Biology subjects

Ishida, Y.

Publications and source records attributed to Ishida, Y..

25 records · Page 2Linked to original sources

bioRxiv2022

Multiphasic hepatitis B virus kinetic patterns in humanized chimeric mice can be explained via stochastic agent-based modeling of intracellular virion production cycles

Serum hepatitis B virus (HBV) kinetics in urokinase-type plasminogen activator/severe combined immunodeficient (uPA-SCID) mice reconstituted with humanized livers from inoculation to steady state is highly dynamic despite the absence of an adaptive immune response. We developed a stochastic agent-based model that includes virion production cycles in individual infected human hepatocytes. The model was calibrated using a genetic algorithm approach with the serum HBV kinetics observed in mice inoculated with 108 HBV genome equivalents and fit the data well when the following viral production parameters were assumed: (1) An eclipse phase lasting 5-50 hours and (2) a post-eclipse phase production rate that is based on increasing production cycles initially starting with a long production cycle of 1 virion per 20 hours that gradually reaches 1 virion per hour after approximately 3-4 days before virion production increases dramatically to reach to a steady state production rate of 4 virions per hour per cell. The model was then validated by showing it could accurately simulate the viral kinetics observed with lower HBV inoculation doses (104-107 genome equivalents) in which similar, but delayed patterns were observed. Together, modeling suggests that it is the cyclic nature of the virus lifecycle combined with an initial slow but increasing rate of HBV production from each cell that plays a role in generating the observed multiphasic HBV kinetic patterns in humanized mice.

systems biology↗

bioRxiv2021

Heterozygous loss of ZBTB38 leads to early embryonic lethality in mice via suppressing Nanog and Sox2

Mammalian DNA methylation is an epigenetic modification which is involved in various biological processes, including gene expression regulation. In mice, methyltransferases are responsible for DNA methylation, which are critical for early embryogenesis. However, the significance of methyl-CpG binding proteins (MBPs) that bind methylated CpG remains largely unknown. We previously demonstrated that ZBTB38/CIBZ-a zinc finger type of MBP-is required for ES cell proliferation by positively regulating Nanog expression. However, the physiological function of ZBTB38 remains unclear. In this study, we generated conditional ZBTB38 knockout mice using Cre-loxP technology. Unexpectedly, our results showed that germline loss of the ZBTB38 single allele resulted in decreased epiblast cell proliferation and increased apoptosis shortly after implantation, leading to early embryonic lethality. We found that heterozygous loss of ZBTB38 reduced the expression of Nanog, Sox2, and the genes responsible for epiblast proliferation, differentiation, and cell viability. Despite this lethal phenotype, ZBTB38 is dispensable for ES cell establishment and identity. Together, these findings indicate that ZBTB38 is essential for early embryonic development, providing new insights into the roles of MBP in implantation.

developmental biology↗

bioRxiv2021

Nasal delivery of single-domain antibodies improve symptoms of SARS-CoV-2 infection in an animal model

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung via a nebulizer could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients. Author summaryVaccines for COVID-19 are now available but therapeutic drugs are still needed to treat life-threatening cases and those who cannot be vaccinated. We discovered a new heavy-chain single-domain antibody that can effectively neutralize the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19. Unlike other drug candidates, which prevent the virus from attaching to the receptor on the host cell, this new antibody acts by blocking the virus membrane from fusing with the host cell membrane. We studied the behavior of the new antibody in vitro using VeroE6/TMPRSS2 cells and human lung organoids. When delivered through the nose to infected Syrian hamsters, we found that this antibody could prevent the typical symptoms caused by SARS-CoV-2. Our results are significant because delivering simple drugs directly to infected lungs using a nebulizer could increase the potency of the drugs while reducing the risk of immune reaction that could occur if the drugs escape or are delivered through the blood stream.

microbiology↗

bioRxiv2021

Multi-Scale LM/EM Neuronal Imaging from Brain to Synapse with a Tissue Clearing Method, ScaleSF

The mammalian brain is organized over sizes that span several orders of magnitude, from synapses to the entire brain. Thus, a technique to visualize neural circuits across multiple spatial scales (multi-scale neuronal imaging) is vital for deciphering brain-wide connectivity. Here, we developed this technique by coupling successive light microscope/electron microscope (LM/EM) imaging with an ultrastructurally-preserved tissue clearing method, ScaleSF. Our multi-scale neuronal imaging incorporates 1) brain-wide macroscopic observation, 2) mesoscopic circuit mapping, 3) microscopic subcellular imaging, and 4) EM imaging of nanoscopic structures, allowing seamless integration of structural information from the brain to synapses. We applied the technique to three neural circuits of two different species, mouse striatofugal, mouse callosal, and marmoset corticostriatal projection systems, and succeeded in the simultaneous interrogation of their circuit structure and synaptic connectivity in a targeted way. Our multi-scale neuronal imaging will significantly advance the understanding of brain-wide connectivity by expanding the scales of objects.

neuroscience↗

bioRxiv2021

Whole-genome sequence analysis of mutations in rice plants regenerated from zygotes, mature embryos, and immature embryos

Somaclonal variation was studied by whole-genome sequencing in rice plants (Oryza sativa L., Nipponbare) regenerated from the zygotes, mature embryos, and immature embryos of a single mother plant. The mother plant and its seed-propagated progeny were also sequenced. A total of 338 variants of the mother plant sequence were detected in the progeny, and mean values ranged from 9.0 of the seed-propagated plants to 37.4 of regenerants from mature embryos. The ratio of single nucleotide variants among the variants was 74.3%, and the natural mutation rate calculated using the variants in the seed-propagated plants was 1.2 x 10-8. The percentage and the mutation rate were consistent with the values reported previously. Plants regenerated from mature embryos had significantly more variants than different progeny types. Therefore, using zygotes and immature embryos can reduce somaclonal variation during the genetic manipulation of rice.

plant biology↗

bioRxiv2020

Understanding hepatitis B virus dynamics and the antiviral effect of interferon-alpha treatment in humanized chimeric mice

BackgroundWhereas the mode of action of lamivudine (LAM) against hepatitis B virus (HBV) is well established, the inhibition mechanism(s) of interferon- are less completely defined. To advance our understanding, we mathematically modelled HBV kinetics during pegylated interferon--2a (pegIFN), LAM and pegIFN+LAM treatment of chronically HBV-infected humanized uPA/SCID chimeric mice. MethodsThirty-nine uPA/SCID mice with humanized livers whose pre-treatment steady-state serum HBV reached 9.2{+/-}0.4 logIU/mL were treated with pegIFN, LAM or pegIFN+LAM for 14 days. Serum HBV DNA and intracellular HBV DNA were measured frequently. We developed a nonlinear mixed effect viral kinetic model and simultaneously fit it to the serum and intracellular HBV DNA data. ResultsUnexpectedly, even in the absence of an adaptive-immune response, a biphasic decline in serum HBV DNA and intracellular HBV DNA was observed in response to all treatments. Modeling predicts that the first phase represents pegIFN inhibiting intracellular HBV DNA synthesis with efficacy of 86%, which was similar under LAM and pegIFN+LAM. In contrast, there were distinct differences in HBV decline during the 2nd phase which was accounted for in the model by a time-dependent inhibition of intracellular HBV DNA synthesis with the steepest decline observed during pegIFN+LAM (0.46/d) and the slowest (0.052/d) during pegIFN mono-treatment. ConclusionsReminiscent of observations in patients treated with pegIFN and/or LAM, a biphasic HBV decline was observed in treated humanized mice in the absence of adaptive immune response. Interestingly, combination treatment does not increase the initial inhibition of HBV production; however, enhancement of second phase decline is observed providing insight into the dynamics of HBV treatment response and the mode of action of interferon- against HBV. Author SummaryChronic hepatitis B virus (HBV) infection remains a global health care problem as we lack sufficient curative treatment options. Elucidating the dynamic of HBV infection and treatment at the molecular level would potentially facilitate the development of novel, more effective HBV antivirals. Currently, the only well-established small animal HBV infection model available is the chimeric uPA/SCID mice with humanized livers; however, the HBV infection kinetics under interferon- (IFN) in this model system have not been determined in sufficient detail to support the in-depth studies of HBV treatment response needed to identify/confirm more effective drug targets. In this study 39 chronic HBV-infected uPA/SCID humanized mice treated with IFN and/or lamivudine were analysed using a mathematical modelling approach. We found that IFN main mode of action is blocking HBV DNA synthesis and that 73% of synthesized HBV DNA per are secreted from infected cells. Our data-driven mathematical modeling study provides novel insights into IFN anti-HBV mechanism(s) and viral-host interplay at the molecular level.

systems biology↗

bioRxiv2019

Pivotal involvement of the CX3CL1-CX3CR1 axis for the recruitment of M2-TAMs in skin carcinogenesis

We previously revealed the crucial roles of CX3CL1-CX3CR1 axis in skin wound healing. Although repeated wounds frequently develop into skin cancer, the roles of CX3CL1 in skin carcinogenesis remain elusive. Here, we proved that CX3CL1 protein expression and CX3CR1+ macrophages were observed in human skin cancer tissues. Similarly, we observed the enhancement of CX3CL1 expression and the abundant accumulation of CX3CR1+ tumor-associated macrophages (TAMs) with M2 phenotypes in the skin carcinogenesis process induced by the combined treatment with 7,12-dimethylbenz-(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA). In this mouse skin carcinogenesis process, CX3CR1+ TAMs exhibited M2 phenotypes with the expression of Wnt3a and angiogenic molecules including vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)-9. Compared to wild-type mice, CX3CR1-deficient mice showed fewer numbers of skin tumors with a lower incidence. Concomitantly, M2-macrophage numbers and neovascularization reduced with the depressed expression of angiogenic factors and Wnt3a. Thus, the CX3CL1-CX3CR1 axis can crucially contribute to skin carcinogenesis by regulating the accumulation and functions of TAMs. Thus, this axis can be a good target for preventing and/or treating skin cancers.

cancer biology↗