常陸 圭介

Lysine methylation is an important epigenetic modification that regulates chromatin structure and gene expression. However, it is still difficult to distinguish its methylated states without labels at the single-molecule level. In this study, we investigate the discrimination of lysine methylation states using single-molecule tunneling measurements with gold nano-gap electrodes. The conductance decreases stepwise as the number of methyl groups increases, even though density functional theory (DFT) shows that all molecules have almost the same HOMO energy levels. This result suggests that conductance is not determined only by the electronic structure, but also by how the molecule is arranged between the electrodes. Statistical analysis of current signals shows that high-conductance events become less frequent after methylation, indicating fewer strongly coupled configurations. The relationship between current and molecular length also supports that transport depends on variations in molecular configurations. Machine learning analysis achieved an F-score of 0.76 for distinguishing methylated from unmethylated lysine. In contrast, distinguishing between mono-, di-, and trimethylated forms gave a lower F-score of 0.49, reflecting overlap in the signals. These results suggest that single-molecule tunneling currents are sensitive to stepwise lysine methylation states through differences in transient molecular configurations. This work demonstrates the potential of single-molecule tunneling measurements for label-free analysis of epigenetic modifications.

Salt stress severely limits wheat growth and seed yield; however, the mechanisms underlying plant-derived smoke (PDS)-induced salt tolerance remain unclear. The present study performs membrane proteomics to clarify how PDS solution enhances salt tolerance in wheat. Immunoblot analysis of subcellular marker proteins confirms successful enrichment of membrane fractions. Principal component analysis shows that 200 mM NaCl markedly alters membrane-protein composition in wheat roots, whereas 2000 ppm PDS solution largely restores these changes even under salt stress. At the protein level, mitochondrial ascorbate peroxidase increases in roots under salt stress but decreases with PDS-solution treatment, while leaves show the opposite trend. Salt stress reduces ATP content and H+-ATPase abundance; PDS-solution treatment restores both to near-control levels. In contrast, aquaporin levels increase under salt stress but decline after PDS-solution application. In addition, the expression of ammonium transporter was downregulated significantly under salt stress but recovered with PDS-solution treatment. These results suggest that PDS solution may confer salt-stress tolerance to wheat by regulating energy metabolism, water permeability, and ammonium absorption in the root membrane.

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.