Biology subjects

Morishita, R.

Publications and source records attributed to Morishita, R..

4 recordsLinked to original sources

bioRxiv2026

Structural determination of human nucleosomes reconstituted by the ExACT platform

The structure and function of eukaryotic chromatin have been extensively studied using conventional salt dialysis-based nucleosome assembly, which has provided fundamental structural insights into nucleosomes as well as a mechanistic understanding of their roles in DNA replication, repair and gene expression. Recently, we developed a labor-saving and time-efficient nucleosome assembly method using a wheat germ cell-free Expression and Assembly Coupled Technology (ExACT), which is emerging as a powerful tool for chromatin research. Here we report cryo-electron microscopy (cryo-EM) structures of human H3.1- and H3.3-containing nucleosomes assembled using this approach and validated by deep-learning-based amino acid-wise model quality (DAQ) scoring. The structures of H3.1- and H3.3-nucleosomes are nearly identical to previously reported models, confirming the structural fidelity of the method. In addition, we determined the previously unreported structure of the primate-specific H3.X-containing nucleosome. Together, these findings validate the cell-free co-expression nucleosome assembly platform and establish this method as a robust framework for biochemically investigating chromatin dynamics.

molecular biology↗

bioRxiv2026

Disruption of the NTCP-EGFR receptor complex as a strategy for selective inhibition of hepatitis B and D virus entry

Hepatitis B and D virus (HBV, HDV) enter hepatocytes through a coordinated process mediated by a receptor complex consisting of sodium taurocholate co transporting polypeptide (NTCP) and its entry cofactors, including epidermal growth factor receptor (EGFR). Here, we established an in vitro assay to evaluate the NTCP-EGFR interaction and identified Oxy229, an oxysterol-based compound that disrupted this molecular interaction. Oxy229 selectively inhibited HBV and HDV infection to HepG2-NTCP cells and primary human hepatocytes. Mechanistic analysis revealed that Oxy229 impaired the relocalization of the HBV-NTCP complex from plasma membrane to intracellular vesicles. Notably, Oxy229 did not compromise the physiological functions of NTCP and EGFR, i.e., bile acid transport and activation of downstream EGFR signaling pathways including Ras-MAPK and PI3K-Akt pathways, indicating selective inhibition of viral entry. Compound derivative analysis identified Oxy283, which acquired dual inhibitory activity against both NTCP-EGFR interaction and NTCP multimerization, resulting in enhanced anti-HBV potency. These findings establish the functional significance of the NTCP-receptor complex formation in HBV/HDV entry and highlight this machinery as a potential target for antiviral intervention.

microbiology↗

bioRxiv2023

Label-free visualization and quantification of the drug-type-dependent response of tumor spheroids by dynamic optical coherence tomography

We demonstrate label-free dynamic optical coherence tomography (D-OCT)-based visualization and quantitative assessment of patterns of tumor spheroid response to three anti-cancer drugs. The study involved treating human breast adenocarcinoma (MCF-7 cell-line) with paclitaxel (PTX), tamoxifen citrate (TAM), and doxorubicin (DOX) at concentrations of 0 (control), 0.1, 1, and 10 M for 1, 3, and 6 days. In addition, fluorescence microscopy imaging was performed for reference. The D-OCT imaging was performed using a custom-built OCT device. Two algorithms, namely logarithmic intensity variance (LIV) and late OCT correlation decay speed (OCDSl) were used to visualize the tissue dynamics. The spheroids treated with 0.1 and 1 M TAM appeared similar to the control spheroid, whereas those treated with 10 M TAM had significant structural corruption and decreasing LIV and OCDSl over treatment time. The spheroids treated with PTX had decreasing volumes and decrease of LIV and OCDSl signals over time at most PTX concentrations. The spheroids treated with DOX had decreasing and increasing volumes over time at DOX concentrations of 1 and 10 M, respectively. Meanwhile, the LIV and OCDSl signals decreased over treatment time at all DOX concentrations. The D-OCT, particularly OCDSl, patterns were consistent with the fluorescence microscopic patterns. The diversity in the structural and D-OCT results among the drug types and among the concentrations are explained by the mechanisms of the drugs. The presented results suggest that D-OCT is useful for evaluating the difference in the tumor spheroid response to different drugs and it can be a useful tool for anti-cancer drug testing.

biophysics↗

bioRxiv2020

Preclinical study of DNA vaccines targeting SARS-CoV-2

To fight against the worldwide COVID-19 pandemic, the development of an effective and safe vaccine against SARS-CoV-2 is required. As potential pandemic vaccines, DNA/RNA vaccines, viral vector vaccines and protein-based vaccines have been rapidly developed to prevent pandemic spread worldwide. In this study, we designed plasmid DNA vaccine targeting the SARS-CoV-2 Spike glycoprotein (S protein) as pandemic vaccine, and the humoral, cellular, and functional immune responses were characterized to support proceeding to initial human clinical trials. After intramuscular injection of DNA vaccine encoding S protein with alum adjuvant (three times at 2-week intervals), the humoral immunoreaction, as assessed by anti-S protein or anti-receptor-binding domain (RBD) antibody titers, and the cellular immunoreaction, as assessed by antigen-induced IFN{gamma} expression, were up-regulated. In IgG subclass analysis, IgG2b was induced as the main subclass. Based on these analyses, DNA vaccine with alum adjuvant preferentially induced Th1-type T cell polarization. We confirmed the neutralizing action of DNA vaccine-induced antibodies via two different methods, a binding assay of RBD recombinant protein with angiotensin-converting enzyme 2 (ACE2), a receptor of SARS-CoV-2, and pseudovirus assay. Further B cell epitope mapping analysis using a peptide array showed that most vaccine-induced antibodies recognized the S2 and RBD subunits, but not the S1 subunit. In conclusion, DNA vaccine targeting the spike glycoprotein of SARS-CoV-2 might be an effective and safe approach to combat the COVID-19 pandemic.

immunology↗