Biology subjects

Honda, Y.

Publications and source records attributed to Honda, Y..

8 recordsLinked to original sources

bioRxiv2026

Sequentially Self-Assembled Supramolecular Nanocomplexes Enable Systemic Cas9 RNP Delivery and In Vivo Tumor Genome Editing

In vivo genome editing with CRISPR-Cas9 ribonucleoproteins (RNPs) holds substantial therapeutic promise, yet rapid bloodstream clearance and the absence of delivery systems capable of systemic tumor targeting have hindered its clinical translation. Herein, a supramolecular ternary complex platform is reported in which Cas9/sgRNA RNPs are co-assembled with tannic acid (TA) and phenylboronic acid (PBA)-conjugated polymers through sequential self-assembly, producing [~]30 nm core-shell ternary complexes that protect RNPs from enzymatic degradation and dissociate selectively at endosomal pH. Upon intravenous administration in subcutaneous tumor-bearing mice, these ternary complexes exhibit prolonged blood circulation and preferential tumor accumulation, achieving 37.2% gene editing at tumor sites compared with only 1.5% for free RNPs. The platform successfully knocks out previously undruggable oncogenes including mutant KRAS and polo-like kinase 1 (PLK1), markedly suppressing tumor growth in vivo. By integrating sequential supramolecular self-assembly with stimuli-responsive cargo release, this strategy establishes a generalizable framework for systemically administered in vivo CRISPR therapeutics.

bioengineering↗

bioRxiv2025

Passive and active demethylation in immune diseases with proliferation driving global hypomethylation

Global DNA hypomethylation is a hallmark of immune-mediated diseases, yet its regulatory significance remains unclear. Replication-associated loss of DNA methylation at solo-WCGW (W = A/T) CpGs has recently been proposed as a consequence of cell division. Here we systematically investigated genome-wide hypomethylation patterns across seven immune-mediated diseases. Most diseases exhibited global hypomethylation, particularly at solo-WCGW CpGs in transcriptionally repressed regions, potentially reflecting increased immune cell proliferation. By contrast, CpG sites whose methylation levels were associated with cytokine exposure or SNP genotypes were predominantly located in transcriptionally active regions. To investigate whether immunological events driving immune cell proliferation may also be imprinted in transcriptionally active regions, we searched for differentially methylated regions (DMRs) correlated with an index reflecting the extent of solo-WCGW hypomethylation. In narcolepsy, we identified a DMR within the T-cell receptor alpha (TRA) locus, and greater hypomethylation was associated with increased clonality of both TRA and TRB repertoires, with a similar pattern in an independent cohort. In multiple sclerosis, a DMR was also detected within the IGH locus encoding the B-cell receptor. Together, these findings suggest that the hypomethylation index captures the impact of disease-specific immune dynamics, while reflecting a shared epigenetic signature of immune cell proliferation across diseases.

genomics↗

bioRxiv2024

Multiomics analysis of narcolepsy T cells: global hypomethylation in solo-WCGW motif linked to T cell proliferation

Narcolepsy type 1 (NT1) is a chronic sleep disorder caused by a loss of orexin-producing cells in the brain and involves autoimmune mechanisms, including the presence of autoreactive T cells. We performed a genome-wide DNA methylation analysis using CD4+ and CD8+ T cells of NT1 patients. Analysis of differentially methylated regions as well as multiomics analysis with genomic and transcriptomic data obtained from the same samples indicated that cell chemotaxis pathways are implicated as a cause in the pathogenesis of NT1. Additionally, we found global hypomethylation in both the T cells of NT1 cases (CD4+: P = 1.69E-67; CD8+: P = 4.83E-12). These NT1-associated hypomethylated sites were significantly more abundant in solo-WCGW (sequences without neighboring CpGs, where W is an A or T base; P = 9.87E-194). Solo-WCGW tends to lose DNA methylation over the course of cell divisions, suggesting enhanced T cell proliferation in NT1.

genomics↗

bioRxiv2023

Bayesian Approach Enabled Objective Comparison of Multiple Human iPSC-derived Cardiomyocytes' Proarrhythmia Sensitivities.

The one-size-fits-all approach has been the mainstream in medicine, and the well-defined standards support the development of safe and effective therapies for many years. Advancing technologies, however, enabled precision medicine to treat a targeted patient population (e.g., HER2+ cancer). In safety pharmacology, computational population modeling has been successfully applied in virtual clinical trials to predict drug-induced proarrhythmia risks against a wide range of pseudo cohorts. In the meantime, population modeling in safety pharmacology experiments has been challenging. Here, we used five commercially available human iPSC-derived cardiomyocytes growing in 384-well plates and analyzed the effects of ten potential proarrhythmic compounds with four concentrations on their calcium transients (CaTs). All the cell lines exhibited an expected elongation or shortening of calcium transient duration with various degrees. Depending on compounds inhibiting several ion channels, such as hERG, peak and late sodium and L-type calcium or IKs channels, some of the cell lines exhibited irregular, discontinuous beating that was not predicted by computational simulations. To analyze the shapes of CaTs and irregularities of beat patterns comprehensively, we defined six parameters to characterize compound-induced CaT waveform changes, successfully visualizing the similarities and differences in compound-induced proarrhythmic sensitivities of different cell lines. We applied Bayesian statistics to predict sample populations based on experimental data to overcome the limited number of experimental replicates in high-throughput assays. This process facilitated the principal component analysis to classify compound-induced sensitivities of cell lines objectively. Finally, the association of sensitivities in compound-induced changes between phenotypic parameters and ion channel inhibitions measured using patch clamp recording was analyzed. Successful ranking of compound-induced sensitivity of cell lines was validated by visual inspection of raw data. Author SummaryCardiac safety is one of the most stringent regulatory risk monitoring during drug development. Regulatory agencies, including the Food and Drug Administration (FDA), require drug developers to conduct thorough preclinical and clinical studies to evaluate drug-induced proarrhythmia risks. The CiPA (Comprehensive in vitro Proarrhythmia Assay) initiative led by the FDA validated the applications of human cardiomyocytes derived from induced pluripotent stem cells in proarrhythmia risk assessments. The assay can be cost effective, and use of high-throughput approaches enables scale-up analysis. However, limited diversity in cell lines to recapitulate heterogeneity of human patients are still lacking in the cardiac safety analysis. We applied various computational tools to maximize capacity for analyzing diverse response of commercially available five cell lines against reference chemical compounds. Limited number of experimental data made it difficult to predict similarities and differences in drug responses among different cell lines. By applying Bayesian approach, our analysis could intuitively grasp the probabilities of expecting different responses from different cell lines.

pharmacology and toxicology↗

bioRxiv2023

Contribution of default mode network to game and delayed-response task performance: power and connectivity analyses of theta oscillation in the monkey

Neuroimaging studies have demonstrated the presence of a default mode network (DMN) which shows greater activity during rest, and an executive network (EN) which is activated during cognitive tasks. DMN and EN are thought to have competing functions. However, recent studies reported that the two networks show coactivation during some cognitive tasks. To clarify how DMN works and how DMN interacts with EN for cognitive control, we recorded EEG activities in the medial prefrontal (anterior DMN: aDMN), posterior cingulate/precuneus (posterior DMN: pDMN), and lateral prefrontal (EN) areas in the monkey. As cognitive tasks, we employed a monkey-monkey competitive video game (GAME) and a delayed-response (DR) task. We focused on theta oscillation because of its importance in cognitive control. We also examined theta band connectivity among the three network areas using the Granger causality analysis. DMN and EN were found to work cooperatively in both tasks. In all the three network areas, we found GAME-task-related, but no DR-task-related, increase in theta power from the resting level, maybe because of the higher cognitive demand associated with the GAME task performance. The information flow conveyed by the theta oscillation was directed more to aDMN than from aDMN for both tasks. The GAME-task-related increase in theta power in aDMN is supposed to be supported by more information flow conveyed by the theta oscillation from EN and pDMN.

neuroscience↗

bioRxiv2022

Germline-specific role for unconventional components of the γ-tubulin complex in Caenorhabditis elegans

The {gamma}-tubulin complex ({gamma}TuC) is a widely conserved microtubule nucleator, but some of its components GCP4-6. Here, we identified two {gamma}TuC-associated proteins in C. elegans, namely GTAP-1 and -2, for which apparent orthologs were detected only in the genus Caenorhabditis. Their centrosomal localization was interdependent. In early C. elegans embryos, whereas the conserved {gamma}TuC component MZT-1/MOZART1 was essential for the localization of centrosomal {gamma}-tubulin, depletion of GTAP-1 and/or -2 caused up to 50% reduction of centrosomal {gamma}-tubulin and precocious disassembly of spindle poles during mitotic telophase. In the adult germline, GTAP-1 and GTP-2 contributed to the efficient recruitment of {gamma}TuC to the plasma membrane. Depletion of GTAP-1, but not GTAP-2, severely disrupted both the microtubule array and the honeycomb-like structure in the adult germline. We propose that GTAP-1 and -2 are unconventional components of {gamma}TuC that contribute to the organization of both centrosomal and non-centrosomal microtubules by targeting the {gamma}TuC to specific subcellular sites in a tissue-specific manner. SUMMARY STATEMENTHaruta et al. show that GTAP-1 and -2, two unconventional components of the {gamma}-tubulin complex ({gamma}TuC) in C. elegans, contribute to targeting the {gamma}TuC to embryonic centrosomes and germ-cell membrane in adults.

cell biology↗

bioRxiv2021

Aberrant localization of CDC42 C-terminal variants to the Golgi apparatus drives pyrin inflammasome-dependent autoinflammation

Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Elevated levels of serum IL-18 in patients and effectiveness of IL-1{beta}-blocking therapy indicate that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1{beta} in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but no that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave way for elucidating the mechanism of pyrin inflammasome formation.

immunology↗

bioRxiv2020

Effect of belt electrode-skeletal muscle electrical stimulation on immobilization-induced muscle fibrosis

PurposeMacrophage accumulation in response to decreasing myonuclei may be the major mechanism underlying immobilization-induced muscle fibrosis in muscle contracture, an intervention strategy suppressing these lesions is necessary. Therefore, this research investigated the effect of belt electrode-skeletal muscle electrical stimulation (B-SES), a new electrical stimulation device, to the macrophage accumulation via myonuclei decrease in immobilization-induced muscle fibrosis. Materials and Methods18 Wistar male rats were divided into the control group, immobilization group (with plaster cast fixation to immobilize the soleus muscles in a shortened position for 2 weeks), and B-SES group (with muscle contractile exercise through B-SES during the immobilization period). B-SES stimulation was performed at a frequency of 50 Hz and an intensity of 4.7 mA, muscle contractile exercise by B-SES was applied to the lower limb muscles for 20 minutes/session (twice a day) for 2 weeks (6 times/week). The bilateral soleus muscles were used for histological, immunohistochemical, biochemical, and molecular biological analyses. ResultsThe number of myonuclei was significantly higher in the B-SES group than in the immobilization group, and there was no significant difference between the B-SES and control groups. The cross-sectional area of type I and II myofibers in the immobilization and B-SES groups was significantly lower than that in the control group, and the cross-sectional area of type I myofibers in the B-SES group was higher than that in the immobilization group. However, Atrogin-1 and MuRF-1 mRNA expression in the immobilization and B-SES groups was significantly higher than those in the control group. Additionally, the number of macrophages, IL-1{beta}, TGF-{beta}1, and -SMA mRNA expression, and hydroxyproline expression was significantly lower in the control and B-SES groups than those in the immobilization group. ConclusionThis research surmised that muscle contractile exercise through B-SES prevented immobilization-induced muscle fibrosis, and this alteration suppressed the development of muscle contracture.

pathology↗