Biology subjects

Kobayashi, H.

Publications and source records attributed to Kobayashi, H..

62 records · Page 4Linked to original sources

bioRxiv2020

Infrared absorptions as indicators for Pseudomonas aeruginosa and Acinetobacter baumannii

There is clinical demand for simple and contact-free diagnosis techniques in medical practice. This study shows that the infrared absorptions of volatile metabolites can be used to distinguish between the air around Pseudomonas aeruginosa, Acinetobacter baumannii, other bacteria, and normal room air. Gas samples were collected from the air surrounding single and mixed laboratory cultures, and preliminary data on human breath samples are also presented. The infrared spectra of a variety of gasses are measured with a high resolution of 0.5 cm-1 to obtain information about the wavenumber position of the key bands. Hence, it is not necessary to specify the molecular species of biomarkers. This work also shows that discrimination rates can be improved by observing additional infrared absorptions caused by different characteristic volatile molecules. The significance of this work is that the specific wavenumber positions of the key bands that allow the application of infrared lasers are provided. As a result, it is considered that Pseudomonas aeruginosa and Acinetobacter baumannii can be monitored more sensitively and easily. With further research and development, this simple approach could be used in future applications to identify infections in healthcare settings.

microbiology↗

bioRxiv2020

High-Throughput Functional Evaluation of BRCA2 Variants of Unknown Significance

Numerous nontruncating missense variants of the BRCA2 gene have been identified, but there is a lack of convincing evidence, such as familial data, demonstrating their clinical relevance and they thus remain unactionable. To assess the pathogenicity of variants of unknown significance (VUSs) within BRCA2, we developed a novel method, the MANO-B method, for high-throughput functional evaluation utilizing BRCA2-deficient cells and poly(ADP-ribose) polymerase (PARP) inhibitors. The estimated sensitivity and specificity of this assay compared to those of the International Agency for Research on Cancer (IARC) classification system were 95% and 95%, respectively. We classified the pathogenicity of 186 BRCA2 VUSs with our original computational pipeline, resulting in the classification of 126 mutations as "neutral/likely neutral", 23 as "intermediate", and 37 as "deleterious/likely deleterious". We further invented a simplified, on-demand annotation system, the Accurate BRCA Companion Diagnostic (ABCD) test, as a companion diagnostic for PARP inhibitors in patients with unknown BRCA2 VUSs. The ABCD test classification was reproducible and consistent with that of a large-scale MANO-B method.

cancer biology↗

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↗

bioRxiv2019

Classification of platelet aggregates by agonist type

Platelets are anucleate cells in blood whose principal function is to stop bleeding by forming aggregates for hemostatic reactions. In addition to their participation in physiological hemostasis, platelet aggregates are also involved in pathological thrombosis and play an important role in inflammation, atherosclerosis, and cancer metastasis. The aggregation of platelets is elicited by various agonists, but these platelet aggregates have long been considered indistinguishable and impossible to classify. Here we present an intelligent method for classifying them by agonist type. It is based on a convolutional neural network trained by high-throughput imaging flow cytometry of blood cells to identify and differentiate subtle yet appreciable morphological features of platelet aggregates activated by different types of agonists. The method is a powerful tool for studying the underlying mechanism of platelet aggregation and is expected to open a window on an entirely new class of clinical diagnostics, pharmacometrics, and therapeutics.

cell biology↗

bioRxiv2019

Evolution of imprinting via lineage-specific insertion of retroviral promoters

Imprinted genes are expressed from a single parental allele. In mammals, this unusual mode of transcription generally depends on the epigenetic silencing of one allele by DNA methylation (DNAme) established in the germline. While many species-specific imprinted orthologues have been documented in eutherians, the molecular mechanisms underlying the evolutionary switch from biallelic to imprinted expression are currently unknown. During mouse oogenesis, gametic differentially methylated regions (gDMRs) acquire DNAme in a process guided by transcription. Here we show that transcription initiating in proximal lineage-specific endogenous retroviruses (ERVs) is likely responsible for DNAme established in oocytes at 4/6 mouse-specific and 17/110 human-specific maternal imprinted gDMRs (igDMRs). The latter can be further divided into Catarrhini (Old World monkeys and apes)- or Hominoidea (ape)-specific igDMRs, which are embedded within transcription units initiating in ERVs specific to these primate lineages. Using CRISPR-Cas9 mutagenesis, we deleted the relevant murine-specific ERVs upstream of the maternally methylated genes Impact and Slc38a4. Strikingly, imprinting at these genes was lost in the offspring of females harboring these deletions and biallelic expression was observed. Our work reveals a novel evolutionary mechanism whereby maternally silenced genes arise from biallelically expressed progenitors.

genetics↗

bioRxiv2019

Paternal age affects offspring's behavior possibly via an epigenetic mechanism recruiting a transcriptional repressor REST

Advanced paternal age has deleterious effects on mental health of next generation. Using a mouse model, we have confirmed that offspring derived from aged fathers showed impairments in behavior and abnormalities in the brain structure and activity. Comprehensive target DNA methylome analyses revealed in aged sperm more hypo-methylated genomic regions, in which REST/NRSF binding motif was enriched. Gene set enrichment analyses also identified enrichment of "REST/NRSF target genes", in addition to "Late-fetal genes" and autism spectrum disorder-related "SFARI genes", in up-regulated genes of developing brains from aged father. Indeed, gene sets near hypo-methylated genomic regions with REST/NRSF binding motif were also enriched in up-regulated genes of developing brains. Taken altogether, DNA hypo-methylation due to paternal aging in sperm will induce leaky expression of REST/NRSF target genes in the developing brain, thereby causing neuronal abnormalities and subsequent behavioral alteration in offspring.

neuroscience↗

bioRxiv2018

Environmental Optimization Enables Maintenance of Quiescent Hematopoietic Stem Cells Ex Vivo

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our novel culture system provides a means to evaluate properties of steady state HSCs and test effects of defined factors in vitro under near-physiological conditions.

cell biology↗

bioRxiv2018

Electron microscopic analysis of the exo-skeleton of hadal zone amphipod Hirondellea gigas

The amphipod Hirondellea gigas inhabits the deepest regions of the oceans in extra high-pressure. However, the mechanisms by which they adapt to their high-pressure environments remain unknown. In this study, we investigated elements of the exoskeleton of H. gigas captured from the deepest points of the Mariana Trench. The H. gigas exoskeleton contained aluminum, as well as a major amount of calcium carbonate. Unlike other accumulated metals, aluminum was distributed on the surface of exoskeletons. To investigate how H. gigas obtains aluminum, we conducted a metabolome analysis and found that gluconic acid/gluconolactone was capable of extracting metals from the sediment under the habitat conditions of H. gigas. The extracted aluminum ions are transformed into the gel state of aluminum hydroxide in alkaline seawater, and this gel covers the body to protect the amphipod. The aluminum gel would be one of good materials to adapt to such high-pressure environment.

zoology↗