Biology subjects

Kurata, M.

Publications and source records attributed to Kurata, M..

6 recordsLinked to original sources

bioRxiv2026

Stromal CTHRC1 protects the valvular interstitium from macrophage-associated inflammatory remodeling and calcification

Background: Calcific aortic valve disease (CAVD) is characterized by progressive inflammatory and fibrocalcific remodeling. Although valvular interstitial cells (VICs) are generally considered to drive fibrosis and osteogenic remodeling, whether injury-activated VICs mount endogenous protective responses that preserve the valvular interstitial microenvironment and restrain calcification remains unknown. Methods: We performed spatial transcriptomic profiling of aortic valves in a mouse model of endothelial injury-induced CAVD to define early injury-responsive programs within the valvular interstitium. The cellular origin and spatial distribution of candidate protective factors were examined by immunohistochemistry and lineage tracing, and their relevance to human disease was assessed using stenotic aortic valves. The functional role of CTHRC1 was investigated using genetic Cthrc1 deficiency combined with longitudinal hemodynamic assessment, histological analysis, and spatial transcriptomic profiling. Results: Spatial transcriptomics identified Cthrc1 as a prominent component of an early injury-induced stromal response in the expanding valvular interstitium. CTHRC1 was strongly expressed in activated VICs within thickened murine valve leaflets and human stenotic aortic valves. Lineage tracing demonstrated that the expanded VIC population arose predominantly from PDGFR{beta}+ resident interstitial cells, with minimal endothelial contribution. Despite comparable early hemodynamic responses to endothelial injury, Cthrc1 deficiency exacerbated chronic valvular calcification. Spatial profiling of Cthrc1-deficient valves revealed pronounced interstitial accumulation of galectin-3+ foamy macrophages, accompanied by mitochondrial respiratory-chain signature loss and cell death-associated pathway activation. These findings indicate that transient CTHRC1 induction after endothelial injury defines an endogenous stromal protective response that preserves the valvular interstitial microenvironment and limits macrophage-associated tissue injury and subsequent dystrophic calcification. Conclusions: Injury-activated VICs are not merely effectors of pathological remodeling, but can engage an endogenous tissue-protective response through CTHRC1. These findings identify a previously unrecognized stromal defense mechanism linking endothelial injury to macrophage-associated inflammatory remodeling and dystrophic calcification and suggest CTHRC1-dependent stromal protection as a potential therapeutic axis for limiting CAVD progression.

molecular biology↗

bioRxiv2026

OAC-PCA: orthogonal adjustment of confounding effects in principal component analysis for metabolomics data mining

Principal component analysis (PCA) is widely used in mass spectrometry-based metabolomics for exploratory data mining. Statistical testing of loading values can extract metabolite features associated with score patterns, but this approach requires principal components (PCs) to remain orthogonal while loadings are defined as correlation coefficients between PC scores and variables. Adjustment for Confounding PCA (AC-PCA) was previously developed to explore biologically meaningful components from data matrices affected by biological and technical confounders. However, AC-PCA does not simultaneously ensure PC orthogonality and a correlation-coefficient definition of loadings, limiting the statistical interpretation of its loadings. Here, we reformulated AC-PCA as Orthogonal Adjustment for Confounding effects in PCA (OAC-PCA). In OAC-PCA, PCs remain orthogonal, and loadings retain this correlation-coefficient interpretation. These properties enable statistical testing of metabolite associations while accounting for confounding effects.

bioinformatics↗

bioRxiv2024

Spatial Clustering Analysis with Spectral Imaging-based Single-Step Multiplex Immunofluorescence (SISS-mIF)

Precision medicine, based on spatial biology, is crucial for accurately diagnosing cancer and predicting drug responses. Here, we introduce the Spectral Imaging-based Single-Step Multiplex Immunofluorescence (SISS-mIF) technique, utilizing hyperspectral imaging to capture fluorescence spectra simultaneously. This approach optimizes tissue autofluorescence spectra for each image automatically, allowing the use of fluorescent direct-labeled antibodies for multicolor staining in a single step. Unlike conventional methods, the images are generated as standardized intensity independent of capture conditions, enabling consistent comparisons under different imaging conditions. This technique allows the detection of CD3, CD5, and CD7 in T-cell lymphoma on a single slide. The use of fluorescent direct-labeled antibodies enables triple staining of CD3, CD5, and CD7 without cross-reactivity, maintaining the same intensity as single stains. Furthermore, we developed a joint Non-Negative Matrix Factorization-based Spatial Clustering Analysis (jNMF-SCA) with a modified spectral unmixing system, highlighting its potential as a supportive diagnostic tool for T-cell lymphoma.

pathology↗

bioRxiv2024

MS-DIAL 5 multimodal mass spectrometry data mining unveils lipidome complexities

Lipidomics and metabolomics communities comprise various informatics tools; however, software programs that can handle multimodal mass spectrometry (MS) data with structural annotations guided by the Lipidomics Standards Initiative are limited. Here, we provide MS-DIAL 5 to facilitate the in-depth structural elucidation of lipids through electron-activated dissociation (EAD)-based tandem MS, as well as determine their molecular localization through MS imaging (MSI) data using a species/tissue-specific lipidome database containing the predicted collision-cross section (CCS) values. With the optimized EAD settings using 14 eV kinetic energy conditions, the program correctly delineated the lipid structures based on EAD-MS/MS data from 96.4% of authentic standards. Our workflow was showcased by annotating the sn- and double-bond positions of eye-specific phosphatidylcholine molecules containing very-long-chain polyunsaturated fatty acids (VLC-PUFAs), characterized as PC n-3-VLC-PUFA/FA. Using MSI data from the eye and HeLa cells supplemented with n-3-VLC-PUFA, we identified glycerol 3-phosphate (G3P) acyltransferase (GPAT) as an enzyme candidate responsible for incorporating n-3 VLC-PUFAs into the sn-1 position of phospholipids in mammalian cells, which was confirmed using recombinant proteins in a cell-free system. Therefore, the MS-DIAL 5 environment, combined with optimized MS data acquisition methods, facilitates a better understanding of lipid structures and their localization, offering novel insights into lipid biology.

bioinformatics↗

bioRxiv2022

Indirect CRISPR screening with photoconversion revealed key factors of drug resistance with cell-cell interactions

Comprehensive screenings to clarify indirect cell-cell interactions, such as those in the tumor microenvironment, especially comprehensive assessments of supporting cells effects, are challenging. Therefore, in this study, indirect CRISPR screening for drug resistance with cell-cell interactions was invented. The photoconvertible fluorescent protein Dendra2 was inducted to determine the drug resistance responsible factors of supporting cells with CRISPR screenings. Random mutated supporting cells co-cultured with leukemic cells induced drug resistance with cell- cell interactions. Supporting cells responsible for drug resistance were isolated with green-to-red photoconversion, and 39 candidate genes were identified. Knocking out C9orf89, MAGI2, MLPH, or RHBDD2 in supporting cells reduced the ratio of apoptosis of cancer cells. In addition, the low expression of RHBDD2 in supporting cells, specifically fibroblasts, of clinical pancreatic cancer showed a shortened prognosis, and a negative correlation with CXCL12 was observed. Indirect CRISPR screening was established to isolate the responsible elements of cell-cell interactions. This screening method could reveal new mechanisms in all kinds of cell-cell interactions by revealing live phenotype-inducible cells, and it could be a new platform for discovering new targets of drugs for conventional chemotherapies.

cancer biology↗

bioRxiv2018

Highly Multiplexed Genome Engineering Using CRISPR/Cas9 gRNA Arrays

The CRISPR/Cas9 system is an RNA guided nuclease system that evolved as a mechanism of adaptive immunity in bacteria. This system has been adopted for numerous genome engineering applications in research and recently, therapeutics. The CRISPR/Cas9 system has been largely implemented by delivery of Cas9 as protein, RNA, or plasmid along with a chimeric crRNA-tracrRNA guide RNA (gRNA) under the expression of a pol III promoter, such as U6. Using this approach, multiplex genome engineering has been achieved by delivering several U6-gRNA plasmids targeting multiple loci. However, this approach is limiting due to the efficiently of delivering multiple plasmids to a single cell at one time. To augment the capability and accessibility of multiplexed genome engineering, we developed an efficient golden gate based method to assemble gRNAs linked by optimal Csy4 ribonuclease sequences to deliver up to 10 gRNAs as a single gRNA array transcript. Here we report the optimal expression of our guide RNA array under a strong pol II promoter. This system can be implemented alongside the myriad of CRISPR applications, allowing users to model complex biological processes requiring numerous gRNAs.

bioengineering↗