Biology subjects

Miyamoto, Y.

Publications and source records attributed to Miyamoto, Y..

15 recordsLinked to original sources

bioRxiv2026

A novel recombinant BCG vaccine using a mycobacteriophage promoter shows improved protection against tuberculosis

The limited efficacy of conventional bacillus Calmette-Guerin (BCG) vaccination against adult pulmonary tuberculosis highlights the need for improved vaccine strategies. Genome integration can overcome the plasmid instability of recombinant BCG, but we hypothesized that this approach would be constrained by reduced transcriptional activity of conventional promoters when present as a single genomic copy. Here we identified P79, a mycobacteriophage-derived promoter that sustains high-level expression from a single genomic locus and used it to construct a genome-integrated recombinant BCG, termed LRC-BCG. LRC-BCG secretes the HSP70-MMPII fusion antigen flanked by the PEST sequences, maintained stable antigen expression over serial passages, and induced strong activation of macrophages and dendritic cells in vitro. In vivo, LRC-BCG inhibited Mycobacterium tuberculosis multiplication more effectively than conventional BCG, even at low doses. These findings demonstrate the feasibility of using P79 for stable genome-integrated recombinant BCG and support LRC-BCG as a promising tuberculosis vaccine candidate.

microbiology↗

bioRxiv2026

Intramuscular adrenaline administration does not improve a survival period on rats with crush syndrome despite short-term hemodynamic support and renal protection

Crush syndrome (CS) is a serious medical condition characterized by damage to the muscle cells due to pressure and is associated with high mortality, even in patients receiving fluid therapy. We focused on adrenaline (Adr), a standard medication administered by medical teams dispatched during disasters. Adr is readily available for use in disaster scenarios owing to its inclusion in standard emergency kits. The effectiveness of Adr in the treatment of CS remains a subject of ongoing debate. This study aimed to evaluate the impact of Adr on acute complications, such as heart failure, shock, and renal failure, and explore whether its influence on inflammatory pathways is correlated with improved survival in rats with CS. The CS model involved subjecting anesthetized rats to bilateral hindlimb compression using a rubber tourniquet for 5 h. Subsequently, the rats were randomly divided into eight groups. Under continuous monitoring and recording of the arterial blood pressure, blood and tissue samples were collected for biochemical analyses at designated time points before and after reperfusion. The survival rate, vital signs, and blood gas parameters were higher in the CS group than in the sham group. They were improved in the Adr-treated group (0.01 or 0.01 mg/kg), which was not significantly different from that in the CS group, despite the improvement in shock and kidney dysfunction. In conclusion, intramuscular Adr provides immediate hemodynamic stabilization and renal protection during the early stages of CS. However, its use requires careful dose titration; low doses may promote the systemic release of lethal toxins, whereas high doses may worsen metabolic acidosis. These findings highlight the importance of combining Adr with other therapies, such as fluid resuscitation, to manage systemic toxemia inherent in CS.

pharmacology and toxicology↗

bioRxiv2025

Gamete release in Ciona robusta: roles of gonadotropin-releasing hormone and the photoreception system

Gamete release, an essential event for animals, is regulated by various factors including environmental stimuli, neural circuit, and endocrine molecules. In this study, we investigated the mechanisms regulating gamete release in the ascidian Ciona robusta, part of the sister group of vertebrates. Ciona is a hermaphrodite, releasing sperm first from orange-pigmented organ (OPO) at the end of the spermiduct, followed by eggs from the oviduct beneath the spermiduct knob-like structure. In this study, behavioral and morphological analyses revealed that this sequential release occurs because the knob-like structure compresses the oviduct opening at the onset of gamete release. Observations of transgenic Ciona expressing Kaede under the gonadotropin-releasing hormone 2 (Gnrh2) promoter showed that Gnrh2-expressing neurons and fibers accumulate in the epithelium around the spermiduct openings. In contrast, Gnrh1-expressing neurons are localized in the cerebral ganglion and project toward the ovary, suggesting distinct roles of GnRH1 and GnRH2 in reproductive regulation. RNA-seq and real-time PCR analyses revealed that Opsin2, Opsin3, beta-carotene-15,15-monooxygenase (Bco), and several ion channel genes are specifically expressed in the OPO, along with Gnrh2. In situ hybridization showed that these genes are localized in the innermost OPO layer, suggesting that the OPO functions as a photoreceptive organ. Although, Gnrh2 expression has been considered low in adult Ciona, our study revealed strong OPO-specific expression with photoreceptive genes. Collectively, these findings suggest that GnRH2 plays a central role in gamete release regulation, potentially in coordination with the photoreceptive system, while GnRH1 may regulate ovarian functions. These results indicate that C. robusta employs distinct GnRH systems in a tissue-specific manner to regulate reproduction.

evolutionary biology↗

bioRxiv2024

Importin α4 deficiency induces psychiatric disorder-related behavioral deficits and neuroinflammation in mice.

Importin 4, which is encoded by the Kpna4 gene, is a well characterized nuclear-cytoplasmic transport factor known to mediate transport of transcription factors including NF-{kappa}B. Here, we report that Kpna4 knock-out (KO) mice exhibit psychiatric disorder-related behavioral abnormalities such as anxiety-related behaviors, deceased social interaction and sensorimotor gating deficits. Contrary to a previous study predicting attenuated NF-{kappa}B activity as a result of Kpna4 deficiency, we observed a significant increase in expression levels of NF-{kappa}B genes and pro-inflammatory cytokines such as TNF, Il1{beta} or Il-6 in the Prefrontal Cortex or Basolateral Amygdala of the KO mice. Moreover, examination of inflammatory responses in primary cells revealed that Kpna4 deficient cells have an increased inflammatory response, which was rescued by addition of not only full-length, but also a nuclear transport deficient truncation mutant of importin 4, suggesting contribution of its non-transport functions. Furthermore, RNAseq of sorted adult Microglia and Astrocytes and subsequent transcription factor analysis suggested increases in Polycomb repressor complex 2 (PRC2) activity in Kpna4 KO cells. Taken together, importin 4 deficiency induces psychiatric disorder-related behavioral deficits in mice, along with an increased inflammatory response and possible alteration of PRC2 activity in glial cells.

neuroscience↗

bioRxiv2023

Importin-7-dependent nuclear localization of the Flavivirus core protein is required for infectious virus production.

Flaviviridae is a family of positive-stranded RNA viruses, including human pathogens such as Japanese encephalitis virus (JEV), dengue virus (DENV), zika virus (ZIKV), and West Nile virus (WNV). Nuclear localization of the viral core protein is conserved among Flaviviridae, and this feature may be targeted for developing broad-ranging anti-flavivirus drugs. However, the mechanism of core protein translocation to the nucleus and the importance of nuclear localization in the viral life cycle remain unknown. We aimed to identify the molecular mechanism underlying core protein nuclear translocation. We identified importin-7 (IPO7), an importin-{beta} family protein, as a nuclear carrier for Flaviviridae core proteins. Nuclear import assays revealed that core protein was transported into the nucleus via IPO7, whereas IPO7 deletion by CRISPR/Cas9 impaired their nuclear localization. To understand the importance of core protein nuclear localization, we evaluated the production of infectious virus or single-round-infectious-particles in wild-type or IPO7-deficient cells; both processes were significantly impaired in IPO7-deficient cells, whereas intracellular infectious virus levels were equivalent in wild-type and IPO7-deficient cells. These data suggested that IPO7-mediated nuclear localization of core proteins plays a role in the release of infectious virus particles of flaviviruses.

microbiology↗

bioRxiv2023

Structural elucidation of recombinant Trichomonas vaginalis 20S proteasome bound to covalent inhibitors

Proteasomes are essential for protein homeostasis in mammalian cells1-4 and in protozoan parasites such as Trichomonas vaginalis (Tv).5 Tv and other protozoan 20S proteasomes have been validated as druggable targets.6-8 However, in the case of Tv 20S proteasome (Tv20S), biochemical and structural studies were impeded by low yields and purity of the native proteasome. We successfully made recombinant Tv20S by expressing all seven and seven {beta} subunits together with the Ump-1 chaperone in insect cells. We isolated recombinant proteasome and showed that it was biochemically indistinguishable from the native enzyme. We confirmed that the recombinant Tv20S is inhibited by the natural product marizomib (MZB)9 and the recently developed peptide inhibitor carmaphycin-17 (CP-17)8,10. Specifically, MZB binds to the {beta}1, {beta}2 and {beta}5 subunits, while CP-17 binds the {beta}2 and {beta}5 subunits. Next, we obtained cryo-EM structures of Tv20S in complex with these covalent inhibitors at 2.8[A] resolution. The structures revealed the overall fold of the Tv20S and the binding mode of MZB and CP-17. Our work explains the low specificity of MZB and higher specificity of CP-17 towards Tv20S as compared to human proteasome and provides the platform for the development of Tv20S inhibitors for treatment of trichomoniasis.

biophysics↗

bioRxiv2023

Importin α as a molecular marker for investigating the microenvironment of micronuclei

Micronuclei (MN) are membrane-enclosed chromatin bodies and hallmarks of genome instability. Here, we report that importin , a key nuclear transport factor, is highly concentrated in a distinct subset of MN in cultured human cancer cells. This selective localization is not governed by classical nuclear transport pathways. Live-cell photobleaching revealed remarkably reduced mobility of importin between MN and cytoplasm. In addition, the subset of importin -positive MN exhibited collapsed nuclear envelopes and compromised barrier functions. Importin was also enriched in euchromatin regions, where it colocalized with chromatin-regulating molecules. Importantly, importin and DNA repair/sensing molecules such as RAD51, RPA2, and cGAS showed mutually exclusive localization in MN, indicating that MN comprise distinct internal environments. These findings identify importin as a molecular marker of the restricted MN state, representing a previously unrecognized microenvironment distinct from subsets characterized by conventional molecular markers of disrupted MN. This framework provides new insights into how MN heterogeneity underlies genome instability and immune evasion during cancer progression. Summary statementAccumulation of importin in micronuclei, followed by modulation of the microenvironment of the micronuclei, suggests a non-canonical function of importin in genomic instability and cancer development.

cell biology↗

bioRxiv2023

Development of subunit selective substrates for Trichomonas vaginalis proteasome

The protozoan parasite, Trichomonas vaginalis (Tv) causes trichomoniasis, the most common, non-viral, sexually transmitted infection in the world. Only two closely related drugs are approved for its treatment. The accelerating emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health. There is an urgent need for novel effective anti-parasitic compounds. The proteasome is a critical enzyme for T. vaginalis survival and was validated as a drug target to treat trichomoniasis. However, to develop potent inhibitors of the T. vaginalis proteasome, it is essential that we understand which subunits should be targeted. Previously, we identified two fluorogenic substrates that were cleaved by T. vaginalis proteasome, however after isolating the enzyme complex and performing an in-depth substrate specificity study, we have now designed three fluorogenic reporter substrates that are each specific for one catalytic subunit. We screened a library of peptide epoxyketone inhibitors against the live parasite and evaluated which subunits are targeted by the top hits. Together we show that targeting of the {beta}5 subunit of T. vaginalis is sufficient to kill the parasite, however, targeting of {beta}5 plus either {beta}1 or {beta}2 results in improved potency.

biochemistry↗

bioRxiv2022

Phase-separated nuclear bodies of nucleoporin fusions, SET-NUP214 and NUP98-HOXA9, promote condensation of MLL1 and CRM1 to activate target genes

Nucleoporins NUP98 and NUP214 form chimeric fusion proteins that assemble into phase-separated nuclear bodies. However, the function and physiological significance of these nuclear bodies remain largely unknown. Previously, we reported that both NUP98-HOXA9 and SET-NUP214 are recruited to HOX cluster regions via chromatin-bound CRM1, a nuclear export receptor (Oka et al., 2019). Here, we show that these nuclear bodies promote the condensation of mixed lineage leukemia 1 (MLL1), a histone methyltransferase which is essential for the maintenance of HOX gene expression. Our analysis revealed that SET-NUP214 and CRM1 robustly associate with MLL1 to form nuclear bodies and are colocalized on chromatin. We also showed that MLL1 and CRM1 are recruited to the nuclear bodies of NUP98-HOXA9 and that the NUP98-HOXA9/CRM1/MLL1 complex accumulates on its target gene loci, including HOX clusters and MEIS1. These phenomena were not observed in phase-separation-deficient mutants or non-DNA-binding mutants of NUP98-HOXA9. Collectively, these results show that both phase separation and proper targeting of nucleoporin fusions to specific sites could enhance the activation of a wide range of target genes by promoting the condensation of MLL1 and CRM1.

cell biology↗

bioRxiv2021

Establishment of a stable SARS-CoV-2 replicon system for application in high-throughput screening.

Experiments with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are limited by the need for biosafety level 3 (BSL3) conditions. A SARS-CoV-2 replicon system rather than an in vitro infection system is suitable for antiviral screening since it can be handled under BSL2 conditions and does not produce infectious particles. However, the reported replicon systems are cumbersome because of the need for transient transfection in each assay. In this study, we constructed a bacterial artificial chromosome vector (the replicon-BAC vector) including the SARS-CoV-2 replicon and a fusion gene encoding Renilla luciferase and neomycin phosphotransferase II, examined the antiviral effects of several known compounds, and then established a cell line stably harboring the replicon-BAC vector. Several cell lines transiently transfected with the replicon-BAC vector produced subgenomic replicon RNAs (sgRNAs) and viral proteins, and exhibited luciferase activity. In the transient replicon system, treatment with remdesivir or interferon-{beta} but not with camostat or favipiravir suppressed the production of viral agents and luciferase, indicating that luciferase activity corresponds to viral replication. VeroE6/Rep3, a stable replicon cell line based on VeroE6 cells, was successfully established and continuously produced viral proteins, sgRNAs and luciferase, and their production was suppressed by treatment with remdesivir or interferon-{beta}. Molnupiravir, a novel coronavirus RdRp inhibitor, inhibited viral replication more potently in VeroE6/Rep3 cells than in VeroE6-based transient replicon cells. In summary, our stable replicon system will be a powerful tool for the identification of SARS-CoV-2 antivirals through high-throughput screening.

microbiology↗

bioRxiv2021

Intestinal Transgene Delivery with Native E. coli Chassis Allows Persistent Physiological Changes

Live bacterial therapeutics (LBT) could reverse disease by engrafting in the gut and providing persistent beneficial functions in the host. However, attempts to functionally manipulate the gut microbiome of conventionally-raised (CR) hosts have been unsuccessful, because engineered microbial organisms (i.e., chassis) cannot colonize the hostile luminal environment. In this proof-of-concept study, we use native bacteria as chassis for transgene delivery to impact CR host physiology. Native Escherichia coli isolated from stool cultures of CR mice were modified to express functional bacterial (bile salt hydrolase) and eukaryotic (Interleukin-10) genes. Reintroduction of these strains induces perpetual engraftment in the intestine. In addition, engineered native E. coli can induce functional changes that affect host physiology and reverse pathology in CR hosts months after administration. Thus, using native bacteria as chassis to "knock-in" specific functions allows mechanistic studies of specific microbial activities in the microbiome of CR hosts, and enables LBT with curative intent.

synthetic biology↗

bioRxiv2021

A stress-reduced passaging technique improves the viability of human pluripotent cells

Xeno-free culture systems have expanded the clinical and industrial application of human pluripotent stem cells (PSCs). However, yet some problems, such as the reproducibility among the experiments, remain. Here we describe an improved method for the subculture of human PSCs. The revised method significantly enhanced the viability of human PSCs by lowering DNA damage and apoptosis, resulting in more efficient and reproducible downstream applications such as gene editing, gene delivery, and directed differentiation. Furthermore, the method did not alter PSC characteristics after long-term culture and attenuated the growth advantage of abnormal subpopulations. This robust passaging method minimizes experimental error and reduces the rate of PSCs failing quality control of human PSC research and application. HighlightsO_LIThe revised passaging method significantly increases the viability of human PSCs. C_LIO_LIThe method triggers less DNA damage and apoptosis signals compared to the conventional method. C_LIO_LIThe stress-reduced method improves the results of downstream applications. C_LIO_LIThe method does not alter PSC characters and attenuates the overgrowth of abnormal subpopulations. C_LI

cell biology↗

bioRxiv2021

Hematopoietic stem and progenitor cells integrate Bacteroides-derived innate immune signals to promote gut tissue repair

Bone marrow (BM)-resident hematopoietic stem and progenitor cells (HSPCs) are often activated by bacterial insults to replenish the host hemato-immune system, but how they integrate the associated tissue damage signals to initiate distal tissue repair is largely unknown. Here, we showed that acute gut inflammation expands HSPCs in the BM through GM-CSFR activation, and directs them to inflamed mesenteric lymph nodes for further differentiation into myeloid cells specialized in gut tissue repair. We also identified that this process is exclusively mediated by Bacteroides, a commensal gram-negative bacteria, that activates innate immune signaling. In contrast, chronic gut inflammation reduces HSC potential for hematopoietic reconstitution and immune response against infection. Similarly, microbial signals contribute to aging-associated HSPC expansion. These findings establish a cross-organ communication that promotes tissue regeneration, but if sustained, impairs tissue homeostasis that may be relevant to aging and chronic disorders. SummaryThe infiltrating microbiota Bacteroides upon acute colitis directed MPP migration from the BM to the MLN for their subsequent expansion and differentiation into tissue-repairing Ly6C+/G+ cells, whereas chronic colitis impairs HSC functionality similarly as aging.

pathology↗

bioRxiv2021

SARS-CoV-2 ORF6 disturbs nucleocytoplasmic trafficking to advance the viral replication

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the coronavirus disease 2019 pandemic. ORF6 is known to antagonize the interferon signaling by inhibiting the nuclear translocation of STAT1. Here we show that ORF6 acts as a virulence factor through two distinct strategies. First, ORF6 directly interacts with STAT1 in an IFN-independent manner to inhibit its nuclear translocation. Second, ORF6 directly binds to importin 1, which is a nuclear transport factor encoded by KPNA2, leading to a significant suppression of importin 1-mediated nuclear transport. Furthermore, we found that KPNA2 knockout enhances the viral replication, suggesting that importin 1 suppresses the viral propagation. Additionally, the analyses of gene expression data revealed that importin 1 levels decreased significantly in the lungs of older individuals. Taken together, SARS-CoV-2 ORF6 disrupts the nucleocytoplasmic trafficking to accelerate the viral replication, resulting in the disease progression, especially in older individuals.

microbiology↗

bioRxiv2019

Relationship between ocean area and incidence of anthropogenic debris ingested by longnose lancetfish (Alepisaurus ferox)

Longnose lancetfish (Alepisaurus ferox) may has been studied as an indicator of marine pollution caused by marine litter. The objectives of this study were to determine the difference in frequency of occurrence of plastics ingested by longnose lancetfish in different ocean area. In this study, we compared the incidence and characteristics of anthropogenic debris in the stomachs of longnose lancetfish. We examined 91 longnose lancetfish caught by pelagic longline fishing in Sagami Bay, the North Pacific Ocean, approximately 200 km south of Shikoku, and in the Indian Ocean. Broken down by ocean area, the incidence of anthropogenic debris ingestion was highest in Sagami Bay (23 of 34 specimens, 68%), followed by the North Pacific Ocean (1 of 9, 11%), and the Indian Ocean (8 of 48, 17%). The frequency of occurrence increased in area close to the sphere of human habitation. The anthropogenic debris collected in this study were more than 70% classified as plastic sheeting. Stomach content analysis revealed that more than 90% of the plastic fragments were composed of PP and PE, which have specific gravities that are less than that of seawater. The results of this study show that some of the plastics flowing from the land into the sea are spreading through under the water surface of the ocean.

ecology↗