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Biology subjects

Kusakabe, T.

Publications and source records attributed to Kusakabe, T..

6 recordsLinked to original sources

Prior Bombyx mori latent virus exposure enhances subsequent BmNPV infection and suppresses antiviral responses in the silkworm, Bombyx mori

Bombyx mori latent virus (BmLV) is a persistent RNA virus frequently detected in silkworm-derived cell lines and has been reported to interact with B. mori nucleopolyhedrovirus (BmNPV). However, whether previous BmLV exposure alters subsequent BmNPV infection in silkworm larvae remains unclear. Here, we examined how BmLV exposure history affects subsequent BmNPV infection in cultured cells and fifth-instar B. mori larvae. Persistent BmLV infection increased BmNPV-mediated reporter protein expression and BmNPV genome abundance in cultured cells, whereas short-term BmLV exposure or simultaneous BmLV and BmNPV coinfection did not reproduce the increases observed in persistently BmLV-positive cultures. In silkworm larvae (n22 strain), 24 h BmLV pre-exposure before BmNPV challenge increased BmNPV-mediated reporter protein expression and viral genome accumulation compared with BmNPV infection alone. BmLV antigenome RNA was detected in multiple larval tissues after BmLV exposure, indicating BmLV replication during the pre-exposure period. Notably, this prior BmLV exposure was associated with altered antiviral responses upon subsequent BmNPV infection. Specifically, BmLV-pre-exposed larvae showed reduced induction of Dicer-2, R2D2, and stimulator of interferon genes (STING) in the fat body. These findings indicate that prior BmLV exposure enhances subsequent BmNPV infection and is associated with attenuated induction of host antiviral genes. This study expands our understanding of how sequential viral infections shape host-virus interactions and suggests a novel approach for increasing recombinant protein production in baculovirus-based expression systems.

microbiology↗

Therapeutic ability of extracellular vesicles derived from angio-miRNA-modified mesenchymal stromal cells in ischemic animal models

BackgroundRegenerative therapy using extracellular vesicles (EVs) is expected to be an option for supportive therapy of chronic limb-threatening ischemia. In this study, we examined the angiogenic potential of EVs derived from genetically modified MSCs, focusing on the angio-miRNAs in these vesicles. MethodsBone marrow mesenchymal stromal cells (BM-MSCs) were transfected with a lentiviral vector containing specific angio-miRNAs (miRNA-126, -135b, or -210). EVs were separated from the culture medium of BM-MSCs via ultracentrifugation. The quality and quantity of the EVs were evaluated using nanoparticle tracking analysis, fluorometry, and an ExoScreen assay. Tube formation assays were conducted using human umbilical vein endothelial cells to assess the angiogenic potency of the EVs in vitro. The EVs were injected into the ischemic hind limb muscles of mice. The grades of limb ischemia, blood perfusion, and muscle tissues were analyzed to assess the angiogenic potency of the EVs in vivo. ResultsAngio-miRNAs were overexpressed in MSCs transfected with a lentiviral vector. Tube formation was significantly enhanced when endothelial cells were cultured with the EVs (P < 0.05). Compared to non-contact co-culture with BM-MSCs, EVs derived from modified BM-MSCs significantly promoted tube formation in endothelial cells (P < 0.05). The severity of limb ischemia on day 7 was alleviated when a combination of two or three EVs derived from modified BM-MSCs was injected compared with native EVs. Toe blood perfusion on day 14 significantly recovered when a combination of two or three EVs derived from modified BM-MSCs was injected (P < 0.05). Tissue analysis revealed that the number of newly formed endothelial cells in ischemic muscles increased when a combination of two or three EVs derived from modified BM-MSCs was injected. ConclusionsThe use of a combination of EVs derived from genetically modified MSCs stimulated angiogenesis both in vitro and in vivo.

cell biology↗

Characterization of high affinity IgM and IgG monoclonal antibodies against norovirus variants GII.4 and GII.17

Human norovirus, a leading cause of viral gastroenteritis, results in significant global health and economic burden, requiring sensitive and accurate diagnosis and effective therapeutics and vaccines. In this study, we immunized mice with the virus like capsid particles of GII.4, a mainstream strain, and GII.17, a modern strain that began to circulate in 2014, and used hybridoma technology to generate hybridoma cells that produce norovirus-binding antibodies against GII.4 and GII.17, respectively. Selection of these hybridoma cells yielded monoclonal IgG and IgM antibodies against these strains. Characterization of these antibodies revealed that avidity effect by multivalent binding is necessary for IgM to bind to norovirus at high efficiency, while IgG achieve high affinity even by monovalent binding. Surface plasmon resonance and ELISA data suggest that the high density of antigen protrusion domain in the norovirus capsid, containing approximately protomers, facilitates IgM to bind to norovirus capsid with high efficiency.

biochemistry↗

Colibactin-induced genotoxicity and colorectal cancer exacerbation critically depends on adhesin-mediated epithelial binding

Various bacteria are suggested to contribute to colorectal cancer (CRC) development, including pks+ E. coli which produce the genotoxin colibactin that induces characteristic mutational signatures in host epithelial cells. It remains unclear how the highly unstable colibactin molecule is able to access host epithelial cells and its DNA to cause harm. Using the microbiota-dependent ZEB2-transgenic mouse model of invasive CRC, we found that pks+ E. coli drives CRC exacerbation and tissue invasion in a colibactin-dependent manner. Using isogenic mutant strains, we further demonstrate that CRC exacerbation critically depends on expression of the E. coli type-1 pilus adhesin FimH and the F9-pilus adhesin FmlH. Blocking bacterial adhesion using a pharmacological FimH inhibitor attenuates colibactin-mediated genotoxicity and CRC exacerbation. Together, we show that the oncogenic potential of pks+ E. coli critically depends on bacterial adhesion to host epithelial cells and is critically mediated by specific bacterial adhesins. Adhesin-mediated epithelial binding subsequently allows production of the genotoxin colibactin in close proximity to host epithelial cells, which promotes DNA damage and drives CRC development. These findings present promising therapeutic avenues for the development of anti-adhesive therapies aiming at mitigating colibactin-induced DNA damage and inhibiting the initiation and progression of CRC, particularly in individuals at risk for developing CRC.

cancer biology↗

Highly efficient protein expression of Plasmodium vivax surface antigen, Pvs25 by silkworm, Bombyx mori, and its biochemical analysis.

Plasmodium vivax ookinete surface protein, Pvs25 is a transmission-blocking vaccine (TBV) candidate for malaria. Pvs25 has four EGF-like domains containing 22 cysteine residues forming 11 intramolecular disulfide bonds and this structural feature makes recombinant expression of Pvs25 difficult. In this study, we report the high expression of recombinant Pvs25 as a soluble form in silkworm, Bombyx mori. The Pvs25 protein was purified from hemolymphs of larvae and pupae by affinity chromatography. In the Pvs25 expressed by silkworm, no isoform with inappropriate disulfide bonds was found, requiring no further purification step which is necessary in case of Pichia pastoris based expressions systems. The Pvs25 from silkworm were confirmed to be the molecularly uniform by sodium dodecyl sulfate gel electrophoresis analysis and size exclusion chromatography analysis. To examine the immunogenicity, the Pvs25 from B. mori, was administered to BALB/c mice by the subcutaneous (s.c.) route with the oil adjuvant. The Pvs25 produced by silkworm induced potent and robust immune response, and the induced antisera correctly recognized P. vivax ookinetes in vitro, demonstrating the potency of Pvs25 from silkworm as a TBV candidate for malaria. This is the first study that to construct a mass production system for malaria TBV antigens by the silkworm to the best of our knowledge.

biochemistry↗

Clomipramine suppresses ACE2-mediated SARS-CoV-2 entry

Myocardial damage caused by the newly emerged coronavirus (SARS-CoV-2) infection is one of key determinants of COVID-19 severity and mortality. SARS-CoV-2 entry to host cells are initiated by binding with its receptor, angiotensin converting enzyme (ACE) 2, and the ACE2 abundance is thought to reflect the susceptibility to infection. Here, we found that clomipramine, a tricyclic antidepressant, potently inhibits SARS-CoV-2 infection and metabolic disorder in human iPS-derived cardiomyocytes. Among 13 approved drugs that we have previously identified as potential inhibitor of doxorubicin-induced cardiotoxicity, clomipramine showed the best potency to inhibit SARS-CoV-2 spike glycoprotein pseudovirus-stimulated ACE2 internalization. Indeed, SARS-CoV-2 infection to human iPS-derived cardiomyocytes (iPS-CMs) and TMPRSS2-expressing VeroE6 cells were dramatically suppressed even after treatment with clomipramine. Furthermore, the combined use of clomipramine and remdesivir was revealed to synergistically suppress SARS-CoV-2 infection. Our results will provide the potentiality of clomipramine for the breakthrough treatment of severe COVID-19.

pharmacology and toxicology↗