Biology subjects

Nagao, M.

Publications and source records attributed to Nagao, M..

15 recordsLinked to original sources

bioRxiv2026

VicMAG, an open-source tool for visualizing circular metagenome-assembled genomes highlighting bacterial virulence and antimicrobial resistance

Bacterial pathogens spread in clinical and environmental settings, and mobile genetic elements (MGEs), such as plasmids and phages, mediate the transfer of virulence factor genes (VFGs) and antimicrobial resistance genes (ARGs) among bacterial communities. Metagenomic analysis of environmental and wastewater samples using highly accurate long-read sequencing technologies, such as PacBio HiFi sequencing, provides valuable insights into monitoring the regional spread of VFGs and ARGs, including dissemination mediated by MGEs. No visualization tool is currently available for the comprehensive display of numerous resulting circular metagenome-assembled genomes (cMAGs) with functional gene annotations. Here, we developed VicMAG, a visualization tool for highly complex cMAGs derived from long-read metagenome assemblies annotated using updated databases of VFGs, ARGs, and MGEs. Using 353 cMAGs from PacBio HiFi sequencing of a wastewater sample, we demonstrated the utility of VicMAG for metagenome visualization. VicMAG provides comprehensive, size-aware visualization of cMAGs representing bacterial chromosomes and plasmids, annotated with VFGs, ARGs, and phages. By simultaneously visualizing all cMAGs in a framework, VicMAG facilitates a holistic understanding of the distribution and genomic context of VFGs and ARGs across complex microbial communities. This tool supports integrated surveillance of bacteria associated with virulence and antimicrobial resistance across clinical, environmental, and One Health contexts.

bioinformatics↗

bioRxiv2025

Functional and structural profiling of circulation via genetically encoded modular fluorescent probes.

Sustained labeling of fluids is crucial for their investigation in animal models. Here, we introduce a mouse line (Alb-mSc-ST), where blood and interstitial fluid are labeled with the red fluorescent protein mScarlet and SpyTag. The SpyTag-SpyCatcher technology is exploited to monitor circulating fluid properties by biosensors or detect blood-brain barrier disruption. This approach represents a valuable tool for studying vascular structure, permeability and microenvironment in body organs in vivo.

neuroscience↗

bioRxiv2025

Evolution of BA.2.86 to JN.1 reveals functional changes in non-structural viral proteins are required for fitness of SARS-CoV-2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is still circulating among humans, leading to the continuous evolution. SARS-CoV-2 Omicron JN.1 evolved from a distinct SARS-CoV-2 lineage, BA.2.86, spread rapidly worldwide. It is unclear why BA.2.86 did not become dominant and was quickly replaced by JN.1, which possesses one amino acid substitution in the spike protein (S:L455S) and two in the non-spike proteins NSP6 and ORF7b (NSP6:R252K and ORF7b:F19L) compared to BA.2.86. Here, we utilized recombinant viruses to elucidate the impact of these mutations on the virological characteristics of JN.1. We found that the mutation in the spike attenuated viral replication, but the non-spike mutations enhanced replication, suggesting the mutations in the non-spike proteins compensate for the one in the spike to improve viral fitness, as the mutations in the spike contribute to further immune evasion. Our findings suggest that functional changes in both the spike and non-spike proteins are necessary in the evolution of SARS-CoV-2 to enable evasion of adaptive immunity within the human population while sustaining replication. IMPORTANCEBecause the spike protein is strongly associated with certain virological properties of SARS-CoV-2, such as immune evasion and infectivity, most previous studies on SARS-CoV-2 variants have focused on spike protein mutations. However, the non-spike proteins also contribute to infectivity, as observed throughout the evolution of Omicron subvariants. In this study, we demonstrate a "trade-off" strategy in SARS-CoV-2 Omicron JN.1 in which the reduced infectivity caused by spike mutation is compensated by non-spike mutations. Our results provide insight into the evolutionary scenario of the emerging virus in the human population.

microbiology↗

bioRxiv2023

Virological characteristics of the SARS-CoV-2 Omicron EG.5.1 variant

In middle-late 2023, a sublineage of SARS-CoV-2 Omicron XBB, EG.5.1 (a progeny of XBB.1.9.2), is spreading rapidly around the world. Here, we performed multiscale investigations to reveal virological features of newly emerging EG.5.1 variant. Our phylogenetic-epidemic dynamics modeling suggested that two hallmark substitutions of EG.5.1, S:F456L and ORF9b:I5T, are critical to the increased viral fitness. Experimental investigations addressing the growth kinetics, sensitivity to clinically available antivirals, fusogenicity and pathogenicity of EG.5.1 suggested that the virological features of EG.5.1 is comparable to that of XBB.1.5. However, the cryo-electron microscopy reveals the structural difference between the spike proteins of EG.5.1 and XBB.1.5. We further assessed the impact of ORF9b:I5T on viral features, but it was almost negligible at least in our experimental setup. Our multiscale investigations provide the knowledge for understanding of the evolution trait of newly emerging pathogenic viruses in the human population.

microbiology↗

bioRxiv2023

Stress-induced vascular remodeling: novel insight into the role of omega-3 fatty acid metabolite, 4-oxoDHA

BackgroundStress has garnered significant attention as a prominent risk factor for inflammation-related diseases, particularly cardiovascular diseases (CVDs). However, the precise mechanisms underlying stress-driven CVDs remain elusive, thereby impeding the development of effective preventive and therapeutic strategies. MethodsTo explore the correlation between plasma lipid metabolites and depressive states, we conducted a study involving healthy volunteers (n=408). Liquid chromatography (LC)/mass spectrometry (MS)/MS-based lipidomics and the self-rating depression (SDS) scale questionnaire were employed for data collection. In addition, we utilized a mouse model by subjecting mice to restraint stress and investigating the impact of stress on plasma lipid metabolites and vascular remodeling following carotid ligation. In vitro functional and mechanistic studies were performed using macrophages, endothelial cells, and neutrophil cells. ResultsOur findings revealed a significant association between depressive state and reduced plasma levels of 4-oxoDHA, a specific omega-3 fatty acid metabolite regulated by 5-lipoxygenase (LO) in neutrophils in healthy volunteers. In mice, restraint stress led to decreased plasma 4-oxoDHA levels and exacerbated vascular remodeling. Moreover, 4-oxoDHA demonstrated the ability to enhance Nrf2-HO-1 pathways, exerting anti-inflammatory effects on endothelial cells and macrophages. Mechanistically, stress-induced noradrenaline triggered the degradation of 5-LO in neutrophils through the proteasome system, facilitated by dopamine D2-like receptor activation. The reduction in circulating 4-oxoDHA resulted in the downregulation of the Nrf2-HO-1 anti-inflammatory axis and an increase in ICAM-1 expression, vascular permeability, and remodeling. ConclusionsOur study unveiled a novel stress-induced pathway of vascular inflammation, mediated through the regulation of omega-3 fatty acid metabolites. Reduced levels of circulating 4-oxoDHA under stress conditions may serve as a promising biomarker for stress. This understanding of the interplay between neurobiology and lipid metabolism provides a potential avenue for the development of treatments aimed at preventing stress-induced systemic neuroinflammation. Highlights- Our study reveals that stress-induced reduction in circulating levels of a specific omega-3 fatty acid metabolite, 4-oxoDHA, contributes to vascular inflammation. - We have identified a novel pathway that explains how stress promotes systemic vascular inflammation by regulating omega-3 fatty acid metabolites in the circulation. - Our findings provide new evidence for the role of 4-oxoDHA in maintaining Nrf2-ARE-related anti-inflammatory functions in endothelial cells and macrophages.

biochemistry↗

bioRxiv2023

Virally induced CRISPR/Cas9-based knock-in of fluorescent albumin allows long-term visualization of cerebral circulation in infant and adult mice

Albumin, a protein produced by liver hepatocytes, represents the most abundant protein in blood plasma. We have previously engineered a liver-targeting adeno-associated viral vector (AAV) that expresses fluorescent protein-tagged albumin to visualize blood plasma in mice. While this approach is versatile for imaging in adult mice, transgene expression vanishes when AAV is administered in neonates due to dilution of the episomal AAV genome in the rapidly growing liver. Here, we use CRISPR/Cas9 genome editing to insert the fluorescent protein mNeonGreen (mNG) gene into the albumin (Alb) locus of hepatocytes to produce fluorescently labeled albumin (Alb-mNG). We constructed a CRISPR AAV that includes [~]1 kb homologous arms around Alb exon 14 to express Alb-mNG. Subcutaneous injection of this AAV with AAV-CMV-Cas9 in postnatal day 3 mice resulted in two-photon visualization of the cerebral cortex vasculature within ten days. The expression levels of Alb-mNG were persistent for at least three months and were so robust that vasomotion and capillary blood flow could be assessed transcranially in early postnatal mice. This knock-in approach provides powerful means for micro- and macroscopic imaging of cerebral vascular dynamics in postnatal and adult mice.

neuroscience↗

bioRxiv2022

Horizontal transmission of penicillin binding protein 1A caused a nationwide spread of β-lactam resistance in pneumococci

The emergence and spread of drug-resistant bacteria continue to be a global crisis. The mechanism of the resistance spread via mobile genetic elements such as plasmids is well known, however, the impact of the natural transformation on the spread remains unclear. Streptococcus pneumoniae is well known to be a transformable pathogen by natural competence and they become {beta}-lactam resistance by the acquisition of chromosomal genetic elements including mutated PBPs by natural transformation. To trace the transmission of pneumococcal PBPs among nationwide pediatric population and analyze the impact of transformed PBPs to {beta}-lactam resistance, we collected and analyzed more than 1300 isolates of S. pneumoniae through nationwide surveillance study for pediatric pneumococcal diseases between 2012-2017 in Japan. We discovered a high prevalence of a specific PBP1A type (pbp1a-13) in {beta}-lactam resistant pneumococci that had a 370SSMK substitution in their {beta}-lactam binding SXXK motif, suggesting that this pbp1a-13 transferred horizontally between different clones resulting in emergence and spread of {beta}-lactam resistant pneumococcal clones. Divergence dating analysis suggested that pbp1a-13 was inserted into major resistant lineages in the early 1990s through the 2000s, before introduction of pneumococcal conjugate vaccines in Japan. Our additional analysis for pneumococcal isolates that were recovered in the 90s in Japan suggested that pbp1a-13 in GPSC1 (serotype 19F-CC236) and GPSC14 (serotype 23F-CC242) isolates were the origin of the currently spread pbp1a-13. We provide evidence of pbp1a horizontal transmission at a nationwide scale and highlight the importance of PBP profile monitoring for identifying the emergence and spread of resistant pneumococci lineages.

microbiology↗

bioRxiv2022

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed passive head motion (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents heads. SUMMARYThe present protocol describes a custom-designed passive head motion system, which reproduces mechanical accelerations at rodents heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

bioengineering↗

bioRxiv2022

Delayed antigen-specific CD4+ T cell induction correlates with impaired immune responses to SARS-COV-2 mRNA vaccination in the elderly

Despite the clinical efficacy of coronavirus disease 2019 mRNA vaccines, the elderly demonstrate lower IgG levels and neutralizing titers and a higher risk of severe diseases. CD4+ T cells play a central role in regulating antigen-specific antibody and CD8+ T-cell responses; however, because their composition and functionality change significantly with age, relationships between age-associated defects in T cells and the immunogenicity of or reactogenicity to mRNA vaccines are unclear. Using a vaccine cohort (n = 216), we found that the elderly (aged [≥]65 years) showed delayed induction and early contraction of vaccine-specific CD4+ T cells, and that the compromised C-X-C motif chemokine receptor 3+ circulating T follicular helper cell response after the first dose was associated with the lower IgG levels. Additionally, the elderly experienced significantly fewer systemic adverse effects (AEs) after the second dose, with those exhibiting few AEs showing lower cytokine+ CD4+ T cells after the first dose and lower antibody levels after the second dose. Furthermore, T helper 1 cells in the elderly expressed higher levels of programmed cell death protein-1, a negative regulator of the T-cell response, which was associated with less production of vaccine-specific CD4+ T cells and impaired CD8+ T-cell expansion. Thus, efficient induction of vaccine-specific effector/memory CD4+ T cells after the first dose may trigger robust cytokine production after the second dose, leading to effective vaccine responses and higher systemic reactogenicity. These results suggested that an enhanced CD4+ T-cell response after the first dose is key to improved vaccination efficacy in the elderly. One Sentence SummaryWe compared immunogenicity and reactogenicity to COVID-19 mRNA vaccine in 107 adults (aged <65 years) and 109 elderly (aged [≥]65) individuals.

immunology↗

bioRxiv2022

The structure of insulin granule core determines secretory capacity being reduced in type-2 diabetes

Exocytosis in excitable cells is essential for their physiological functions. Although the exocytotic machinery controlling cellular secretion has been well investigated, the function of the vesicular cargo, i.e. secretory granular content remains obscure. Here we combine dSTORM imaging and single-domain insulin antibody, to dissect the in situ structure of insulin granule cores (IGCs) at nano level. We demonstrate that the size and shape of the IGCs can be regulated by the juxta-granular molecules Nucleobindin-2 and Enolase-1, that further contribute to the stimulated insulin secretion. IGCs located at the plasma membrane are larger than those in the cytosol. The IGCs size is decreased by [~]20% after glucose stimulation due to the release of the peripheral part of IGCs through incomplete granule fusion. Importantly, the reduction of the IGCs size is also observed in non-stimulatory pancreatic {beta}-cells from diabetic db/db mice, Akita (Ins2+/-) mice and human Type-2 diabetic donors, in accordance with impaired secretion. These findings overall highlight the structure of exocytotic insulin cores as a novel modality amenable to targeting in the stimulated exocytosis in {beta}-cells with impaired insulin secretion.

physiology↗

bioRxiv2020

Mechanical impact on the head has an antihypertensive effect

Physical exercise is known to be beneficial for various brain functions. However, the mechanisms behind the positive effects of exercise on the brain remain to be elucidated. Here we show that passive head motion in hypertensive rats, which reproduces the mechanical accelerations generated in their heads during moderate-velocity treadmill running, decreases the expression of angiotensin II type 1 receptor (AT1R) in astrocytes in the rostral ventrolateral medulla (RVLM), thereby lowering blood pressure. Passive head motion generates interstitial fluid movement that is estimated to exert shear stress with an average magnitude of <1 Pa on the cells in the rat medulla. Fluid shear stress of a sub-Pa magnitude decreases AT1R expression in cultured astrocytes. In hypertensive rats, inhibition of interstitial fluid movement following hydrogel introduction to the RVLM eliminates the antihypertensive effects of passive head motion and treadmill running. Furthermore, vertically oscillating chair riding by hypertensive adult humans, which reproduces the mechanical accelerations generated in their heads during light jogging or fast walking, lowers their blood pressure. Our findings indicate that moderate mechanical intervention can have antihypertensive effects by modulating the function of RVLM astrocytes through interstitial fluid shear stress. We anticipate that mechanical regulation is responsible for a variety of the positive effects of physical exercise on human health, particularly those related to brain functions.

bioengineering↗

bioRxiv2020

Smad3 Regulates Smooth Muscle Cell Fate and Governs Adverse Remodeling and Calcification of Atherosclerotic Plaque

Atherosclerotic plaques consist mostly of smooth muscle cells (SMC), and genes that influence SMC biology can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD, and is associated with expression of SMAD3 in SMC, but the mechanism by which this gene modifies CAD risk remains poorly understood. SMC-specific deletion of Smad3 in a murine atherosclerosis model resulted in greater plaque burden, positive remodeling, and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of Smad3 altered SMC progeny phenotype toward the previously described chondromyocyte fate, but importantly also promoted transition to a novel cell-state that governs remodeling and recruitment of inflammatory cells. This new remodeling population was marked by uniquely high Mmp3 and Cxcl12 expression, and its appearance correlated with higher-risk plaque features such as increased positive remodeling and macrophage content. Further, investigation of transcriptional mechanisms by which Smad3 alters SMC cell-fate revealed novel roles for Hox and Sox transcription factors whose direct interaction with Smad3 regulate an extensive transcriptional program balancing remodeling and vascular ECM with significant implications for human Mendelian aortic aneurysmal diseases. Together, these data suggest that Smad3 expression in SMC inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including positive remodeling, monocyte recruitment, and vascular calcification.

cell biology↗

bioRxiv2020

Comparison of 12 molecular detection assays for SARS-CoV-2

Molecular testing for SARS-CoV-2 is the mainstay for accurate diagnosis of the infection, but the diagnostic performances of available assays have not been defined. We compared 12 molecular diagnostic assays, including 8 commercial kits using 155 respiratory samples (65 nasopharyngeal swabs, 45 oropharyngeal swabs, and 45 sputum) collected at 2 Japanese hospitals. Sixty-eight samples were positive for more than one assay and one genetic locus and were defined as true positive samples. All the assays showed a specificity of 100% (95% confidence interval, 95.8 to 100). The N2 assay kit of the US Centers for Disease Control and Prevention (CDC) and the N2 assay of the Japanese National Institute of Infectious Disease (NIID) were the most sensitive assays with 100% sensitivity (95% confidence interval, 94.7 to 100), followed by the CDC N1 kit, E assay by Corman, and NIID N2 assay multiplex with internal control reactions. These assays are reliable as first-line molecular assays in laboratories when combined with appropriate internal control reactions.

microbiology↗

bioRxiv2020

The environment-sensing aryl-hydrocarbon receptor inhibits the chondrogenic fate of modulated smooth muscle cells in atherosclerotic lesions

IntroductionSmooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. MethodsWe combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. ResultsGenomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term "chondromyocytes" (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. ConclusionOverall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.

genomics↗

bioRxiv2019

Exposure to maternal obesity per se programs sex-differences in pancreatic islets of the offspring

Maternal obesity increases type 2 diabetes (T2D) risk in the offspring. Given that nearly half of women of child-bearing age in many populations are currently overweight/obese, it is key that we improve our understanding of the impact of the in utero/early life environment on offspring islet function. Using a well-established mouse model of diet-induced obesity, we examined offspring islets before the onset of metabolic dysfunction. This allowed us to determine inherent changes, in males and females, which are distinct from the response of islets to an existing obesogenic, insulin resistant milieu hence identifying islet dysregulation reflecting very early manifestation of the disease before the onset of disrupted glucose homeostasis. Female offspring of obese dams displayed higher glucose-stimulated insulin secretion and mitochondrial respiration, increased expression of estrogen receptor and decreased cleaved-caspase 3 and Bax:Bcl-2 reflecting reduced susceptibility to apoptosis. In contrast, male offspring of obese dams displayed compromised mitochondrial respiration characterised by decreased ATP synthesis-driven respiration and increased \"uncoupled\" respiration and reduced docked insulin granules in {beta}-cells. Thus, maternal obesity \"programs\" sex-differences in offspring islet function. Islets of female but not male offspring appear primed to cope with a nutritionally-rich postnatal environment, which may reflect differences in future T2D risk.

physiology↗