonstrated that the adaptive method outperformed the conventional one in terms of estimation precision of minor-state parameters based on equal numbers of measurements. Because the algorithm used for the evaluation of the utility function is independent of the type of experiment, the proposed system can be applied to other instruments, as well as other NMR experiments, if the forward model or its approximation can be calculated sufficiently quickly. Author SummaryIn scientific experiments, experimental conditions are usually optimized to maximize the amount of information obtained about the target system. However, this optimization often involves measuring unknown system parameters. In nuclear magnetic resonance (NMR) spectroscopy, chemical exchange saturation transfer (CEST) is an effective method for observing minor states of certain molecules, such as proteins, indirectly via an easily observable major state. The optimal conditions for CEST depend on the resonant frequency of the minor state, which is not known before the experiment. Therefore, conventional CEST requires repeated measurements over a wide frequency range, most of which contain little information regarding minor populations. This problem may be resolved by adopting a step-by-step strategy that selects the most informative experimental condition for each subsequent step based on past data. In this study, we implemented this strategy for the CEST analysis of proteins. It can be also applied to other NMR experiments.

biophysics↗

bioRxiv2022

Pulmonary dust foci as rat pneumoconiosis lesion induced by titanium dioxide nanoparticles in 13-week inhalation study

BackgroundMost toxicological studies on titanium dioxide (TiO2) particles to date have concentrated on carcinogenicity and acute toxicity, with few studies focusing of pneumoconiosis, which is a variety of airspace and interstitial lung diseases caused by particle-laden macrophages. The present study examined rat pulmonary lesions associated with pneumoconiosis after inhalation exposure to TiO2 nanoparticles (NPs). MethodsMale and female F344 rats were exposed to 6.3, 12.5, 25, or 50 mg/m3 anatase type TiO2 NPs for 6 hours/day, 5 days/week for 13 weeks using a whole-body inhalation exposure system. After the last exposure the rats were euthanized and blood, bronchoalveolar lavage fluid, and all tissues including lungs and mediastinal lymph nodes were collected and subjected to biological and histopathological analyses. ResultsNumerous milky white spots were present in the lungs after exposure 25 and 50 mg/m3 TiO2 NPs. Histopathological analysis revealed that the spots were alveolar lesions, characterized predominantly by the agglomeration of particle-laden macrophages and the presence of reactive alveolar epithelial type 2 cell (AEC2) hyperplasia. We defined this characteristic lesion as pulmonary dust foci (PDF). The PDF is an inflammatory niche, with decreased vascular endothelial cells in the interstitium, and proliferating AEC2 transformed into alveolar epithelial progenitor cells. The AEC2 in the PDF had acquired DNA damage. Based on PDF induction, the lowest observed adverse effect concentration for pulmonary disorders in male and female rats in this study was 12.5 mg/m3 and 6.3 mg/m3, respectively. The no observed adverse effect concentration for male rats was 6.3 mg/m3. There was a sex difference in lung lesion development, with females showing more pronounced lesion parameters than males. ConclusionsInhalation exposure to TiO2 NPs caused PDF, an air-space lesion which is an alveolar inflammatory niche containing particle-laden macrophages and proliferating AEC2. This PDF histopathologically resembles some pneumoconiosis lesions (pulmonary siderosis and hard metal pneumoconiosis) in workers and lung disease in smokers, suggesting that it is an early pneumoconiosis lesion caused by exposure to TiO2 NPs in rats and a common alveolar reaction in mammals.

pathology↗

bioRxiv2021

Lack of pulmonary fibrogenicity and carcinogenicity of titanium dioxide nanoparticles in 26-week inhalation study in rasH2 mouse model

BackgroundWith the rapid development of alternative methods based on the spirit of animal welfare, the publications of animal studies evaluating endpoints such as cancer have been extremely reduced. There have been no systemic inhalation exposure studies of titanium dioxide nanoparticles (TiO2 NPs) using CByB6F1-Tg(HRAS)2Jic (rasH2) 26-week study mice model for detecting carcinogenicity. MethodsMale and female rasH2 mice were exposed to 2, 8 or 32 mg/m3 of TiO2 NPs for 6 hours/day, 5 days/week for 26 weeks using a whole-body inhalation exposure system, with reference to the Organization for Economic Co-operation and Development principles of Good Laboratory Practice. All tissues including lungs, and blood were collected and subjected to biological and histopathological analyses. Additionally, Ki67 positive index were evaluated in mice lung alveolar epithelial type 2 cell (AEC2). ResultsThis study established a stable method for generating and exposing TiO2 NPs aerosol, and clarified the dose-response relationship by TiO2 NPs inhalation to rasH2 mice. TiO2 NPs exposure induced deposition of particles in lungs and mediastinal lymph nodes in a dose-dependent manner in each exposure group. Additionally, alveolar inflammation was only observed in 32 mg/m3 exposure group in both the sexes. Exposure to TiO2 NPs, as well as other organs, did not increase the incidence of lung tumors in any group, and pulmonary fibrosis and pre-neoplastic lesions were not observed in all groups. Finally, the cell proliferative activity of AEC2 was examined, and it was not increased by exposure to TiO2 NPs. ConclusionsThis is the first report showing the lack of pulmonary fibrogenicity and carcinogenicity (no evidence of carcinogenic activity) of TiO2 NPs in 26-week inhalation study in rasH2 mice exposed up to 32 mg/m3, which is considered to be a high concentration. Macrophages undergoing phagocytosis due to TiO2 NPs exposure formed inflammatory foci in the alveolar regions of exposed mice but did not develop fibrosis or hyperplasia or tumors. Moreover, the cell proliferative ability of AEC2 in lesions was not increased. In addition, no carcinogenicity was observed for any organs other than the lungs in this study.

pharmacology and toxicology↗

bioRxiv2021

Movements of Mycoplasma mobile gliding machinery detected by high-speed atomic force microscopy

Mycoplasma mobile, a parasitic bacterium, glides on solid surfaces, such as animal cells and glass by a special mechanism. This process is driven by the force generated through ATP hydrolysis on an internal structure. However, the spatial and temporal behaviors of the internal structures in living cells are unclear. In this study, we detected the movements of the internal structure by scanning cells immobilized on a glass substrate using high-speed atomic force microscopy (HS-AFM). By scanning the surface of a cell, we succeeded in visualizing particles, 2 nm in hight and aligned mostly along the cell axis with a pitch of 31.5 nm, consistent with previously reported features based on electron microscopy. Movements of individual particles were then analyzed by HS-AFM. In the presence of sodium azide, the average speed of particle movements was reduced, suggesting that movement is linked to ATP hydrolysis. Partial inhibition of the reaction by sodium azide enabled us to analyze particle behavior in detail, showing that the particles move 9 nm right, relative to the gliding direction, and 2 nm into the cell interior in 330 ms, then return to their original position, based on ATP hydrolysis. IMPORTANCEThe Mycoplasma genus contains bacteria generally parasitic to animals and plants. Some Mycoplasma species form a protrusion at a pole, bind to solid surfaces, and glide by a special mechanism linked to their infection and survival. The special machinery for gliding can be divided into surface and internal structures that have evolved from rotary motors represented by ATP synthases. This study succeeded in visualizing the real-time movements of the internal structure by scanning from the outside of the cell using an innovative high-speed atomic force microscope, and then analyzing their behaviors.

microbiology↗

bioRxiv2019

Brainstem organoids from human pluripotent stem cells contain neural crest population

The brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases.

developmental biology↗