Biology subjects

Mukai, H.

Publications and source records attributed to Mukai, H..

10 recordsLinked to original sources

bioRxiv2026

Repeated swim exposure and PKN1a knockout enhance group I mGluR-dependent excitability associated with reduced EAAT3 expression in mouse dentate granule cells

Stress-related experiences alter glutamatergic signaling and neuronal excitability, but the mechanisms that couple experience to dentate granule cell function remain incompletely understood. Here, we examined how protein kinase N1a (PKN1a), a protein kinase C-like serine/threonine kinase, and repeated swim exposure regulate mouse hippocampal dentate granule cell excitability, with a focus on the neuronal glutamate transporter excitatory amino acid transporter 3 (EAAT3) and group I metabotropic glutamate receptors (mGluRs). Five days of repeated swim exposure increased spike firing in mature dentate granule cells from wild-type mice. PKN1a knockout produced a similar increase, and repeated swim did not further enhance firing in knockout mice. The enhanced firing observed after repeated swim exposure and in PKN1a knockout mice was reduced by co-application of an mGluR1 antagonist (LY367385) and an mGluR5 antagonist (MPEP). Inhibition of glutamate transporters with DL-TBOA increased granule cell firing in control wild-type mice but did not further increase firing in repeated-swim wild-type or PKN1a knockout mice, suggesting occlusion of transporter-dependent regulation of excitability. Repeated swim exposure and PKN1a knockout also reduced total and surface expression of EAAT3 in the hippocampus, whereas expression of the glial glutamate transporter EAAT2 was not significantly altered. Finally, PKN1a knockout and repeated swim exposure reduced anxiety-related behavior in the elevated plus maze test. Thus, PKN1a-dependent regulation of EAAT3 may restrain group I mGluR-dependent excitability in dentate granule cells, whereas repeated swim exposure and PKN1a knockout shift this system toward a lower-EAAT3, higher-excitability state accompanied by reduced anxiety-related behavior.

neuroscience↗

bioRxiv2026

Large-scale dimensional behavioral profiling dissociates fear memory from locomotor confounds in mice: The necessity of baseline-normalized metrics

Fear conditioning is widely used to assess associative memory in mice, yet percent freezing conflates memory with baseline locomotor and anxiety-related traits. A systematic survey of recent studies (2020-2025) found that fewer than 1% statistically integrate locomotor activity into freezing analyses. Here, we address this gap using a large-scale dataset of >10,000 mice across >160 comparisons, including genetic mutations, pharmacological interventions and aging, tested in 15 standardized behavioral paradigms. Conventional freezing scores covaried strongly with general locomotor activity, obscuring memory-related phenotypes. Multiple factor analysis identified two principal behavioral dimensions, locomotor activity and learning/memory: conventional freezing aligned with the locomotor dimension, whereas freezing subtraction and the activity suppression ratio mapped onto the memory dimension and improved detection of synaptic plasticity phenotypes. These analyses show that baseline locomotor normalization is essential for interpreting fear conditioning as a memory assay and provide an open framework for selecting and reporting locomotor-normalized metrics.

neuroscience↗

bioRxiv2025

The relationship between passive ankle joint stiffness and the stiffness of muscles, nerve, and tendon

Passive joint stiffness reflects the stiffness of various soft tissues across a joint. However, no previous studies have investigated the relationship between passive joint stiffness and muscle, nerve, and tendon stiffness. This study aimed to clarify whether passive ankle joint stiffness is related to stiffness in the triceps surae muscles, tibial nerve, and Achilles tendon. Thirty-eight healthy adults participated in the study. The passive ankle joint stiffness (slope of angle-passive torque curve) and shear wave velocities, which indicate soft tissue stiffness, of the triceps surae muscles and tibial nerve were measured at 5{degrees} of ankle plantarflexion and 5{degrees}, 15{degrees}, and 25{degrees} of ankle dorsiflexion. The shear wave velocity of the Achilles tendon was measured only at 5{degrees} of plantarflexion. A multiple regression model (forced-entry method) was constructed at each angle, specifying the shear wave velocities as the independent variables and passive joint stiffness as the dependent variable. At 5{degrees} of plantarflexion, no shear wave velocities were significantly related to passive joint stiffness (all p [≥] 0.05). At 5{degrees} and 15{degrees} of dorsiflexion, only the shear wave velocities of the tibial nerve were significantly positively related to passive joint stiffness (p = 0.024 and 0.008, respectively). At 25{degrees} of dorsiflexion, the shear wave velocities of the lateral gastrocnemius muscle and tibial nerve were significantly positively related to passive joint stiffness (p = 0.002 and 0.001, respectively). It can be concluded that both triceps surae muscles stiffness and tibial nerve stiffness are related to passive ankle joint stiffness.

biophysics↗

bioRxiv2025

ATP-independent phosphate recycling on AGC kinase activation loops induced by alkali metal ions

Changes in extracellular Na and K concentrations have traditionally been considered to influence intracellular signal transduction through alterations in cell volume or membrane potential. However, whether intracellular ion concentration changes directly regulate signaling molecules, independent of these conventional pathways, remains largely unexplored. In this study, we demonstrate that even in the absence of cellular membranes, an increase in Na or K concentration rapidly reduces activation-loop phosphorylation of multiple AGC kinases, including PKN, PKC{zeta}/{lambda}, and p70 S6 kinase. When ion concentrations were reduced, the activation-loop phosphorylation, which had initially decreased, recovered within a short period. Notably, this recovery occurred in the absence of PDK1, a known kinase responsible for the phosphorylation of these activation loops, and did not require ATP or Mg{superscript 2} in lysate assays. 3{superscript 2}P tracing experiments revealed a novel reacquisition of phosphate group mechanism, in which phosphate groups transiently dissociate from the activation loops under high Na or K conditions and are subsequently re-incorporated into the activation loops when ion concentrations are reduced. These findings indicate that elevated Na or K concentrations directly and rapidly reduce the activity of multiple AGC kinases, and that the activity rapidly recovers upon reduction in ion concentrations, through an unconventional phosphate transfer mechanism distinct from canonical protein phosphorylation reactions. Our study suggests the existence of a robust phosphorylation homeostasis mechanism independent of conventional kinase-phosphatase systems, providing new insights into signaling pathways regulated by intracellular Na/K ion dynamics.

biochemistry↗

bioRxiv2025

Age-related decline of synaptic plasticity is regulated by neuro-androgen and neuro-estrogen in normal aging of hippocampus

We revealed a good relationship between age-dependent decrease in the hippocampal dendritic spine density and age-dependent decrease in hippocampal androgen and estrogen levels with normal aging of male rats. Approximately 25% decrease in the spine density was observed in hippocampal CA1 region by going from 3 month-old (3m; young adult) to 24 month-old (24m; aged). We found a significant age-induced decrease in hippocampal neuro-androgen levels by going from 3m to 24 m using mass-spectrometric analysis. The hippocampal levels of testosterone (T) and dihydrotestosterone (DHT) dramatically decreased from 17 nM T and 7 nM DHT at 3 m to 17/100 nM T and 7/15 nM DHT at 24 m. On the other hand, hippocampal estradiol (E2) was moderately decreased with aging, from 8 nM at 3 m to 2 nM at 24m. Comprehensive analysis of mRNAs of hippocampal steroidogenic enzymes and receptors showed an age-dependent decrease in their expression levels by approximately 50% (P450(17)), 25% (17-hydroxysteroid dehydrogenase) and 0% (5-reductase and P450arom). Androgen receptor AR was moderately decreased but estrogen receptor ER was not decreased with aging. The 25% decrease in the spine density with aging may be due to a balance between considerably decreased T and DHT levels (spine decrease factor) and remained moderately high E2 level (spine increase factor) in the 24m hippocampus. Aged hippocampus still has moderate capacity of sex-steroid synthesis and their functions. Interestingly, DHT-supplementation and T-supplementation recovered the spine density at 24m.

neuroscience↗

bioRxiv2025

Tibial nerve stiffness is related to maximum angle of ankle dorsiflexion

The maximum angle of ankle dorsiflexion is affected by the triceps surae muscle stiffness and stretch tolerance, which may be strongly reflected by the tibial nerve stiffness. However, no study has evaluated the triceps surae muscle and tibial nerve stiffness simultaneously or clarified their association with the maximum angle. The purpose of this study was to investigate the association between the maximum angle and both the stiffness. Forty-one healthy adults participated. The shear wave velocities of the triceps surae muscles and tibial nerve were measured at 5{degrees}, 15{degrees}, and 25{degrees} ankle dorsiflexion. Multiple linear regression analysis was performed using the forced entry method, specifying the shear wave velocities of the four tissues as the independent variables and the maximum angle as the dependent variable. This analysis was performed at each angle where the shear wave velocity was measured. Multiple linear regression analysis was also performed using the stepwise method, specifying the shear wave velocities of the tibial nerve at the three angles as the independent variables and the maximum angle as the dependent variable. Using the forced entry method, the shear wave velocity of the tibial nerve at each angle was significantly negatively associated with the maximum angle, whereas those of the muscles were not. Using the stepwise method, only the shear wave velocity of the tibial nerve at 25{degrees} was significantly negatively associated with the maximum angle. These results suggest that the tibial nerve stiffness in a greatly lengthened position determines the maximum angle of ankle dorsiflexion.

physiology↗

bioRxiv2024

Defensive fungal symbiosis on insect hindlegs

Tympanal organs as "insect ears" have evolved repeatedly. Dinidorid stinkbugs were reported to possess a conspicuous tympanal organ on females hindlegs. Here we report an unexpected discovery that the stinkbugs "tympanal organ" is actually a novel symbiotic organ. The stinkbugs "tympanum" is not membranous but a porous cuticle, where each pore connects to glandular secretory cells. In reproductive females, the hindleg organ is covered with fungal hyphae growing out of the pores. Upon oviposition, the females skillfully transfer the fungi from the organ to the eggs. The eggs are quickly covered with hyphae and physically protected against wasp parasitism. The fungi are mostly benign Cordycipitaceae entomopathogens and show considerable diversity among insect individuals and populations, indicating environmental acquisition of specific fungal associates. These results uncover a novel external fungal symbiosis in which hosts elaborate morphological, physiological and behavioral specializations underpin the selective recruitment of benign entomopathogens for a defensive purpose.

microbiology↗

bioRxiv2023

Surface-engineered extracellular vesicles to modulate antigen-specific T cell expansion for cancer immunotherapy

Extracellular vesicles (EVs), including exosomes, are emerging as novel mediators of cell-cell communications, involved in various processes such as immune activation and immunosuppression. Despite the recent development of several EVs-based cancer immunotherapies, their clinical efficacy remained limited. Here, using fusion with tetraspanin as one of the EV engineering techniques, we created antigen-presenting extracellular vesicles (AP-EVs) to reproduce the functional characteristics of professional antigen-presenting cells (APCs). AP-EVs were also equipped with surface-bound IL-2, a feature not inherent to APCs, which facilitated selective delivery of IL-2 to antigen-specific CD8+ T cells. AP-EVs were engineered to express a peptide-major histocompatibility class I (pMHCI) complex, a costimulatory CD80 molecule, and IL-2, allowing the simultaneous presentation of multiple immune modulators to antigen-specific CD8+ T cells. This promoted the clonal expansion and differentiation of antigen-specific cytotoxic T lymphocytes, leading to potent anticancer immune responses. Combination therapy with AP-EVs and anti-PD-1 demonstrated enhanced anticancer immunity against established tumors compared with anti-PD-1 monotherapy. Our engineered EVs represent a novel effective strategy for cancer immunotherapy.

bioengineering↗

bioRxiv2021

Ultra-small cells and DPANN genome unveiled inside an extinct vent chimney

Chemosynthetic organisms flourish around deep-sea hydrothermal vents where energy-rich fluids are emitted from metal sulfide chimneys. In contrast to actively venting chimneys, the nature of microbial life in extinct chimneys without fluid venting remains largely unknown. Here, the occurrence of ultra-small cells in silica-filled grain boundaries inside an extinct chimney is demonstrated by high-resolution bio-signature mapping. The ultra-small cells are associated with extracellularly precipitated Cu2O nanocrystals. Single-gene analysis shows that the chimney interior is dominated by a member of Pacearchaeota known as a major phylum of DPANN. Genome-resolved metagenomic analysis reveals that the chimney Pacearchaeota member is equipped with a nearly full set of genes for fermentation-based energy generation from nucleic acids, in contrast to previously characterized Pacearchaeota members lacking many genes for nucleic acid fermentation. We infer that the ultra-small cells associated with silica and extracellular Cu2O nanocrystals in the grain boundaries are Pacearchaeota, on the basis of the experimentally demonstrated capability of silica to concentrate nucleic acids from seawater and the presence of Cu-exporting genes in a reconstructed Pacearchaeota genome. Given the existence of ~3-billion-year-old submarine hydrothermally deposited silica, proliferation of microbial life using silica-bound nucleic acids might be relevant to the primitive vent biosphere.

microbiology↗

bioRxiv2020

Efficient Development of Platform Cell Lines Using CRISPR-Cas9 1 and Transcriptomics Analysis

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

bioengineering↗