Biology subjects

Miyashita, A.

Publications and source records attributed to Miyashita, A..

13 recordsLinked to original sources

bioRxiv2025

N-acetylchitohexaose induces immune priming against Pseudomonas aeruginosa infection in the silkworm, Bombyx mori

Lysine Motif (LysM) domain-containing receptors are evolutionarily conserved pattern recognition receptors (PRRs) that serve as key mediators of glycan sensing and innate immune activation in plants and mammals. In invertebrates, however, their role in activating innate immunity remains poorly understood, although some evidence for immunosuppressive functions exists. In this study, we performed in silico structural analyses and identified a putative B. mori LYSMD3 homolog (XP_004933441.1). This protein exhibits high structural similarity in the LysM domain to human LYSMD3, with a root-mean-square deviation (RMSD) of 0.559 [A], indicating close structural alignment. RNA-seq analysis of hemocytes isolated from silkworm larvae injected with N-acetylchitohexaose (GN6), a chitin-derived oligosaccharide and known ligand of human LYSMD3, revealed transcriptional activation of innate immune effectors, including antimicrobial peptide (AMP) genes such as cecropins. GN6 also induced cecropin transcription in isolated hemocytes in vitro, and western blotting of hemolymph confirmed elevated Cecropin B protein levels. Furthermore, GN6 and chitin significantly improved survival against P. aeruginosa infection, with median effective doses (ED) values of 0.62 and 0.48 {micro}g/larva, respectively. In contrast, N-acetylglucosamine (GlcNAc) and shorter oligosaccharides (GN2-GN5) were ineffective. These findings provide the first molecular-level evidence of a putative glycan receptor in silkworms based on structural similarity to known LysM domains. Moreover, GN6-induced antimicrobial peptide expression and enhanced infection resistance demonstrate immune priming in this model, supporting an evolutionarily conserved glycan-sensing pathway in invertebrates.

immunology↗

bioRxiv2025

Leaf Mold Agar Facilitates Recovery of Soil Bacterial Diversity Beyond Bacillus Dominance

Dominance of Bacillus species on conventional agar media often hampers the isolation of taxonomically diverse soil bacteria (i.e., "Bacillus dominance"). To overcome this Bacillus dominance, we developed a novel agar medium containing leaf mold extract (LME) and assessed its utility for isolating microorganisms from soil samples. For comparison, conventional yeast malt extract agar (YME agar) was used as a control. 16S rRNA gene sequencing revealed that colonies cultured on YME agar predominantly consisted of Bacillus-related bacteria, whereas LME agar enabled the growth of a wider variety of taxa, particularly actinomycetes such as Streptomyces. Notably, LME agar supported a higher diversity of soil bacterial isolates even from samples with a low abundance of Bacillus-related species. Among the 138 isolates recovered from LME agar, some either exhibited very low 16S rRNA gene sequence identity to known species in public databases, or failed to yield 16S rRNA amplicons with conventional universal primers, suggesting the presence of previously uncharacterized or novel taxa. Furthermore, one unidentified isolate failed to grow on standard nutrient media (YME, BHI, LB10, or TSB), but proliferated in liquid leaf mold extract, indicating a specific nutrient dependency. These findings demonstrate that LME agar can facilitate the isolation of a broader spectrum of soil microorganisms (including rare and previously uncultured species) not readily recoverable on common basal media. Incorporation of ecosystem-derived substrates into culture media may greatly enhance the discovery and study of novel microbial diversity from environmental samples. ImportanceSoil contains a vast number of microorganisms, many of which remain undiscovered due to limitations in standard laboratory methods. A major issue is the overgrowth of Bacillus species on conventional media, which suppresses the growth of other bacteria. To address this, we developed a new agar medium using leaf mold extract (LME) and found it successfully supported the growth of a much wider variety of soil bacteria, including rare species. Some of the isolated microbes could not be identified using standard genetic tools, and one unique strain only grew in LME-based media, indicating a special nutrient requirement. These findings show that using natural environmental components like leaf mold can help scientists grow and study bacteria that were previously missed. This approach can expand our understanding of microbial diversity and may lead to the discovery of new microbes with potential applications in agriculture, medicine, or environmental science.

microbiology↗

bioRxiv2024

Selective agonism of GPR34 stimulates microglial uptake and clearance of amyloid β fibrils

Microglia, the primary immune cells of the central nervous system, play a crucial role in maintaining brain homeostasis through phagocytosis of various substrates, including amyloid-{beta} (A{beta}) fibrils, a hallmark of Alzheimer disease (AD) pathology. However, the molecular mechanisms regulating microglial A{beta} uptake remain poorly understood. Here, we identified GPR34, a Gi/o-coupled receptor highly expressed in microglia, as a novel regulator of fibrillar A{beta} phagocytosis. Treatment with a selective GPR34 agonist, M1, specifically enhanced uptake of A{beta} fibrils, but not its monomer or oligomer, in both mouse and human microglia. Mechanistically, M1 reduced intracellular cAMP levels, which inversely correlated with A{beta} uptake activity. Importantly, a single intrahippocampal injection of M1 in an AD mouse model significantly increased microglial A{beta} uptake in vivo. Furthermore, single-nucleus RNA-sequencing analysis of Japanese AD patient samples revealed a significant reduction of GPR34 expression in microglia from AD patients compared to controls. We also observed an age-dependent decline in microglial GPR34 expression in both human and mouse datasets, suggesting a potential contribution of GPR34 downregulation to age-related A{beta} accumulation and AD risk. Collectively, our findings identify GPR34 as a promising target for modulating microglial A{beta} clearance and highlight the therapeutic potential of GPR34 agonists in AD. Significance statementAlzheimer disease (AD) is characterized by amyloid-{beta} (A{beta}) accumulation in the brain. Microglia, the brains immune cells, play a crucial role in the metabolism of A{beta}. We discovered that activating the microglial receptor GPR34 with a selective agonist enhances the phagocytosis of A{beta} fibrils, a key pathogenic form of A{beta}. Importantly, GPR34 expression decreases with aging and AD progression, potentially contributing to impaired A{beta} clearance. Our findings highlight GPR34 as a promising therapeutic target for AD, as boosting its activity could promote A{beta} clearance and slow disease progression. This study provides valuable insights into microglial function in AD and offers a novel strategy for developing disease-modifying therapies.

neuroscience↗

bioRxiv2024

Advancing microbial isolation: The impact of leaf mold extract agar on soil samples

In this study, we developed a new agar medium using leaf mold extract and evaluated its microbial cultivation performance with soil samples. As a control for performance evaluation, the general-purpose nutrient medium YME agar was used. For YME agar, isolated microbes were confirmed to belong mainly to the Bacillus genus through 16S rRNA gene sequencing. In contrast, for the leaf mold agar, bacteria belonging to either Streptomyces or Rhizobium were frequently isolated. Also, of the 51 sequenced isolates on the leaf mold agar medium, an unidentified species (i.e., not listed in databases) with 16S rRNA sequence identity below 98.7% was found. The unidentified species did not grow in standard nutrient media (YME, BHI, LB10, or TSB) but grew in 10% leaf mold extract. The findings of this study suggest that the use of leaf mold agar medium can effectively isolate soil microbes that are difficult to culture in general-purpose nutrient media such as YME. Moreover, this approach indicates a viable method for discovering unknown, previously-uncultured microbial species from soil samples.

microbiology↗

bioRxiv2022

Adult ticks (Ixodes scapularis) infected with B. burgdorferi) have increased overwintering survival, with implications for tick population growth

The tick, Ixodes scapularis, vectors pathogens such as Borrelia burgdorferi, the bacterium that causes Lyme Disease. Over the last few decades I. scapularis has expanded its range, introducing a novel health threat into these areas. Warming temperatures appear to be one cause of its range expansion to the north. However, other factors are also involved. We show that unfed adult female ticks infected with B. burgdorferi have greater overwintering survival than uninfected female ticks. Locally collected adult female ticks were placed in individual microcosms and allowed to overwinter in both forest and dune grass environments. In the spring we collected the ticks and tested both dead and living ticks for B. burgdorferi DNA. Infected ticks had greater overwintering survival compared with uninfected ticks every winter for three consecutive winters in both forest and dune grass environments. We discuss the most plausible explanations for this result. The increased winter survival of adult female ticks could enhance tick population growth. Our results suggest that, in addition to climate change, B. burgdorferi infection itself may be promoting the northern range expansion of I. scapularis. Our study highlights how pathogens could work synergistically with climate change to promote host range expansion.

zoology↗

bioRxiv2022

Intracellular FUS protein accumulation leads to cytoskeletal, organelle and cellular homeostasis perturbations

The molecular mechanisms that connect the formation of aberrant cytoplasmic FUS condensates to biological malfunction are incompletely understood. Here, we develop an approach to determine the intracellular FUS viscosity in live mammalian cells and find that ALS-related mutant P525L-FUS forms the most viscous condensates and has impaired cytoskeletal mechanoproperties and increased euchromatin formation. We further show that some of the main cellular organelles, e.g., actin/tubulin, lysosomes, mitochondria, the endoplasmic reticulum, are significantly functionally/structurally impaired in the presence of FUS. These may be related to defects in the tubulin network, as the latter facilitates transport, formation, fusion and fission of organelles. We observe significant increases in lysosomal biogenesis, size and pH; moreover, intracellular FUS accumulation significantly promotes cytoplasmic-to-nuclear translocation of TFEB, i.e., the master gene for inducing autophagy. However, despite these, increased autophagy needed for protein aggregate clearance is not observed to occur. Our study reveals that the formation of highly viscous FUS condensates significantly impacts cytoskeletal/organelle function and cellular homeostasis, which are closely associated with cell ageing. This raises the intriguing question as to whether mutant FUS activates similar cell processes as those during cellular senescence.

biophysics↗

bioRxiv2021

Purification of immunostimulatory glucose homopolymer from broccoli (Brassica oleracea var. italica)

We prepared broccoli (Brassica oleracea var. italica) neutral polysaccharides (flow-through fractions of anion exchange column chromatography from hot water extracts) from different broccoli cultivars and compared their immunostimulatory effects in the silkworm muscle contraction assay. The cultivars showed a wide range of activity, with the cultivar Winter dome showing the highest specific activity (more than 100 times higher than curdlan). Furthermore, the active substance was purified by gel filtration column chromatography. The active substance showed heterogeneous molecular weights of more than 270 kDa. Sugar composition analysis of the purified fraction revealed that more than 95% of its sugar component was glucose, suggesting that the immunostimulatory neutral polysaccharide from broccoli cultivar Winter dome was a homopolymer of glucose. The purified fraction also induced TNF production in cultured mouse macrophage cells. These results suggest that the glucose homopolymer in broccoli has an immunostimulatory effect on both arthropod and mammalian immune system.

biochemistry↗

bioRxiv2021

Activation of innate immunity by neutral polysaccharides from broccoli buds

Edible substances that stimulate the innate immune system are good candidates for functional foods to improve human health. We have previously reported that acidic polysaccharides from broccoli extract exhibit immunostimulatory effects, but neutral polysaccharides have been overlooked. In the present study, we found that neutral polysaccharides have significantly stronger (higher specific activity) immunostimulatory activity than acidic polysaccharides. The hot water extract of broccoli showed the immunostimulatory activity in the silkworm muscle contraction assay, suggesting that it stimulates innate immunity via paralytic peptide pathway. The activity was concentrated in the buds, but not in the stems and stalk. The active substance was recovered in the flow-through fraction of diethylaminoethyl-cellulose column chromatography with neutral polysaccharides. The specific activity of the fraction was significantly higher than that of the acidic polysaccharides from broccoli reported previously. These results suggest that the neutral polysaccharide present in broccoli buds stimulates innate immunity and can be semi-purified by one-step chromatography.

biochemistry↗

bioRxiv2019

Disruption of a RAC1-centred protein interaction network is associated with Alzheimer’s disease pathology and causes age-dependent neurodegeneration

The molecular biological mechanisms of Alzheimers disease (AD) involve disease-associated cross-talk through many genes and include a loss of normal as well as a gain of abnormal interactions among genes. A protein domain network (PDN) is a collection of physical bindings that occur between protein domains, and the states of the PDNs in patients with AD are likely to be perturbed compared to those in normal healthy individuals. To identify PDN changes that cause neurodegeneration, we analysed the PDNs that occur among genes co-expressed in each of three brain regions at each stage of AD. Our analysis revealed that the PDNs collapsed with the progression of AD stage and identified five hub genes, including Rac1, as key players in PDN collapse. Using publicly available gene expression data, we confirmed that the mRNA expression level of the RAC1 gene was downregulated in the entorhinal cortex (EC) of AD brains. To test the causality of these changes in neurodegeneration, we utilized Drosophila as a genetic model and found that modest knockdown of Rac1 in neurons was sufficient to cause age-dependent behavioural deficits and neurodegeneration. Finally, we identified a microRNA, hsa-miR-101-3p, as a potential regulator of RAC1 in AD brains. As the Braak neurofibrillary tangle (NFT) stage progressed, the expression levels of hsa-miR-101-3p were upregulated specifically in the EC. Furthermore, overexpression of hsa-miR-101-3p in the human neuronal cell line SH-SY5Y caused RAC1 downregulation. These results highlight the utility of our integrated network approach for identifying causal changes leading to neurodegeneration in AD.

genomics↗

bioRxiv2018

Terminal Reproductive Investment, Physiological Trade-offs and Pleiotropic Effects: Their effects produce complex immune/reproductive interactions in the cricket Gryllus texensis

O_LIShould females increase or decrease reproduction when attacked by pathogens? Two hypotheses provide opposite predictions. Terminal reproductive investment theory predicts an increase in reproduction, but hypothesized physiological trade-offs between reproduction and immune function might be expected to produce a decrease. There is evidence for both hypotheses. What determines the choice between the two responses remains unclear. We examine the effect of age on the reproductive response to immune challenge in long-wing females of the Texas field cricket, Gryllus texensis, when fed an ecologically valid (limited) diet.\nC_LIO_LIThe limited diet reduced reproductive output. However, immune challenge had no effect on their reproductive output either in young or middle-aged crickets, which is contrary to either prediction.\nC_LIO_LIFlight muscle maintenance correlated negatively with reproductive output, suggesting a physiological trade-off between flight muscle maintenance and reproduction. Within the long-wing variant there was considerable variability in flight muscle maintenance. This variability may mask physiological trade-offs between immunity and reproduction.\nC_LIO_LIMiddle-aged crickets had higher total phenoloxidase (PO) activity in their hemolymph, compared to young females, which is contrary to the terminal investment theory. Given that PO is involved in both immunity and reproduction, the increased PO may reflect simultaneous investment in both functions.\nC_LIO_LIWe identified four proPO transcripts in a published RNA-seq dataset (transcriptome). Three of the proPO genes were expressed either in the fat body or the ovaries (supporting the hypothesis that PO is bifunctional); however, the two organs expressed different subsets. The possible bifunctionality of PO suggests that it may not be an appropriate immune measure for immune/reproductive trade-offs in some species.\nC_LIO_LIIncreasing age may not cue terminal reproductive investment prior to senescence.\nC_LI

ecology↗

bioRxiv2018

Enhancer variants associated with Alzheimer’s disease affect gene expression via chromatin looping

BackgroundGenome-wide association studies (GWASs) have identified single-nucleotide polymorphisms (SNPs) that may be genetic factors underlying Alzheimers disease (AD). However, how these AD-associated SNPs (AD SNPs) contribute to the pathogenesis of this disease is poorly understood because most of them are located in non-coding regions, such as introns and intergenic regions. Previous studies reported that some disease-associated SNPs affect regulatory elements including enhancers. We hypothesized that non-coding AD SNPs are located in enhancers and affect gene expression levels via chromatin loops.\n\nResultsWe examined enhancer locations that were predicted in 127 human tissues or cell types, including ten brain tissues, and identified chromatin-chromatin interactions by Hi-C experiments. We report the following findings: (1) nearly 30% of non-coding AD SNPs are located in enhancers; (2) expression quantitative trait locus (eQTL) genes affected by non-coding AD SNPs within enhancers are associated with amyloid beta clearance, synaptic transmission, and immune responses; (3) 95% of the AD SNPs located in enhancers co-localize with their eQTL genes in topologically associating domains suggesting that regulation may occur through chromatin higher-order structures; (4) rs1476679 spatially contacts the promoters of eQTL genes via CTCF-CTCF interactions; (5) the effect of other AD SNPs such as rs7364180 is likely to be, at least in part, indirect through regulation of transcription factors that in turn regulate AD associated genes.\n\nConclusionOur results suggest that non-coding AD SNPs may affect the function of enhancers thereby influencing the expression levels of surrounding or distant genes via chromatin loops. This result may explain how some non-coding AD SNPs contribute to AD pathogenesis.

genomics↗

bioRxiv2017

Calling song maturity in two-spotted cricket, Gryllus bimaculatus: its patterns and a possible physical explanation

Males of Gryllus bimaculatus (two-spotted cricket) emit acoustic signals by stridulating two forewings. One of their songs, calling song, plays a role in attracting females to mate, yet the significance of each song trait in attracting conspecific females remains unclear. Among such traits, the relevance of frequency component (i.e. song pitch) has been underestimated, as orthopterans had long been believed incapable of song pitch recognition. However, recent literatures suggested that ears of orthopteran species are capable of frequency recognition as mammalians do. My previous report demonstrated that their calling song recorded from mature adults has constant and pure peak frequency component around 5.7 kHz, further supporting a biological significance of the frequency component. In this study, I tracked its change over time in sexual maturity phase (i.e. from early adult phase). 300 calling songs were recorded over time from a pool of 122 adult crickets, as it required large number of animals because the crickets rarely sing at very early adult stage. A maturation process of calling song was observed, where the peak frequency distributed lower and more variable frequency in early adult phase (e.g. mean peak value was 4.9 kHz on day 3), then it gradually increased and converged to 5.8 kHz with two weeks. The coefficient of variance also decreased over the process, showing minimum around day 20. Also, I found that the young crickets (supposed to emit immature song), emit perfectly tuned calling song with 5.8 kHz peak in helium-substituted (80% Helium and 20% Oxygen) environment. These findings suggest that the robustly regulated frequency of the calling song is acquired during the early-to-mid adult stage, and it may be associated with sexual maturity of males. Also, the helium-substitution experiment suggests that physical resistance from surrounding gas molecules negatively impacts stability of calling songs of young males, implying that a muscle development and/or forewing hardening may help song maturation. This study highlights a biological significance of the frequency component, such that females may adaptively select sexually mature males based on the song trait.

zoology↗

bioRxiv2017

A fast immune priming that confers a complete infection resistance on silkworm (Bombyx mori)

I have previously reported an immune priming in silkworm triggered by peptidoglycans, which is long-lasting but slow-acting. Here I report a faster immune priming, which can be triggered by an injection of gelatin or collagen, that confers a complete infection resistance to Gram-negative bacteria on silkworms. Gelatin-injected silkworms showed 100% viability in a lethal dose of a Gram-negative bacterial infection within two hours after the gelatin injection. Injection of collagen showed a similar effect. Whereas, an injection of non-gelatin protein (bovine serum albumin) solution did not induce such reaction. These results suggest that the silkworm possesses a fast and gelatin-inducible pathway that confers infection resistance to Gram-negative bacteria, which may act as a front-line defense. This finding highlights the potency of gelatin as a tool for investigating the primed immune responses in insect species.

immunology↗