Biology subjects

Watanabe, Y.

Publications and source records attributed to Watanabe, Y..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Harnessing the Biosynthetic Diversity of Actinomycetes: Discovery of Unique Natural Products through Comparative Genomic and Metabolic Analysis

Actinomycetes, rich in biosynthetic gene clusters (BGCs), are important sources of natural products (NPs). However, the rate of discovering novel NPs from actinomycetes has declined, indicating the need for a new strategy to obtain NPs. Herein, we present a strategy for the efficient discovery of novel NPs. First, we performed a comprehensive analysis of BGCs in actinomycetes, evaluating the average number and types of BGCs per genus to identify prolific NP producers. Our analysis revealed that certain actinomycetes strains, such as those in the family Pseudonocardiaceae, possess a greater number of BGCs than Streptomyces. In addition, these strains tend to possess strain-specific BGCs compared with others. To facilitate the identification of strain-specific compounds, we developed a comparative metabolic analysis method using molecular networking. Applying this method to eight Pseudonocardiaceae strains, we successfully discovered two novel peptides, lentindoles A (1) and B (2), featuring a 6/5/6 and 6/5/5 tricyclic ring system, respectively, along with their possible biosynthetic precursor, lentindole C (3). Our result demonstrates that the effectiveness of our developed strategy, which is expected to accelerate the discovery of novel NPs.

microbiology↗

bioRxiv2024

Structural basis for phosphatidylcholine synthesis by bacterial phospholipid N-methyltransferases

In phosphatidylcholine (PC)-containing bacteria, PC is synthesized by phospholipid N-methyltransferases (Pmts) and plays an important role in the interactions between symbiotic and pathogenic bacteria and their eukaryotic host cells. Pmts catalyze the SAM dependent three methylation reactions of the head group of phosphatidylethanolamine (PE) to form PC through monomethyl PE and dimethyl PE. However, the precise molecular mechanisms underlying PC biosynthesis by PmtA remain largely unclear, owing to the lack of structural information. Here, we determined the crystal structures of Agrobacterium tumefaciens Pmt (AtPmtA) in complex with SAH or 5'-methylthioadenosine. Crystal structures and NMR analysis revealed the binding mode of AtPmtA to SAH in solution. Structure-based mutational analyses showed that a conserved tyrosine residue in the substrate-binding groove is involved in methylation. Furthermore, we showed that differences in substrate specificity among Pmt homologs were determined by whether the amino acid residues comprising the substrate-binding groove were isoleucine or phenylalanine. These findings provide a structural basis for understanding the mechanisms underlying Pmts-mediated PC biosynthesis.

biochemistry↗

bioRxiv2024

Analysis of the high-order conformational changes in glyceraldehyde-3-phosphate dehydrogenase induced by nicotinamide adenine dinucleotide, adenosine triphosphate, and oxidants

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the sixth step of glycolysis. As a moonlighting protein, GAPDH interacts with other molecules, seemingly unrelated to glycolysis, to exert additional functions, such as inducing apoptosis. However, specific mechanisms underlying these interactions remain unclear. Therefore, in this study, we aimed to elucidate these mechanisms by analyzing human and porcine GAPDHs based on their three-dimensional (3D) structures using various biophysical techniques, including nuclear magnetic resonance spectroscopy, gel filtration chromatography, and thermal shift assay. Although GAPDH became unstable when nicotinamide adenine dinucleotide (NAD+) was removed (apo state), the 3D structure of the tetramer was maintained regardless of the temperature. However, in the presence of adenosine triphosphate (ATP), GAPDH split into dimers at low temperatures, exposing the interface between the dimers and increasing the flexibility of the side chains at the site. Moreover, subunit splitting also occurred upon exposure to oxidizing agents, such as hydrogen peroxide. These findings suggest that GAPDH maintains a stable tetramer in the presence of NAD+ but becomes unstable and easily oxidized upon NAD+ depletion. When multiple residues, including those other than the cysteine residue at the active site, are oxidized by reactive oxygen species or nitric oxide, or when it interacts with ATP, GAPDH splits into dimers. This subunit splitting may trigger interactions with other molecules.

biochemistry↗

bioRxiv2024

ALS-associated RNA binding proteins converge on UNC13A transcription through regulation of REST

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective loss of motor neurons. Although multiple pathophysiological mechanisms have been identified, a unified molecular basis for ALS has remained elusive. The ALS-associated RNA binding protein (RBP) TDP-43 has previously been shown to stabilize UNC13A mRNA by preventing cryptic exon inclusion. We here show that three ALS-associated RBPs--MATR3, FUS, and hnRNPA1--regulate UNC13A expression by targeting the silencing transcription factor REST. These three RBPs bind to and downregulate REST mRNA and thereby promote UNC13A transcription. REST overexpression was detected not only in response to the loss of each of these three RBPs in cultured cells but also in motor neurons of individuals with familial or sporadic ALS. The functional convergence of four RBPs on the regulation of UNC13A, a gene essential for synaptic transmission, highlights a pivotal contribution of these proteins to maitainance of synaptic integrity. Our findings thus provide key insight into ALS pathogenesis and a basis for the development of new therapeutic agents.

neuroscience↗

bioRxiv2024

Evolution of Key Factors Influencing Performance Across Phases in Junior Short Sprints

Sprint performance plays a crucial role in various sports. Short sprints vary in court size and competitive characteristics but are common in many sports. Although the relationship between age and muscle strength has been explored in short sprints, there is limited understanding of how various physical factors interact, particularly concerning differences in the acceleration phase. This study examined the relationship between sprint times at 0-2.5 m, 2.5-5 m, and 5-10 m intervals and various factors in junior athletes. Results indicated that sprint times increased with age, and is correlated with muscle strength and flexibility. Partial correlation analysis showed that faster times in the 0-2.5 m interval were associated with higher hip flexibility; in the 2.5-5 m interval, faster times were associated with higher core flexibility; and in the 5-10 m interval, a relationship with standing long jump performance was confirmed. Furthermore, lower fat-free body weight translated to higher performance. In the acceleration phase of 10 m, flexibility immediately after the start and the subsequent horizontal propulsive force is important factors that are strongly related to performance change in each interval. The study results illuminate the mechanism behind short sprints in junior athletes and emphasize the importance of daily training, considering the sprint distance required for each sport.

animal behavior and cognition↗

bioRxiv2024

SENP2-based N-terminal truncation of α-synuclein in Lewy pathology propagation

-Synuclein (Syn) is a major component of Lewy bodies (LBs) and Lewy neurites (LNs) which are pathological features of Parkinsons disease (PD) and Dementia with Lewy bodies. In the PD brain, with disease progression, LB/LN formation is propagated from the lower brainstem to the cerebral cortex. Prion-like cell-to-cell seed-transmission has been implicated as an underlying mechanism for Lewy-pathology propagation. However, the biochemical properties and production mechanism of those pathogenic seeds are unelucidated. In this study, we ascertained that the seeds released from pathological neurons that harbour LB/LN-like aggregates have the N-terminally truncated form of Syn. This N-terminal truncation is directly catalysed by SENP2, which is a well-known deSUMOylation enzyme. After SENP2 processing of recombinant Syn, the SDS-resistant high-molecular oligomer formation was promoted in vitro. Inhibition of SENP2 activity suppressed aggregate formation and propagation in cultured neurons and mouse brains. Thus, SENP2 might be a novel therapeutic target in LB diseases.

neuroscience↗

bioRxiv2024

Endemic demographics of the Jomon people estimated based on complete mitogenomes reveal their regional diversity

The Jomon culture that spread across Japanese archipelago began about 16,000 years ago and lasted for over 10,000 years. The genetic diversity of the Jomon people, prehistoric hunter-gatherers bearing the Jomon culture, is of great interest in understanding prehistoric East Eurasians. Until now, their demographic history has been estimated using archaeological sites and present-day genomes, but detailed studies using Jomon genomes have been insufficient. To investigate the Jomon demography, we determined the complete mitochondrial genome (mitogenome) sequences from 13 Jomon individuals and conducted population genetic analysis on 40 Jomon genomes including previously published data. Our results revealed an effective population size increase during the Incipient - Initial phase of the Jomon period, which had not been observed in analysis of mitogenome sequences from present-day Japanese populations. This endemic demographic pattern is pronounced in the eastern part of the archipelago, under the assumption of no gene flow between the Eastern and Western Jomon.

genomics↗

bioRxiv2024

Cold adaptation in Upper Paleolithic hunter-gatherers of eastern Eurasia

Previous genomic studies understanding the dispersal of Homo sapiens have suggested that present-day East Eurasians and Native Americans can trace their ancestry to migrations from Southeast Asia. However, ineluctable adaptations during the Last Glacial Maximum (LGM) remain unclear. By analyzing 42 genomes of up to 30-fold coverage from prehistoric hunter-gatherers, Jomon, we reveal their descent from Upper Paleolithic (UP) foragers who migrated to and isolated in the Japanese archipelago during Late Pleistocene. We provide compelling evidence suggesting that these UP people underwent positive selection for cold environments, aiding their survival through the LGM facilitated by non-shivering thermogenesis and detecting it polygenically across multiple loci in the Jomon lineage. Our study pioneers the close estimation of the physiological adaptation of ancient humans by the paleogenomic approach.

evolutionary biology↗

bioRxiv2024

Flow zoometry of Drosophila

Drosophila serves as a highly valuable model organism across numerous fields including genetics, immunology, neuroscience, cancer biology, and developmental biology. Central to Drosophila-based biological research is the ability to perform comprehensive genetic or chemical screens. However, this research is often limited by its dependence on laborious manual handling and analysis, making it prone to human error and difficult to discern statistically significant or rare events amid the noise of individual variations resulting from genetic and environmental factors. In this article we present flow zoometry, a whole-animal equivalent of flow cytometry for large-scale, individual-level, high-content screening of Drosophila. Our flow zoometer automatically clears the tissues of Drosophila melanogaster, captures three-dimensional (3D) multi-color fluorescence tomograms of single flies with single-cell volumetric resolution at an unprecedented throughput of over 1,000 animals within 48 hours (24 hr for clearing; 24 hr for imaging), and performs AI-enhanced data-driven analysis - a task that would traditionally take months or years with manual techniques. To demonstrate its broad applications, we employed the flow zoometer in various laborious screening assays, including those in toxicology, genotyping, and tumor screening. Flow zoometry represents a pivotal evolution in high-throughput screening technology: previously from molecules to cells, now from cells to whole animals. This advancement serves as a foundational platform for "statistical spatial biology", to improve empirical precision and enable serendipitous discoveries across various fields of biology.

bioengineering↗

bioRxiv2023

Age-associated aberrations of cumulus-oocyte interaction and microfilamentous structure in the zona pellucida decline female fertility

One of the major age-related declines in female reproductive function is the reduced quantity and quality of oocytes. Here we demonstrate that structural changes in the zona pellucida (ZP) decrease fertilization rates from 34- to 38-week-old female mice, equivalent to the mid-reproductive of human females. In middle-age ovaries, the decline in the number of transzonal projections was accompanied by a decrease in cumulus cell-oocyte interactions, resulting in a deterioration of the oocyte quality. Scanning electron microscopy showed the ZP surface microfilament structure transitioning from rugged to smooth with age, leading to decreased fertilization rates due to impaired sperm binding to the ZP. Moreover, the fertilization rate of middle-age mice was restored to a comparable level to that of young mice by destabilizing the ZP in the presence of glutathione. These results suggest that the age-related structural changes in the ZP is a key for successful fertilization at reproductive age.

developmental biology↗

bioRxiv2023

Artificial environment impact: O2 concentration changes between IVM and IVF alter embryo production, metabolism, and epigenetic marks

Creating an optimal in vitro cell culture environment requires careful simulation of all critical components, especially the gaseous atmosphere. Although it is well-documented that embryonic culture under low oxygen tension promotes embryonic development, little is known about the effect of changes in in vitro maturation (IVM) and fertilization (IVF) on the epigenome. This study explores the role of oxygen tension variation in the early stages of in vitro production of bovine embryos and its impact on oxidative stress and epigenetic remodeling. We initially validated our system by scrutinizing the epigenetic effects on bovine fibroblasts. We observed that cell cultures under 20% O2 exhibited reduced H3K9me2 levels in early passages, which stabilized with prolonged cultivation and elevated gene expression of HIF2a and KDM5C. Our results reveal that oocytes maturing in 20% O2 environments have heightened levels of reactive oxygen species (ROS) and glutathione (GSH), whereas blastocyst embryos maturing under reduced oxygen tension exhibit increased oxidative stress markers (NRF2, SOD1, SOD2), with upregulated transcripts observed in epigenetic remodelers (KDM5A, TET1). Elevated O2 levels in both IVM and IVF processes showcase improved embryo production. Maintaining consistent O2 levels at either 5% or 20% between IVM and IVF results in heightened GSH, reduced ROS, and increased levels of H3K9me2/3 in embryos. Finally, distinct DNA methylation patterns emerge, indicating higher levels in groups matured under low O2 tension and increased DNA hydroxymethylation in groups fertilized under low O2 tension. In conclusion, our comprehensive investigation underscores the critical role of oxygen concentration in shaping the epigenetic landscape during the early stages of in vitro culture. These findings provide valuable insights for optimizing conditions in assisted reproductive technologies.

developmental biology↗

bioRxiv2023

A Tetravalent Dengue Virus-like Particle Vaccine Induces High levels of Neutralizing Antibodies and Inhibits Dengue Replication in Non-Human Primates

Dengue virus (DENV) represents a significant global health burden, with 50% of the worlds population at risk of infection, and there is an urgent need for next-generation vaccines. Virus-like particle (VLP)-based vaccines, which mimic the antigenic structure of the authentic virus but lack the viral genome, are an attractive approach. Here we describe a dengue VLP (DENVLP) vaccine which generates a robust and long-lasting neutralizing antibody response against all four DENV serotypes in non-human primates. Importantly, DENVLP vaccination produced no ADE response against any of four DENV serotypes. Finally, we demonstrate in a non-human primate challenge model that DENVLP vaccination substantially reduces viral replication. We also transfer the purified IgG from the immunized monkeys into immunodeficient mice, where they protect against subsequent lethal dengue virus challenge, indicating a humoral mechanism of protection. These results indicate that a DENVLP vaccine is a safe and effective vaccine candidate. One Sentence Summary: Immunization of non-human primates with a tetravalent dengue VLP vaccine induces high levels of neutralizing antibodies and reduces the severity of infection for all four dengue serotypes.

microbiology↗

bioRxiv2023

MpmiR319 promotes gemma/gemma cup formation in the Liverwort Marchantia polymorpha

After microRNAs (miRNAs) were initially detected as approximately 21-nt sequences in plants, miRNA-mediated regulation of target gene expression was characterized in many cases. The sequences of miR159/319 family miRNAs are highly conserved and their development-related roles have been comprehensively characterized in dicot and monocot plants. However, their effects in basal land plants remain relatively unknown. We used a CRISPR/Cas9 system to edit genome sequences at MpMIR319a and/or MpMIR319b loci. Mutant mir319b lines developed relatively few gemma cups, while mutant mir319a lines produced relatively few gemmae in each gemma cup. Earlier 5'-RACE or degradome analyses suggested that MpmiR319 targets MpRKD and MpR2R3-MYB21 transcripts and suppresses expression. To determine whether these candidate genes influence the mir319a/mir319b phenotype we constructed miR319-resistant MpRKD (mMpRKD) and MpR2R3-MYB21 (mMpR2R3-MYB21) by decreasing the complementarity to miR319. We also introduced the genes into wild-type liverwort and screened for phenotypic changes. We observed that mMpRKD resulted in gemma/gemma cup-less liverwort mutants, but mMpR2R3-MYB21 did not. No major adverse effects were detected during male and female reproductive development induced by far-red illumination. These results indicate that miR319 influences gemma formation mainly by repressing MpRKD expression rather than MpR2R3-MYB21 expression. HighlightThis study demonstrates MpmiR319 expression affects gemma cup and gemma formation during Marchantia polymorpha vegetative propagation by suppressing its target mRNAs.

plant biology↗

bioRxiv2023

Opioidergic activation of descending pain inhibitory system underlies placebo analgesia

Placebo analgesia is caused by inactive treatment, implicating endogenous brain function involvement. However, the underlying neurobiological mechanisms remain unclear. We found that -opioid signals in the medial prefrontal cortex (mPFC) activate the descending pain inhibitory system to initiate placebo analgesia in neuropathic pain rats. Chemogenetic manipulation demonstrated that specific activation of -opioid receptor-positive (MOR+) neurons in the mPFC or suppression of the mPFC-ventrolateral periaqueductal gray (vlPAG) circuit inhibited placebo analgesia in rats. MOR+ neurons in the mPFC are monosynaptically connected and directly inhibit L5 pyramidal neurons that project to the vlPAG via GABAA receptors. Thus, intrinsic opioid signaling in the mPFC disinhibits excitatory outflow to the vlPAG by suppressing MOR+ neurons, leading to descending pain inhibitory system activation that initiates placebo analgesia. One Sentence SummarySugar pills relieve pain by activating the intrinsic pain inhibitory system via opioidergic signals in the prefrontal cortex.

neuroscience↗

bioRxiv2023

Isoxanthohumol improves obesity and glucose metabolism via inhibiting intestinal lipid absorption with a bloom of Akkermansia muciniphila in mice

AimsDysbiosis is an important factor that leads to metabolic disorders by disrupting energy balance and insulin sensitivity. A decrease in Akkermansia muciniphila is a phenotype of obesity-induced dysbiosis. Although interventions to increase A. muciniphila are expected to improve glucose metabolism, the underlying mechanism has not been fully understood. MethodsIsoxanthohumol (IX), a prenylated flavonoid found in beer hops was administered to high fat diet-fed mice. We analyzed glucose metabolism, gene expression profiles and histology of liver, epididymal adipose tissue and colon. Lipase activity, fecal lipid profiles and plasma metabolomic analysis were assessed. Fecal 16s rRNA sequencing was obtained and selected bacterial species were used for in vitro studies. Fecal microbiota transplantation and monocolonization were conducted to antibiotic-treated or germ-free (GF) mice. ResultsThe administration of IX lowered weight gain, decreased steatohepatitis and improved glucose metabolism. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption by decreasing the expression of the fatty acid transporter CD36 in the small intestine, which was confirmed by increased lipid excretion in feces. IX administration improved the gut barrier function and reduced metabolic endotoxemia. In contrast, the effects of IX were nullified by antibiotics. As revealed using 16S rRNA sequencing, the microbial community structure changed with a significant increase in the abundance of A. muciniphila in the IX-treated group. An anaerobic chamber study showed that IX selectively promoted the growth of A. muciniphila while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct effect of A. muciniphila on lipid and glucose metabolism, we monocolonized either A. muciniphila or Bacteroides thetaiotaomicron to GF mice. A. muciniphila monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism, with decreased levels of multiple classes of fatty acids determined using plasma metabolomic analysis. ConclusionOur study confirmed a direct role of A. muciniphila in energy metabolism, which was induced by microbial actions of IX. These highlight new treatment strategies for preventing metabolic syndrome by boosting the gut microbiota with food components.

microbiology↗

bioRxiv2023

Atg15 is a vacuolar phospholipase that disintegrates organelle membranes

Atg15 (autophagy-related 15) is a vacuolar phospholipase essential for the degradation of cytoplasm-to-vacuole targeting (Cvt) bodies and autophagic bodies, hereinafter referred to as intravacuolar autophagic compartments (IACs), but it remains unknown if Atg15 directly disrupts IAC membranes. Here we show that the recombinant Chaetomium thermophilum Atg15 lipase domain (CtAtg15(73-475)) possesses phospholipase activity that in acidic environments digests phospholipid to generate lysophospholipid and free fatty acid. The activity of CtAtg15(73-475) was markedly elevated by limited digestion with proteinase K. Proteinase K-treated CtAtg15(73-475) was detected as three fragments, with cleavage between S159 and V160, and between F209 and N210. We inserted the human rhinovirus 3C protease recognition sequence into the two sites and found that cleavage between S159 and V160 was important to activate CtAtg15(73-475). We confirmed that CtAtg15 compensated for the absence of ATG15 in Saccharomyces cerevisiae cells, indicating that CtAtg15 could disintegrate S. cerevisiae IAC in vivo. Further, both mitochondria and IAC were disintegrated by CtAtg15 in S. cerevisiae. This study suggests that Atg15 can degrade any organelle membrane, indicating that Atg15 plays a role in disrupting organelle membranes delivered to vacuoles by autophagy.

molecular biology↗

bioRxiv2023

The cholesterol pathway of the Golgi stress response induces cell death and transcription of Golgi-related genes through metabolic dysregulation of phosphatidylinositol-4-phosphate

The Golgi stress response is an important cytoprotective system that enhances Golgi function in response to cellular demand, while cells damaged by prolonged Golgi stress undergo cell death to ensure the survival of organisms. OSW-1, a natural compound with anticancer activity, acts as a potent inhibitor of OSBP that transports cholesterol and phosphatidylinositol-4-phosphate (PI4P) at contact sites between the endoplasmic reticulum and the Golgi apparatus. Previously, we reported that OSW-1 induces the Golgi stress response, resulting in Golgi stress-induced transcription and cell death. However, the underlying molecular mechanism has been unknown. To reveal the mechanism of a novel pathway of the Golgi stress response regulating transcriptional induction and cell death (the cholesterol pathway), we performed a genome-wide knockout screen and found that transcriptional induction as well as cell death induced by OSW-1 was repressed in HeLa cells deficient in factors involved in the PI4P metabolism, such as PITPNB and PI4KB genes. Our data indicate that OSW-1 induces Golgi stress-dependent transcriptional induction and cell death through dysregulation of the PI4P metabolism in the Golgi apparatus.

cell biology↗

bioRxiv2023

Downregulation of CYP7B1 caused by lipotoxicity associates with the progression of non-alcoholic steatohepatitis

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide, with an incidence of >25% of the adult population. NAFLD ranges from benign simple steatosis to non-alcoholic steatohepatitis (NASH). However, its transition mechanisms underlying the pathogenesis remain to be clarified. The expression of Cyp7b1 gene is downregulated in the liver of leptin-deficient mice and methionine and choline-deficient diet-fed mice based on previous microarray data. Thus, in this study, we investigated the effect of CYP7B1 restoration on the progression of NASH in mice fed MCD diet and its association with oxidative and lipid stresses. Our results suggest that restoration of CYP7B1 expression attenuates hepatitis and fibrosis and that lipid and oxidative stresses observed in the early stage of NASH suppresses Cyp7b1 transcription in hepatocytes.

biochemistry↗