Biology subjects

Kobayashi, Y.

Publications and source records attributed to Kobayashi, Y..

52 records · Page 3Linked to original sources

bioRxiv2020

Asymbiotic mass production of the arbuscular mycorrhizal fungus Rhizophagus clarus

Arbuscular mycorrhizal (AM) symbiosis is a mutually beneficial interaction between fungi and land plants and promotes global phosphate cycling in terrestrial ecosystems. AM fungi are recognised as obligate symbionts that require root colonisation to complete a life cycle involving the production of propagules, asexual spores. Recently it has been shown that Rhizophagus irregularis can produce infection-competent secondary spores asymbiotically by adding a fatty acid, palmitoleic acid. Further, asymbiotic growth can be supported using myristate as a carbon and energy source for their asymbiotic growth to increase fungal biomass. However, spore production and the ability of these spores to colonise host roots were still limited compared to co-culture of the fungus with plant roots. Here we show that a combination of two plant hormones, strigolactone and methyl jasmonate, induces production of a large number of infection-competent spores in asymbiotic cultures of Rhizophagus clarus HR1 in the presence of myristate and organic nitrogen. Inoculation of asymbiotically-generated spores promoted the growth of Welsh onions, as observed for spores produced by symbiotic culture system. Our findings provide a foundation for elucidation of hormonal control of the fungal life cycle and development of new inoculum production schemes.

microbiology↗

bioRxiv2020

Inter-breeder differences in prepulse inhibition deficits of C57BL/6J mice in a maternal infection model for schizophrenia

Genetic and environmental factors interact with each other to influence the risk of various psychiatric diseases; however, the intensity and nature of their interactions remain to be elucidated. We used a maternal infection model using polyinosinic-polycytidylic acid (Poly(I:C)) to determine the relationship between the maternal breeding environment and behavioral changes in the offspring. We purchased pregnant C57BL/6J mice from three breeders and administered Poly(I:C) (2 mg/kg) intravenously in their tail vein on gestation day 15. The offspring were raised to 8-12 weeks old and subjected to the acoustic startle tests to measure their startle response intensity, prepulse inhibition levels, and degree of the adaptation of the startle response. No statistical interaction between Poly(I:C) administration and sex was observed for prepulse inhibition; thus, male and female mice were analyzed together. The Poly(I:C) challenge significantly decreased prepulse inhibition levels of the offspring born to the pregnant dams from Breeder A but not those from the other breeders. However, there were no significant inter-breeder differences in Poly(I:C) effects on startle response and on startle adaptation. The rearing environment of mouse dams has a prominent impact on the Poly(I:C)-induced prepulse inhibition deficits in this maternal infection model.

neuroscience↗

bioRxiv2020

Light-dependent induction of Edn2 expression and attenuation of retinal pathology by endothelin receptor antagonists in Prominin-1- deficient mice

Retinitis pigmentosa (RP) and macular dystrophy (MD) are prevalent retinal degenerative diseases associated with gradual photoreceptor death. These diseases are often caused by genetic mutations that result in degeneration of the retina postnatally after it has fully developed. The Prominin-1 gene (Prom1) is a causative gene for RP and MD, and Prom1- knockout (KO) mice recapitulate key features of these diseases including light-dependent retinal degeneration and stenosis of retinal blood vessels. The mechanisms underlying progression of such degeneration have remained unknown, however. We here analysed early events associated with retinal degeneration in Prom1-KO mice. We found that photoreceptor cell death and glial cell activation occur between 2 and 3 weeks after birth. High-throughput analysis revealed that expression of the endothelin-2 gene (Edn2) was markedly up-regulated in the Prom1-deficient retina during this period. Expression of Edn2 was also induced by light stimulation in Prom1-KO mice that had been reared in the dark. Finally, treatment with endothelin receptor antagonists attenuated photoreceptor cell death, gliosis, and retinal vessel stenosis in Prom1-KO mice. Our findings suggest that inhibitors of endothelin signalling may delay the progression of RP and MD and therefore warrant further study as potential therapeutic agents for these diseases.

neuroscience↗

bioRxiv2020

COUNTEN an AI-driven tool for rapid, and objective structural analyses of the Enteric Nervous System

Healthy gastrointestinal functions require a healthy Enteric Nervous System (ENS). ENS health is often defined by the presence of normal ENS structure. However, we currently lack a comprehensive understanding of normal ENS structure as current methodologies of manual enumeration of neurons within tissue and ganglia can only parse limited tissue regions; and are prone to error, subjective bias, and peer-to-peer discordance. Thus, there is a need to craft objective methods and robust tools to capture and quantify enteric neurons over a large area of tissue and within multiple ganglia. Here, we report on the development of an AI-driven tool COUNTEN which parses HuC/D-immunolabeled adult murine myenteric ileal plexus tissues to enumerate and classify enteric neurons into ganglia in a rapid, robust, and objective manner. COUNTEN matches trained humans in identifying, enumerating and clustering myenteric neurons into ganglia but takes a fraction of the time, thus allowing for accurate and rapid analyses of a large tissue region. Using COUNTEN, we parsed thousands of myenteric neurons and clustered them in hundreds of myenteric ganglia to compute metrics that help define the normal structure of the adult murine ileal myenteric plexus. We have made COUNTEN freely and openly available to all researchers, to facilitate reproducible, robust, and objective measures of ENS structure across mouse models, experiments, and institutions.

neuroscience↗

bioRxiv2020

Conservation and host-specific expression of non-tandemly repeated heterogenous ribosome RNA gene in arbuscular mycorrhizal fungi

The ribosomal RNA-encoding gene (rDNA) has a characteristic genomic nature: tens to thousands of copies in a genome, tandemly repeated structure, and intragenomic sequence homogeneity. These features contribute to ribosome productivity via physiological and evolutionary processes. We reported previously the exceptional absence of these features in the model arbuscular mycorrhizal (AM) fungus Rhizophagus irregularis. Here we examine the phylogenetic distribution of the exceptional rDNA features in the genus Rhizophagus via improving the genome sequence of R. clarus. Cross-species comparison indicated similarity of their rDNAs not only in the genomic features but also in the distribution of intragenomic polymorphic sites on the paralogs. Ribosomal RNA comprises multiple domains with different functions. The two Rhizophagus species commonly exhibited a variation enrichment site, ES27L, which is related to translational fidelity and antibiotic sensitivity. Variation enrichment on ES27L has not been observed in other organisms lacking the three rDNA features such as malaria parasites and Cyanidioschyzon merolae. Expression profiling of rDNAs in R. irregularis revealed that rDNA paralogs are expressed differently in association with host plant species. Our results suggest a broad distribution of the disarranged rDNA across AM fungi and its involvement in the successful association with the broad range of host species.

genomics↗

bioRxiv2020

Revisiting the gastrin-releasing peptide/bombesin system: A reverse-evolutionary study considering Xenopus

Gastrin-releasing peptide (GRP), first isolated from the porcine stomach, is a neuropeptide that modulates the autonomic system in mammals and has previously been considered to be the mammalian equivalent of bombesin, a fourteen amino acid peptide first isolated from the skin of the European fire-bellied toad, Bombina bombina. Bombesin-like peptides and the related neuromedin B (NMB) have since been identified in mammals. However, the orthologous relationships among GRP/NMB/bombesin and their receptors in vertebrates are still not well understood. Our studies have focused on the GRP system that is widely conserved among vertebrates. We have used phylogenetic analysis and reverse transcription-PCR, quantitative PCR, immunohistochemistry, and Western blotting experiments to examine the expression of both GRP and its receptor (GRPR) in a clawed frog (Xenopus tropicalis) and to understand the derivation of GRP system in the ancestor of mammals. We demonstrate, by phylogenetic and synteny analyses, that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frog species. In Xenopus, we found the expression of the mRNA for both GRP and GRPR in the brain and stomach. In addition, our quantitative PCR analysis shows that, in Xenopus, the expression of GRP mRNA is highest in the brain, whereas expression of GRPR mRNA is highest in the spinal cord. Our immunohistochemical analysis shows that GRP-immunoreactive cell bodies and fibers are distributed in several telencephalic, diencephalic, and rhombencephalic regions and spinal cord of Xenopus. Our Western blotting analysis also indicates the presence of GRPR protein in the brain and spinal cord of Xenopus. We conclude that GRP peptides and their receptors have evolved to play multiple roles in both the gut and brain of amphibians as one of the gut-brain peptide systems. Author SummaryBombesin is a putative antibacterial peptide isolated from the skin of the frog, Bombina bombina. Two related (bombesin-like) peptides, gastrin-releasing peptide (GRP) and neuromedin B (NMB) have been found in mammals. The history of GRP/bombesin discovery has caused little attention to be paid to the evolutionary relationship of GRP/bombesin and their receptors in vertebrates. We have classified the peptides and their receptors from the phylogenetic viewpoint using a newly established genetic database and bioinformatics. We demonstrate, by phylogenetic and synteny analyses, that GRP is not a mammalian counterpart of bombesin and also that, whereas the GRP system is widely conserved among vertebrates, the NMB/bombesin system has diversified in certain lineages, in particular in frogs. Gene expression analyses combined with immunohistochemistry and Western blotting experiments indicate that GRP peptides and their receptors have evolved from ancestral (GRP) homologues to play multiple roles in both the gut and the brain as one of the gut-brain peptide systems of vertebrates, which is distinct from the frog bombesin lineage.

evolutionary biology↗

bioRxiv2020

Integrated analyses of single-cell atlases reveal age, gender, and smoking status associations with cell type-specific expression of mediators of SARS-CoV-2 viral entry and highlights inflammatory programs in putative target cells

The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, creates an urgent need for identifying molecular mechanisms that mediate viral entry, propagation, and tissue pathology. Cell membrane bound angiotensin-converting enzyme 2 (ACE2) and associated proteases, transmembrane protease serine 2 (TMPRSS2) and Cathepsin L (CTSL), were previously identified as mediators of SARS-CoV2 cellular entry. Here, we assess the cell type-specific RNA expression of ACE2, TMPRSS2, and CTSL through an integrated analysis of 107 single-cell and single-nucleus RNA-Seq studies, including 22 lung and airways datasets (16 unpublished), and 85 datasets from other diverse organs. Joint expression of ACE2 and the accessory proteases identifies specific subsets of respiratory epithelial cells as putative targets of viral infection in the nasal passages, airways, and alveoli. Cells that co-express ACE2 and proteases are also identified in cells from other organs, some of which have been associated with COVID-19 transmission or pathology, including gut enterocytes, corneal epithelial cells, cardiomyocytes, heart pericytes, olfactory sustentacular cells, and renal epithelial cells. Performing the first meta-analyses of scRNA-seq studies, we analyzed 1,176,683 cells from 282 nasal, airway, and lung parenchyma samples from 164 donors spanning fetal, childhood, adult, and elderly age groups, associate increased levels of ACE2, TMPRSS2, and CTSL in specific cell types with increasing age, male gender, and smoking, all of which are epidemiologically linked to COVID-19 susceptibility and outcomes. Notably, there was a particularly low expression of ACE2 in the few young pediatric samples in the analysis. Further analysis reveals a gene expression program shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues, including genes that may mediate viral entry, subtend key immune functions, and mediate epithelial-macrophage cross-talk. Amongst these are IL6, its receptor and co-receptor, IL1R, TNF response pathways, and complement genes. Cell type specificity in the lung and airways and smoking effects were conserved in mice. Our analyses suggest that differences in the cell type-specific expression of mediators of SARS-CoV-2 viral entry may be responsible for aspects of COVID-19 epidemiology and clinical course, and point to putative molecular pathways involved in disease susceptibility and pathogenesis.

bioinformatics↗

bioRxiv2020

Subepidermal blisters are healed by wedge-shaped keratinocytes of hair follicle origin

Injury in adult tissue generally reactivates developmental programs to foster regeneration, but it is not known whether this paradigm applies to growing tissue. Here, by employing blisters, we show that epidermal wounds heal at the expense of skin development. The regenerated epidermis suppresses the expression of tissue morphogenesis genes accompanied by delayed hair follicle (HF) growth. Lineage tracing experiments, cell proliferation dynamics, and mathematical modeling reveal that the progeny of HF junctional zone stem cells, which undergo a morphological transformation, repair the blisters while not promoting HF development. In contrast, the contribution of interfollicular stem cell progeny to blister healing is small. These findings demonstrate that tissue development can be sacrificed for the sake of wound regeneration and suggest that tissue repair does not coincide with the reactivation of developmental programs in all regenerative contexts. Our study elucidates the key cellular mechanism of wound healing in skin blistering diseases.

cell biology↗

bioRxiv2020

Two-photon voltage imaging of spontaneous activity from multiple neurons reveals network activity in brain tissue

Recording the electrical activity of multiple neurons simultaneously would greatly facilitate studies on the structure and function of neuronal circuits. Using fluorescent genetically encoded voltage indicators (GEVI) would be especially desirable, as it would allow cell type-selectivity, longitudinal recordings, and further optical manipulations. By expressing the GEVI ASAP3 via in utero electroporation and rapidly imaging neurons in densely labelled tissues via random-access multi-photon microscopy, we achieve voltage recording of multiple neurons in brain slice with single-trial single-voxel resolution. This approach enables monitoring of subthreshold membrane potential changes and action potentials from multiple locations in soma and dendrites for tens of minutes. By optically recording spontaneous electrical activities in somatosensory cortex neurons, we provide evidence for the development of intralaminar horizontal connections in layer 2/3 with greater sensitivity than calcium imaging. Single-trial optical voltage recordings using ASAP3 thus enables the investigation of network connectivity at cellular resolution.

neuroscience↗

bioRxiv2020

Molecular Choreography of Acute Exercise

Exercise testing is routinely used in clinical practice to assess fitness - a strong predictor of survival - as well as causes of exercise limitations. While these studies often focus on cardiopulmonary response and selected molecular pathways, the dynamic system-wide molecular response to exercise has not been fully characterized. We performed a longitudinal multi-omic profiling of plasma and peripheral blood mononuclear cells including transcriptome, immunome, proteome, metabolome and lipidome in 36 well-characterized volunteers before and after a controlled bout of acute exercise (2, 15, 30 min and 1 hour in recovery). Integrative analysis revealed an orchestrated choreography of biological processes across key tissues. Most of these processes were dampened in insulin resistant participants. Finally, we discovered biological pathways involved in exercise capacity and developed prediction models revealing potential resting blood-based biomarkers of fitness.

systems biology↗

bioRxiv2019

Persistence of a novel regeneration-associated transitional cell state in pulmonary fibrosis

Stem cell senescence is often seen as an age associated pathological state in which cells acquire an abnormal and irreversible state. Here, we show that alveolar stem cell differentiation during lung regeneration involves a unique previously uncharacterized transitional state that exhibits cardinal features normally associated with cell senescence. Specifically, using organoid cultures, multiple in vivo injury models coupled with single cell transcriptomics and lineage tracing analysis, we find that alveolar stem cell differentiation involves a novel, pre-alveolar type-1 transitional state (PATS) en route to their terminal maturation. PATS can be distinguished based on their unique transcriptional signatures, including enrichment for TP53, TGF{beta}, and DNA damage repair signaling, and cellular senescence in both in vivo and ex vivo regenerating tissues. Significantly, PATS undergo extensive cell stretching, which makes them vulnerable to DNA damage, a feature commonly associated with most degenerative lung diseases. Importantly, we find enrichment of PATS-like state in human fibrotic lung tissues, suggesting that persistence of such transitional states underlies the pathogenesis of pulmonary fibrosis. Our study thus redefines senescence as a state that can occur as part of a normal tissue maintenance program, and can be derailed in human disease, notably fibrosis.

cell biology↗

bioRxiv2019

Yolk-Sac-Derived Macrophages Progressively Expand in the Mouse Kidney with Age

Renal macrophages represent a highly heterogeneous and specialized population of myeloid cells with mixed developmental origins from the yolk-sac and hematopoietic stem cells (HSC). They promote both injury and repair by regulating inflammation, angiogenesis, and tissue remodeling. Recent reports highlight differential roles for ontogenically distinct renal macrophage populations in disease. However, little is known about how these populations change over time in normal, uninjured kidneys. Prior reports demonstrated a high proportion of HSC-derived macrophages in the young adult kidney. Unexpectedly, using genetic fate-mapping and parabiosis studies, we find that yolk-sac-derived macrophages progressively expand in number with age and become a major contributor to the renal macrophage population in older mice. This chronological shift in macrophage composition involves cellular proliferation and recruitment from circulating progenitors and may contribute to the distinct immune responses, limited reparative capacity, and increased disease susceptibility of kidneys in the elderly population.

immunology↗

bioRxiv2019

Genetic factor for twisting legume pods identified by fine-mapping of shattering-related traits in azuki bean and yard-long bean

Legumes have evolved a unique manner of seed dispersal in that the seed pods explosively split open with helical tension generated by sclerenchyma on the endocarp. During domestication, azuki bean (Vigna angularis) and yard-long bean (Vigna unguiculata cv-gr. Sesquipedalis) have reduced or lost the sclerenchyma and lost the shattering behavior of seed pods. Here we performed fine-mapping with back-crossed populations and narrowed the candidate genomic region down to 4 kbp in azuki bean and 13 kbp in yard-long bean. Among genes located in these regions, we found MYB26 genes encoded truncated proteins in both the domesticated species. We also found in azuki bean and other legumes that MYB26 is duplicated and only the duplicated copy is expressed in seed pods. Interestingly, in Arabidopsis MYB26 is single copy and is specifically expressed in anther to initiate secondary wall thickening that is required for anther dehiscence. These facts indicated that, in legumes, MYB26 has been duplicated and acquired a new role in development of pod sclerenchyma. However, pod shattering is unfavorable phenotype for harvesting and thus has been selected against by human.

genetics↗

bioRxiv2018

Genome-wide association meta-analysis identifies novel GP2 gene risk variants for pancreatic cancer in the Japanese population

The etiology of pancreatic cancer remains largely unknown. Here, we report the results of a meta-analysis of three genome-wide association studies (GWASs) comprising 2,039 pancreatic cancer cases and 32,592 controls, the largest sample size in the Japanese population. We identified 3 (13q12.2, 13q22.1, and 16p12.3) genome-wide significant loci (P<5.0x10-8) and 4 suggestive loci (P<1.0x10-6) for pancreatic cancer. Of these risk loci, 16p12.3 is novel; the lead SNP maps to rs78193826 (odds ratio (OR)=1.46, 95% CI=1.29-1.66, P=4.28x10-9), an Asian-specific, nonsynonymous glycoprotein 2 (GP2) gene variant predicted to be highly deleterious. Additionally, the gene-based GWAS identified a novel gene, KRT8, which is linked to exocrine pancreatic and liver diseases. The identified GP2 gene variants were pleiotropic for multiple traits, including type 2 diabetes, hemoglobin A1c (HbA1c) levels, and pancreatic cancer. Mendelian randomization analyses corroborated causality between HbA1c and pancreatic cancer. These findings suggest that GP2 gene variants are associated with pancreatic cancer susceptibility in the Japanese population, prompting further functional characterization of this locus.

genetics↗

bioRxiv2017

Evidence of non-tandemly repeated rDNAs and their intragenomic heterogeneity in Rhizophagus irregularis

Arbuscular mycorrhizal fungus (AMF) species are one of the most widespread symbionts of land plants. Our substantially improved reference genome assembly of a model AMF, Rhizophagus irregularis DAOM-181602 (total contigs = 210), facilitated discovery of repetitive elements with unusual characteristics. R. irregularis has only ten or eleven copies of complete 45S rDNAs, whereas the general eukaryotic genome has tens to thousands of rDNA copies. R. irregularis rDNAs are highly heterogeneous and lack a tandem repeat structure. These findings provide evidence for the hypothesis that rDNA heterogeneity depends on the lack of tandem repeat structures. RNA-Seq analysis confirmed that all rDNA variants are actively transcribed. Observed rDNA/rRNA polymorphisms may modulate translation by using different ribosomes depending on biotic and abiotic interactions. The non-tandem repeat structure and intragenomic heterogeneity of AMF rDNA/rRNA may facilitate adaptation to a various environmental condition including the broad host range.

genomics↗

bioRxiv2017

Development of honeybee waggle dance and its differences between recruits and scouts

The lifetime development of the waggle dance of 14 honeybees was automatically recorded just after the imaginal molt using high-definition camera modules connected with a Raspberry Pi computer and numbered radio-frequency identification tags fitted to the back of each bee. For most honeybees, waggle dance follow preceded the appearance of the first waggle dance from 1 week after the imaginal molt. The duration per trip increased just after waggle dance follow. Before the appearance of the first waggle dance, the honeybee repeatedly follows waggle dances that indicate a limited number (2-6) of food source locations. We discriminated between two types of foragers with different roles, recruits and novice scouts, by comparing the vectors indicated by the bees first waggle dance (sending vectors) with dances they had previously followed (received vectors). Of 14 tagged honeybees, 11 were categorized as recruits and 2 as novice scouts. For recruits (but not for novice scouts), the duration per trip increased significantly after waggle dances follow and substantially increased just before the appearance of the first waggle dance. Moreover, recruits increased the number of times they followed waggle dances indicating the same location, and their first waggle dance indicated this location. These results suggest that the differentiation of these two types of foragers is partly related to behavioral differences after waggle dance follows: whether trip is activated or not by follows a waggle dance.\n\nSummary statementBecause of technological difficulties, there are no studies comparing the development of recruit and scout waggle dances. Using miniature radio frequency identification tags, we observed and clarified these developmental processes.\n\nList of Abbreviations

animal behavior and cognition↗