Biology subjects

Yokoyama, M.

Publications and source records attributed to Yokoyama, M..

11 recordsLinked to original sources

bioRxiv2026

Organ-resolved endothelial regulatory programs link aging and metabolic overload to vascular immune remodeling

Systemic aging and metabolic overload remodel the vasculature; however, how endothelial cells integrate these stresses across organs remains unclear. Using multi-organ single-cell and spatial transcriptomics with functional validation, we mapped endothelial and hematopoietic responses in adipose tissue, skeletal muscle, liver, and heart. Organ-specific endothelial transcriptional features were relatively preserved, whereas chronic stress selectively reconfigured regulatory programs: aging induced a conserved Irf/Stat-centered endothelial program, while high-fat diet engaged organ-biased lipid and remodeling programs. Spatial analysis revealed perivascular niches centered on aging-associated interferon-stimulated endothelial activation, with neighboring immune and stromal cells expressing C3 and LRP1-associated signals. Rather than simply amplifying inflammation, these niches contained mechanisms that restrained IFN activation, as C3 depletion upregulated vascular IRF7 expression. In parallel, the IFN downstream effector BST2 promoted anti-inflammatory macrophage differentiation and suppressed atherosclerosis. These findings define vascular inflammaging as an organ-resolved niche process in which endothelial IFN activation is coupled to local inflammatory restraint. HighlightsO_LIAging induces a shared endothelial type I IFN program across organs. C_LIO_LIA high-fat diet triggers organ-biased endothelial remodeling programs. C_LIO_LIPerivascular interferon niches couple inflammation with local restraint. C_LIO_LIIFN-induced endothelial BST2 promotes CD200R-associated macrophage regulatory features. C_LI

cell biology↗

bioRxiv2023

SLC26A2-mediated sulfate metabolism is essential for the tooth development

The sulfate transporter gene SLC26A2 is responsible for diastrophic dysplasia, which represents skeletal dysplasia in humans. This highlights the importance of sulfate metabolism in skeletal formation. SLC26A2-related chondrodysplasia is also known to exhibit abnormalities in craniofacial and tooth development. Although the function of SLC26A2 in mammals has been investigated using genetic mouse models, the essential role of SLC26A2 during craniofacial and tooth development has not been elucidated. In this study, we demonstrate the pivotal roles of SLC26A2-mediated sulfate metabolism during tooth development. Analysis of Slc26a2 expression reveals that it is predominantly expressed in dental tissues, including odontoblasts and ameloblasts, during tooth development. Slc26a2 knockout mice (Slc26a2-KO-{Delta}exon2) exhibit a retrognathic upper jaw, small upper incisors, and hypoplasia of upper molars. Additionally, upper incisors and molars in Slc26a2-KO-{Delta}exon2 mice display flattened odontoblasts and nuclei that lack intracellular polarity. In contrast, tooth phenotype is not remarkable in lower incisors and molars. Furthermore, the expression of odontoblast differentiation markers, Dspp and Dmp1, is significantly decreased in the upper molars of Slc26a2-deficient mice. Ex vivo organ culture of tooth germs by implantation of Slc26a2-deficient tooth germs under the kidney capsule reveals hypoplasia of the dentin matrix as well as tooth root shortening. In vitro studies using human dental pulp stem cells (hDPSCs) show that the expression levels of Dspp and Dmp1 in shSlc26a2 knockdown cells are significantly decreased compared to control cells. Collectively, our data demonstrate that SLC26A2-mediated sulfate metabolism is essential for tooth development. This study may provide insight into the mechanisms underlying tooth abnormalities in patients with recessively inherited chondrodysplasias caused by mutations in the SLC26A2 gene.

developmental biology↗

bioRxiv2022

Proteomic discovery of chemical probes that perturb protein complexes in human cells

Most human proteins lack chemical probes, and several large-scale and generalizable small-molecule binding assays have been introduced to address this problem. How compounds discovered in such "binding-first" assays affect protein function, nonetheless, often remains unclear. Here, we describe a "function-first" proteomic strategy that uses size exclusion chromatography (SEC) to assess the global impact of electrophilic compounds on protein complexes in human cells. Integrating the SEC data with cysteine-directed activity-based protein profiling identifies changes in protein-protein interactions that are caused by site-specific liganding events, including the stereoselective engagement of cysteines in PSME1 and SF3B1 that disrupt the PA28 proteasome regulatory complex and stabilize a dynamic state of the spliceosome, respectively. Our findings thus show how multidimensional proteomic analysis of focused libraries of electrophilic compounds can expedite the discovery of chemical probes with site-specific functional effects on protein complexes in human cells.

biochemistry↗

bioRxiv2022

Spt4 drives cellular senescence by activating non-coding RNA transcription in ribosomal RNA gene clusters

Genome instability can drive aging in many organisms. The ribosomal RNA gene (rDNA) cluster is one of the most unstable regions in the genome. Replicative lifespan in budding yeast is correlated to rDNA stability, suggesting that the rDNA locus produces an aging signal. To understand the underlying mechanism, we looked for yeast mutants with more stable rDNA and longer lifespan than wild-type cells. We reveal that absence of a transcription elongation factor, Spt4, resulted in an increased rDNA stability, a reduced activity of the regulatory E-pro promoter in the rDNA, and extended replicative lifespan in a SIR2-dependent manner. Spt4-dependent lifespan restriction was abolished in the absence of non-coding RNA transcription at the E-pro locus. The amount of Spt4 increases and its function becomes more important as cells age. These findings suggest that Spt4 is a promising aging factor that accelerates cellular senescence through rDNA instability driven by non-coding RNA transcription

molecular biology↗

bioRxiv2022

Extension of bacterial rDNA sequencing to concurrent epigenetic analysis and its application to 16S meta-epigenetics

Although polymerase chain reaction (PCR) amplification and sequencing of the 16S rDNA region has been used in a wide range of scientific fields, it does not provide DNA methylation information. We describe a simple add-on method to investigate 5-methylcytosine residues in the bacterial 16S rDNA region from clinical samples or flora. Single-stranded bacterial DNA after bisulfite conversion was preferentially amplified with multiple displacement amplification (MDA) at pH neutral, and the 16S rDNA region was analyzed using nested bisulfite PCR and sequencing. 16S rDNA bisulfite sequencing can provide clinically important bacterial DNA methylation status concurrently with intact 16S rDNA sequence information. We used this approach to identify novel methylation sites and a methyltransferase (M. MmnI) in Morganella morganii. Next, we analyzed bacterial flora from clinical specimens of small amount and identified different methylation motifs among Enterococcus faecalis strains. The method developed here, referred to as "add-on" to the conventional 16S rDNA analysis, is the most clinically used bacterial identification genetic test, which provides additional information that could not be obtained with the conventional method. Since the relationship between drug resistance in bacteria and DNA methylation status has been reported, bacterial epigenetic information would be useful in clinical testing as well. Our analysis suggests that M. MmnI has a promotive effect on erythromycin resistance. 16S rDNA bisulfite PCR and sequencing coupled with MDA at pH neutral is a useful add-on tool for analyzing 16S meta-epigenetics.

microbiology↗

bioRxiv2020

Microtubule Defect Involved in 'Mitophagy Resistance' Under Subacute Oxidative Stress - Potential Mechanism for Cellular Inflammation

IntroductionOxidative stress is considered an essential mechanism in ICU-acquired weakness. The roles of oxidative stress in autophagy/mitophagy dysfunction remains elusive. Microtubule serves as an essential guide rail for auto/mitophagosome trafficking required for proper maturation of auto/mitophagosomes in normal circumstances, and microtubules network formation is regulated by signal transduction mechanisms involving Akt, GSK3{beta}, and the microtubule plus-end tracking molecule, EB1. We have investigated (1) whether oxidative stress affects this pathway, leading to the defective mitophagy response, and (2) whether trehalose, an auto/mitophagy modulator, can ameliorate these pathological conditions. MethodsBy stably transfecting markers for auto/mitophagy or MT synthesis, we have established a few new C2C12 myocyte cell lines, expressing, GFP-LC3, EB1-GFP, and/or tandem-fluorescence LC3 (tfLC3). To monitor microtubule network, the cells were stained by SiR-tubulin. The cells were cultured in the presence or absence of oxidative stress by hydrogen peroxide (H2O2) and treated with or without trehalose. The response of mitophagy parameters including vesicle motion and the maturation status was monitored by stimulating the cells with carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an established mitophagy inducer, under a time-lapse confocal microscopy. Signal transduction mechanisms linking mitophagy to microtubule formation was analyzed by Western Blotting against Akt and GSK3{beta}. ResultsCells under the oxidative stress, showed abolished MT network formation, decreased microtubule synthesis by EB1, and a decrease in CCCP-invoked response of mitophagosome motion, perturbed mitophagosome maturation, and increased superoxide production. Signal resistance of Akt/GSK3{beta} pathway to mitophagic stimulation, was documented. Trehalose treatment reversed signal resistance, diminished MT synthesis, ameliorated the disturbed MT network, and improved maturation defects, suppressing the production of superoxide. ConclusionsOxidative stress decreases the response of mitophagy and abolishes microtubule network. Trehalose improves the synthetic ability of microtubule and normalized the disturbed microtubule network, resulting in the improvement of the perturbed mitophagosomes maturation under the oxidative stress.

cell biology↗

bioRxiv2019

Dynamic rotation of the protruding domain enhances the infectivity of norovirus

Norovirus is the major cause of epidemic nonbacterial gastroenteritis worldwide. Lack of structural information on infection and replication mechanisms hampers the development of effective vaccines and remedies. Here, using cryo-electron microscopy, we show that the capsid structure of murine noroviruses changes in response to aqueous conditions. By twisting the flexible hinge connecting two domains, the protruding (P) domain reversibly rises off the shell (S) domain in solutions of higher pH, but rests on the S domain in solutions of lower pH. Metal ions help to stabilize the latter conformation in this process. Furthermore, in the resting conformation, the cellular receptor CD300lf is readily accessible, and thus infection efficiency is significantly enhanced. Two P domain conformations were also found in the human norovirus GII.3 capsid. These results provide new insights into the infection mechanisms of the non-envelope viruses that function in dramatic environmental changes such as the digestive tract. Significance StatementThe capsid structures of caliciviruses have been reported to be classified into two different types, according to species and genotype. One is the rising P domain type as shown in human norovirus GII.10 and rabbit hemorrhagic disease virus, where the P domain rises from the S domain surface. The other is the resting P domain type as shown in human norovirus GI.1, sapovirus and San Miguel sea lion virus, where the P domain rests upon the S domain. Here, we demonstrate that the P domain of the murine norovirus infectious particles changes reversibly between the rising and resting P domain types in response to aqueous conditions. Our findings provide new insights into the mechanisms of viral infection of caliciviruses.

pathology↗

bioRxiv2018

Logistic regression analysis of multiple interosseous hand muscle activation using surface electromyography during isometric finger tasks

Intrinsic hand muscles are densely located in the hand, and the myoelectric observation from the surface is sometimes unreliable because of some outside influences that may interfere with the signals. In the present study, we evaluated the activities of multiple interosseous hand-muscles which densely located in the hand, through analyzing the surface electromyographic signals during finger-oriented tasks using univariate and multivariate logistic regression models. Ten healthy subjects participated in our experiment, and isometrically exercised each finger one by one in flexed form. The result of a univariate analysis with the power and amplitude domain predictor variables of the surface electromyographic signals showed significant consistency between the activated finger and the inserted finger of the dorsal interosseous muscles to the proximal phalanx (P < 0.001). Meanwhile, the results of a multivariate analysis showed a higher correlation of the regression model of the fourth dorsal interosseous muscle during the action of the ring finger using frequency-domain variables (the Nagelkerke R2 = 0.716 when the median frequency was used), compared to the model without the frequency-domain variables (the Nagelkerke R2 = 0.583). Our result showed that the logistic regression models have a particular possibility for the analysis of the surface electromyographic signals of densely located hand-muscle activities related to the finger-oriented tasks.

bioengineering↗

bioRxiv2018

In vitro characterization of multidrug-resistant influenza A(H1N1)pdm09 viruses carrying a dual amino acid substitution associated with reduced susceptibility to neuraminidase inhibitors

We detected influenza A(H1N1)pdm09 viruses carrying dual H275Y/I223R, H275Y/I223K, or H275Y/G147R substitutions in their neuraminidase protein, respectively. These viruses showed cross-resistance to oseltamivir and peramivir and reduced susceptibility to zanamivir. The H275Y/G147R virus retained its replication capability at least in vitro, but the H275Y/I223R and H275Y/I223K viruses did not.

microbiology↗

bioRxiv2017

Global phylogenomics of multidrug-resistant Staphylococcus aureus sequence type 772: the Bengal Bay clone

The global spread of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here we demonstrate the recent origin and global spread from the Indian subcontinent of a multidrug resistant Staphylococcus aureus lineage, sequence type 772 (Bengal Bay clone). Short-term outbreaks occurred following intercontinental transmission, typically associated with travel and family contacts, but ongoing endemic transmission was uncommon. Instrumental in the emergence of a single dominant clade in the early 1990s was the acquisition of a multidrug resistance integrated plasmid that did not appear to incur a significant fitness cost. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological and virulence potential of community-associated clones.

genomics↗

bioRxiv2017

Epistasis mediated alleviation of the cost of antibiotic resistance for MRSA

Understanding how multi-drug resistant pathogens evolve is key to identifying means of curtailing their further emergence and dissemination. Fitness costs imposed on bacteria by resistance mechanisms are believed to hamper their dissemination in an antibiotic free environment, however, some have been reported to have little or no cost, which suggests there are few barriers preventing their global spread. One such apparently cost-free resistance mechanism acquired by the major human pathogen Staphylococcus aureus is to the clinically important antibiotic mupirocin, which is mediated by mutation of the highly-conserved and essential isoleucyl-tRNA synthethase (ileS) gene. In Genome Wide Association Studies (GWAS) on two genetically and geographically distinct MRSA lineages we have found this mutation to be associated with changes in bacterial virulence, driven through epistatic interactions with other loci. Given the potential dual effect of this mutation on both antibiotic resistance and virulence we adopted a proteomic approach and observed pleiotropic effects. This analysis revealed that the activity of the secretory apparatus of the PSM family of cytolytic toxins, the Pmt system, is affected in the mupirocin resistant mutant, which explains why it is less toxic. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production is required. Given the widespread use of this antibiotic, and that this resistance often results from a single nucleotide substitution in the ileS gene, these hidden fitness costs provide an explanation for why this resistance mechanism is not more prevalent. This work also demonstrates how population-based genomic analysis of virulence and antibiotic resistance can contribute to uncovering hidden features of the biology of microbial pathogens.

microbiology↗