Biology subjects

Tsuchida, K.

Publications and source records attributed to Tsuchida, K..

8 recordsLinked to original sources

bioRxiv2026

Repeated intermittent clenbuterol administration induces exposure history-dependent proteomic remodeling in mouse skeletal muscle

Clenbuterol (CB), a {beta}2-adrenergic receptor agonist, is known to increase skeletal muscle mass. However, because its effects are influenced by treatment duration, dose, and {beta}-adrenergic receptor responsiveness, it remains unclear how extended intermittent CB exposure alters the skeletal muscle proteome. In this study, we established a repeated intermittent CB administration model in mice and compared the effects of single and repeated CB exposure on plantaris muscle using label-free quantitative proteomics. Repeated CB exposure produced a more evident muscle-weight response than single exposure under the same long-term experimental timeline. Proteomic profiling revealed that repeated CB exposure was not a simple reproduction or amplification of the single CB response, but was associated with distinct changes in sarcomere-associated, tissue-remodeling-related, membrane-trafficking-related, and metabolic proteins. Integrated response-class analysis further identified multiple patterns of protein regulation, including shared CB-responsive proteins and proteins preferentially identified as changed under repeated CB conditions. Immunoblotting validated representative proteins from these response classes: Klhl40 and Napa as shared CB-responsive proteins, Galectin-3 as a strongly CB-responsive remodeling-related protein, and Rab18 as a protein change more clearly detected under repeated CB conditions. Together, these findings indicate that repeated intermittent CB exposure induces exposure history-dependent proteomic remodeling in skeletal muscle. This study provides a resource for understanding the molecular consequences of repeated CB exposure and for future mechanistic studies of {beta}2-adrenergic stimulation-induced skeletal muscle adaptation.

molecular biology↗

bioRxiv2026

Cardiac-Detargeted MyoAAV Enables Systemic Nrl-Mediated Fast Myofiber Remodeling and Hypertrophy Across Multiple Skeletal Muscles

Inducing fast myofiber programs offers therapeutic potential for skeletal muscle disorders such as sarcopenia, where fast myofibers are preferentially lost. Engineered muscle-specific AAV (MyoAAV) vectors enable efficient transduction of skeletal muscles after systemic administration; however, cardiac transgene expression limits applications requiring skeletal muscle-selective delivery. We generated modified MyoAAV vectors by incorporating cardiac-specific miR-208a target sequences into the transgene 3'UTR. This design markedly suppressed cardiac expression while preserving skeletal muscle output, with target-site variation enabling tunable trade-offs between cardiac detargeting and skeletal muscle expression levels. We validated this platform using neural retina leucine zipper (Nrl), a large Maf transcription factor regulating type IIb myofiber identity. Systemic delivery of conventional MyoAAV-Nrl caused severe cardiac hypertrophy and uniform lethality within one month. Conversely, incorporating miR-208a target sequences prevented detectable hypertrophy and eliminated mortality during the experimental observation period. This modification significantly reduced cardiac Nrl expression while maintaining skeletal muscle levels, successfully promoting type IIb myofiber formation and hypertrophy across multiple skeletal muscles. These findings demonstrate that miR-208a-mediated cardiac detargeting combined with MyoAAV-Nrl enables safe systemic induction of fast myofiber remodeling and hypertrophy, establishing a platform for gene therapies targeting skeletal muscle disorders associated with fast myofiber loss.

cell biology↗

bioRxiv2025

Therapeutic Knock-in Genome Editing Using Single AAV Vectors in Mouse Models of Inherited Liver Disease

Gene knock-in therapy has the potential to cure inherited liver diseases but is limited by low efficiency and delivery complexity. Here, we developed a single adeno-associated virus (AAV) vector system comprising a compact CRISPR effector, enAsCas12f, a guide RNA, and a donor template to enable therapeutic genome editing via non-homologous end joining (NHEJ). We targeted the system to the murine Alb locus and applied it to mouse models of hemophilia B, protein C (PC) deficiency, and ornithine transcarbamylase (OTC) deficiency. NHEJ-mediated knock-in showed higher efficiency than homology-directed repair, with successful therapeutic gene insertion in both neonatal and adult mice. The strategy restored plasma factor IX activity in hemophilia B (F9-/-) mice, prolonged survival of PC-deficient (Proc-/-) mice, and prevented hyperammonemia and weight loss in OTC-deficient (Otcspf-ash) mice upon high protein challenge. Importantly, gene integration was restricted to the liver, with no evidence of germline transmission. This compact, all-in-one AAV knock-in platform simplifies vector production, enables efficient delivery, and achieves reliable transgene expression in vivo. Our findings highlight the potential of liver-targeted knock-in genome editing as a transplant-independent treatment for neonatal-onset metabolic diseases, offering a clinically feasible path towards curative gene therapies for a wide range of monogenic liver disorders.

bioengineering↗

bioRxiv2024

High microsatellite but no mitochondrial DNA variation in an invasive Japanese mainland population of the parasitoid wasp Melittobia sosui

Theory predicts that bottleneck events reduce genetic diversity in invasive populations. The parasitoid wasp Melittobia sosui was only identified in the subtropical area of Japanese south islands and Taiwan, but recently found also in the temperate area of the Japanese mainland. The species may expand their distribution northward due to factors such as recent global warming. We investigated population genetics in both the native and invasive areas using mitochondrial and nuclear microsatellite DNA. As expected, there was mitochondrial variation in the native area, but not in the invasive area, which only had one haplotype. However, the two areas had a similar level of microsatellite variation, with averagely 43% and 38% alleles uniquely found in the native and invasive populations, respectively. The difference of genetic variation between mitochondrial and microsatellite DNA in the invasive populations may be explained by the faster mutation rate of microsatellites, as well as the population structure of Melittobia, in which subdivided small inbreeding lineages may facilitate the accumulation of mutations. The high proportion of private alleles suggests that the mainland population diverged from the native populations at least 100 years ago, ruling out the possibility that the mainland population was established recently. Instead, the present study suggests that M. sosui might have already existed in the mainland but with a low frequency, or the mainland population was derived from a third population which diverged from the native populations over 100 years ago.

ecology↗

bioRxiv2024

Dual expansion routes likely underlie the present-day population structure in a Parnassius butterfly across the Japanese Archipelago

The Japanese Archipelago, comprising a series of isolated yet interconnected islands, had been geographically separated from the Eurasian continent. The linear topography presents a unique biogeographic context for dispersing organisms from the continent. In this study, we utilized mitochondrial DNA (mtDNA) and single nucleotide polymorphism (SNP) variation to elucidate the dispersal history of the Japanese clouded butterfly Parnassius glacialis across the Japanese Archipelago, including North Island (Hokkaido), Main Island (Honshu) and Shikoku Island. Our analysis of mtDNA (COI, COII) at 1192 base pairs (bps) revealed 49 haplotypes and identified three distinct haplotype groups in the network. These groups correspond geographically to East Japan, West Japan, and Chugoku-Shikoku. The Chugoku-Shikoku group is the most ancient lineage. Interestingly, the Chugoku-Shikoku lineage showed a closer network connection to the East Japan lineage than the geographically proximate West Japan lineage. Divergence time estimates suggest that the Chugoku-Shikoku lineage diverged from the continental P. glacialis approximately 3.05 million years ago (MYA). Subsequently, from the Chugoku-Shikoku lineage, the East Japan and West Japan lineages diverged around 1.05 MYA, with the subsequent divergence between the East and West Japan lineages occurring at approximately 0.62 MYA. Based on the 3067 SNP genotypes, population structure analysis revealed five distinct genetic structures within the Japanese Archipelago, indicating geographical differentiation. From the analyses by mtDNA and SNP variations, four primary genetic barriers were identified: between Hokkaido and Honshu, between East and Central Japan, within the Kansai region, and within the Chugoku region. The former three lines corresponded to the Blakiston Line, the Itoigawa-Shizuoka Tectonic Line, and Lake Biwa, respectively. These findings suggest that P. glacialis diverged from the continental P. glacialis and expanded its range across the Japanese Archipelago via the North and South routes, establishing its current distribution.

molecular biology↗

bioRxiv2021

Multiple roles of heterogeneous nuclear ribonucleoprotein K during skeletal muscle cell differentiation

RNA-binding proteins (RBPs) regulate cell physiology via the formation of ribonucleic-protein complexes with coding and non-coding RNAs. RBPs have multiple functions in the same cells; however, the precise mechanism through which their pleiotropic functions are determined remains unknown. In this study, we revealed the multiple inhibitory functions of heterogeneous nuclear ribonucleoprotein K (hnRNPK) for myogenic differentiation. We first identified hnRNPK as a lncRNA Myoparr binding protein. Gain- and loss-of-function experiments showed that hnRNPK repressed the expression of myogenin at the transcriptional level. The hnRNPK-binding region of Myoparr was required to repress myogenin expression. Moreover, hnRNPK repressed the expression of a set of genes coding for aminoacyl-tRNA synthetases in a Myoparr-independent manner. Mechanistically, hnRNPK regulated the eIF2/Atf4 pathway, one branch of the intrinsic pathways of the endoplasmic reticulum sensors, in differentiating myoblasts. Thus, our findings demonstrate that hnRNPK plays lncRNA-associated and - independent multiple roles during myogenic differentiation, indicating that the analysis of lncRNA-binding proteins will be useful for elucidating both the physiological functions of lncRNAs and the multiple functions of RBPs.

cell biology↗

bioRxiv2020

Solving the sex ratio scandal in Melittobia wasps

The scandalous sex ratio behaviour of Melittobia wasps has long posed one of the greatest problems for the field of sex allocation. In contrast to the predictions of theory, and the behaviour of numerous other organisms, laboratory experiments have found that Melittobia females do not produce less female-biased offspring sex ratios when more females lay eggs on a patch. We resolve this scandal, by showing that, in nature, females of M. australica have sophisticated sex ratio behaviour, where their strategy also depends upon whether they have dispersed from the patch where they emerged. When females have not dispersed, they will be laying eggs with close relatives, which keeps local mate competition high, even with multiple females, and so they are selected to produce consistently female-biased sex ratios. Laboratory experiments mimic these conditions. In contrast, when females disperse, they will be interacting with non-relatives, and so they adjust their sex ratio depending upon the number of females laying eggs. Consequently, females appear to use dispersal status as an indirect cue of relatedness, and whether they should adjust their sex ratio in response to the number of females laying eggs on the patch.

evolutionary biology↗

bioRxiv2019

Polyandry and Paternity influence disease resistance in a eusocial wasp - support for the pathogen parasite hypothesis

Polyandry (multiple mating by females) is a central challenge for understanding the evolution of eusociality. Several hypotheses have been proposed to explain its observed benefits in eusocial Hymenoptera, and one, the parasite-pathogen hypothesis, proposes that high genotypic variance among workers for disease resistance prevents catastrophic colony collapse. We tested the parasite-pathogen hypothesis in the polyandrous wasp Vespula shidai. We infected isolated workers with the entomopathogenic fungus Beauveria bassiana and quantified their survival in the laboratory. Additionally, we conducted paternity analysis of the workers using nine microsatellite loci to investigate the relationship between survival and matriline/patriline of the workers. As predicted by the parasite-pathogen hypothesis, nestmate workers of different patrilines showed differential resistance to B. bassiana. We also demonstrated variations in virulence among strains of B. bassiana. Our results are the first to directly support the parasite-pathogen hypothesis in eusocial wasps and suggest that similar evolutionary pressures drove the convergent origin and maintenance of polyandry in ants, bees, and wasps.

animal behavior and cognition↗