Biology subjects

Takeda, M.

Publications and source records attributed to Takeda, M..

30 records · Page 2Linked to original sources

bioRxiv2021

Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding -catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of -catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of -catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that hypersensitive mutants affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive -catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that proper AJ plasticity requires appropriate tension sensitivity of -catenin.

cell biology↗

bioRxiv2021

Evaluation of the Efficacy and Safety of a Clinical Grade Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Patch: A Pre-Clinical Study

AimsCardiomyocyte-derived induced pluripotent stem cells (iPSCs) may represent a promising therapeutic strategy for severely damaged myocardium. This study aimed to assess the efficacy and safety of clinical grade human iPSC-derived cardiomyocyte (hiPSC-CM) patches and conduct a pre-clinical proof-of-concept analysis. Methods and resultsA clinical grade hiPSC line was established from peripheral blood mononuclear cells collected from a healthy volunteer homozygous for human leukocyte antigens and differentiated into cardiomyocytes using cytokines and chemical compounds. hiPSC-CMs were cultured on temperature-responsive culture dishes to fabricate the hiPSC-CM patch. The hiPSC-CMs expressed cardiomyocyte-specific genes and proteins while electrophysiological analyses revealed that hiPSC-CMs were similar to the human myocardium. In vitro safety studies using cell growth, soft agar colony formation, and undifferentiated cell assays indicated that tumourigenic cells were not present. Moreover, no genomic mutations were discovered using whole genome and exome sequencing analysis. Tumour formation was not detected in an in vivo tumourigenicity assay using NOG mice. General toxicity tests also showed no adverse events due to hiPSC-CM patch transplantation. An efficacy study using a porcine model of myocardial infarction demonstrated significantly improved cardiac function with angiogenesis and a reduction in interstitial fibrosis, which was enhanced by cytokine secretion from hiPSC-CM patches after transplantation. No lethal arrhythmias were observed. ConclusionhiPSC-CM patches show promise for future translational research and clinical trials for ischaemic heart failure. One-sentence summaryThis pre-clinical study provides a proof-of-concept of the safety and efficacy of hiPSC-CM patches for the treatment of heart failure. Translational PerspectiveRegenerative therapy using cells and tissues is attractive as a novel approach for treating severe heart failure. We focused on human iPS cell-derived cardiomyocytes (hiPSC-CMs) as a cell source. Using basic research, the characteristics of hiPSC, hiPSC-CMs, and hiPSC-CM patches were determined in vitro and in vivo. We also conducted a pre-clinical study using a porcine model of myocardial infarction that confirmed the safety and efficacy of the hiPSC-CM patch, highlighting its potential for clinical application.

cell biology↗

bioRxiv2021

Identification of anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) oxysterol derivatives in vitro

Development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are urgently needed to combat the coronavirus disease 2019 (COVID-19). Oxysterols, defined as oxidized derivatives of cholesterol, include endogenous (naturally occurring) cholesterol metabolites as well as semi-synthetic oxysterol derivatives. We have previously studied the use of semi-synthetic oxysterol derivatives as drug candidates for inhibition of cancer, fibrosis, and bone regeneration. In this study, we have screened a panel of naturally occurring and semi-synthetic oxysterol derivatives for anti-SARS-CoV-2 activity, using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy186 displayed antiviral activity comparable to natural oxysterols. In addition, related oxysterol analogues Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 M and 99% at 15 M, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fall into a therapeutically relevant range (19 M), based on the dose-dependent curve for antiviral activity in our cell culture infection assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 with disrupting the formation of double membrane vesicles (DMVs), intracellular membrane compartments associated with viral replication. Oxy210 also inhibited the replication of hepatitis C virus, another RNA virus whose replication is associated with DMVs, but not the replication of the DMV-independent hepatitis D virus. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk developing COVID-19.

microbiology↗

bioRxiv2020

Fitness selection of hyperfusogenic measles virus F proteins associated with neuropathogenic phenotypes

Measles virus (MeV) is resurgent and caused >200,000 deaths in 2019. MeV infection can establish a chronic latent infection of the brain that can recrudesce months to years after recovery from the primary infection. Recrudescent MeV leads to fatal subacute sclerosing panencephalitis (SSPE) or measles inclusion body encephalitis (MIBE) as the virus spreads across multiple brain regions. Most clinical isolates of SSPE/MIBE strains show mutations in the fusion (F) gene that result in a hyperfusogenic phenotype in vitro and allow for efficient spread in primary human neurons. Wild-type MeV receptor binding protein (RBP) is indispensable for manifesting these mutant F phenotypes, even though neurons lack canonical MeV receptors (CD150/SLAMF1 or Nectin-4). How such hyperfusogenic F mutants are selected for, and whether they confer a fitness advantage for efficient neuronal spread is unresolved. To better understand the fitness landscape that allows for the selection of such hyperfusogenic F mutants, we conducted a screen of [≥]3.1x105 MeV-F point mutants in their genomic context. We rescued and amplified our genomic MeV-F mutant libraries in BSR-T7 cells under conditions where MeV-F-T461I (a known SSPE mutant), but not wild-type MeV can spread. We recovered known SSPE mutants but also characterized at least 15 novel hyperfusogenic F mutations with a SSPE phenotype. Structural mapping of these mutants onto the pre-fusion MeV-F trimer confirm and extend our understanding of the fusion regulatory domains in MeV-F. Our list of hyperfusogenic F mutants is a valuable resource for future studies into MeV neuropathogenesis and the regulation of paramyxovirus fusion. SignificanceMeasles remains a major cause of infant death globally. On rare occasions, measles virus infection of the central nervous system (CNS) leads to a fatal progressive inflammation of the brain many years after the initial infection. MeV isolates from such CNS infections harbor fusion (F) protein mutations that result in a hyperfusogenic phenotype. The small number of hyperfusogenic MeV-F mutants identified thus far limits our ability to understand how these mutations are selected in the context of CNS infections. We performed a saturating mutagenesis screen of MeV-F to identify a large set of mutants that would mimic the hyperfusogenic phenotype of MeV-F in CNS infection. Characterization of these mutants shed light on other paramyxoviruses known to establish chronic CNS infections.

microbiology↗

bioRxiv2020

Anti-severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) potency of Mefloquine as an entry inhibitor in vitro

Coronavirus disease 2019 (COVID-19) has caused serious public health, social, and economic damage worldwide and effective drugs that prevent or cure COVID-19 are urgently needed. Approved drugs including Hydroxychloroquine, Remdesivir or Interferon were reported to inhibit the infection or propagation of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), however, their clinical efficacies have not yet been well demonstrated. To identify drugs with higher antiviral potency, we screened approved anti-parasitic/anti-protozoal drugs and identified an anti-malarial drug, Mefloquine, which showed the highest anti-SARS-CoV-2 activity among the tested compounds. Mefloquine showed higher anti-SARS-CoV-2 activity than Hydroxychloroquine in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 1.28 M, IC90 = 2.31 M, and IC99 = 4.39 M in VeroE6/TMPRSS2 cells. Mefloquine inhibited viral entry after viral attachment to the target cell. Combined treatment with Mefloquine and Nelfinavir, a replication inhibitor, showed synergistic antiviral activity. Our mathematical modeling based on the drug concentration in the lung predicted that Mefloquine administration at a standard treatment dosage could decline viral dynamics in patients, reduce cumulative viral load to 7% and shorten the time until virus elimination by 6.1 days. These data cumulatively underscore Mefloquine as an anti-SARS-CoV-2 entry inhibitor.

microbiology↗

bioRxiv2020

Curing genetic skin disease through altered replication stress response

Revertant mosaicism, or natural gene therapy, refers to the spontaneous in vivo reversion of an inherited mutation in a somatic cell1. Only [~]50 human genetic disorders exhibit revertant mosaicism, implicating a distinctive role played by mutant proteins in somatic correction of a pathogenic germline mutation2. However, the process by which mutant proteins induce somatic genetic reversion in these diseases remains unknown. Here we show that heterozygous pathogenic CARD14 mutations causing autoinflammatory skin diseases, including psoriasis and pityriasis rubra pilaris, are repaired mainly via homologous recombination. Rather than altering the DNA damage response to exogenous stimuli such as X-irradiation or etoposide treatment, mutant CARD14 increased DNA double-strand breaks under conditions of replication stress. Furthermore, mutant CARD14 suppressed new origin firings without promoting crossover events in the replication stress state. Together, these results suggest that mutant CARD14 alters the replication stress response and preferentially drives break-induced replication (BIR), which is generally suppressed in eukaryotes3. Our results highlight the involvement of BIR in reversion events, thus revealing a previously undescribed role of BIR that could potentially be exploited to develop therapeutics for currently intractable genetic diseases.

molecular biology↗

bioRxiv2020

Poliovirus-nonsusceptible Vero cell line for the World Health Organization global action plan

Polio or poliomyelitis is a disabling and life-threatening disease caused by poliovirus (PV). As a consequence of global polio vaccination efforts, wild PV serotype 2 has been eradicated, and wild PV serotypes 1- and 3-transmitted cases have been largely eliminated except for in limited regions around the world. However, vaccine-derived PV, pathogenically reverted live PV vaccine strains in vaccinated humans, has become a serious issue. For the global eradication of polio, the World Health Organization is conducting the third edition of the Global Action Plan, which is requesting stringent control of potentially PV-infected materials. To facilitate the mission, we generated a PV-nonsusceptible Vero cell subline, which may serve as an ideal replacement of standard Vero cells to isolate emerging/re-emerging viruses without the risk of generating PV-infected materials.

microbiology↗

bioRxiv2020

Flexible decision under ambiguous cues reverses neural signaling from occipitotemporal to prefrontal cortex

Adaptation to changing environments involves the appropriate extraction of environmental information to achieve a behavioral goal. It remains unclear how behavioral flexibility is guided under situations where the relevant behavior is ambiguous. Using functional brain mapping of machine-learning decoders and directional functional connectivity, we show that brain-wide reversible neural signaling underpins task encoding and behavioral flexibility in ambiguously changing environments. When relevant behavior is cued ambiguously during behavioral shifting, neural coding is attenuated in distributed cortical regions, but top-down signals from the prefrontal cortex complement the coding. When behavioral shifting is cued more explicitly, modality-specialized occipitotemporal regions implement distinct neural coding about relevant behavior, and bottom-up signals from the occipitotemporal region to the prefrontal cortex supplement the behavioral shift. These results suggest that our adaptation to an ever-changing world is orchestrated by the alternation of top-down and bottom-up signaling in the fronto-occipitotemporal circuit depending on the availability of environmental information.

neuroscience↗

bioRxiv2020

The anticoagulant nafamostat potently inhibits SARS-CoV-2 infection in vitro: an existing drug with multiple possible therapeutic effects

Although infection by SARS-CoV-2, the causative agent of COVID-19, is spreading rapidly worldwide, no drug has been shown to be sufficiently effective for treating COVID-19. We previously found that nafamostat mesylate, an existing drug used for disseminated intravascular coagulation (DIC), effectively blocked MERS-CoV S protein-initiated cell fusion by targeting TMPRSS2, and inhibited MERS-CoV infection of human lung epithelium-derived Calu-3 cells. Here we established a quantitative fusion assay dependent on SARS-CoV-2 S protein, ACE2 and TMPRSS2, and found that nafamostat mesylate potently inhibited the fusion while camostat mesylate was about 10-fold less active. Furthermore, nafamostat mesylate blocked SARS-CoV-2 infection of Calu-3 cells with an EC50 around 10 nM, which is below its average blood concentration after intravenous administration through continuous infusion. These findings, together with accumulated clinical data regarding its safety, make nafamostat a likely candidate drug to treat COVID-19.

microbiology↗

bioRxiv2020

Multidrug treatment with nelfinavir and cepharanthine against COVID-19

Antiviral treatments targeting the emerging coronavirus disease 2019 (COVID-19) are urgently required. We screened a panel of already-approved drugs in a cell culture model of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and identified two new antiviral agents: the HIV protease inhibitor Nelfinavir and the anti-inflammatory drug Cepharanthine. In silico modeling shows Nelfinavir binds the SARS-CoV-2 main protease consistent with its inhibition of viral replication, whilst Cepharanthine inhibits viral attachment and entry into cells. Consistent with their different modes of action, in vitro assays highlight a synergistic effect of this combined treatment to limit SARS-CoV-2 proliferation. Mathematical modeling in vitro antiviral activity coupled with the known pharmacokinetics for these drugs predicts that Nelfinavir will facilitate viral clearance. Combining Nelfinavir/Cepharanthine enhanced their predicted efficacy to control viral proliferation, to ameliorate both the progression of disease and risk of transmission. In summary, this study identifies a new multidrug combination treatment for COVID-19.

microbiology↗

bioRxiv2019

Brainstem organoids from human pluripotent stem cells contain neural crest population

The brainstem controls heartbeat, blood pressure and respiration, which are life-sustaining functions, therefore, disorders of the brainstem can be lethal. Brain organoids derived from human pluripotent stem cells recapitulate the course of human brain development and are expected to be useful for medical research on central nervous system disorders. However, existing organoid models have limitations, hampering the elucidation of diseases affecting specific components of the brain. Here, we developed a method to generate human brainstem organoids (hBSOs), containing neural crest stem cells as well as midbrain/hindbrain progenitors, noradrenergic and cholinergic neurons, and dopaminergic neurons, demonstrated by specific electrophysiological signatures. Single-cell RNA sequence analysis, together with proteomics and electrophysiology, revealed that the cellular population in these organoids was similar to that of the human brainstem and neural crest, which raises the possibility of making use of hBSOs in grafting for transplantation, efficient drug screenings and modeling the neural crest diseases.

developmental biology↗

bioRxiv2019

Plasmodium caprae infection is negatively correlated with infection of Theileria ovis and Anaplasma ovis in goats

Theileria, Babesia, and Anaplasma are tick-borne pathogens affecting livestock industries worldwide. In this study, we surveyed the presence of Babesia ovis, Theileria ovis, Theileria lestoquardi, Anaplasma ovis, Anaplasma phagocytophilum, and Anaplasma marginale in 200 goats from 3 different districts in Sistan and Baluchestan province, Iran. Species-specific diagnostic PCR and sequence analysis revealed that 1.5%, 12.5%, and 80% of samples were positive for T. lestoquardi, T. ovis, and A. ovis, respectively. Co-infections of goats with up to 3 pathogens were seen in 22% of the samples. We observed a positive correlation between A. ovis and T. ovis infection. In addition, by analyzing the data with respect to Plasmodium caprae infection in these goats, a negative correlation was found between P. caprae and A. ovis and between P. caprae and T. ovis. This study contributes to understanding the epidemiology of vector-borne pathogens and their interplay in goats.\n\nImportanceTick-borne pathogens include economically important pathogens restricting livestock farming worldwide. In endemic areas livestock are exposed to different tick species carrying various pathogens which could result in co-infection with several tick-borne pathogens in a single host. The co-infection and interaction among pathogens are important in determining the outcome of disease. Little is known about pathogen interactions in the vector and the host. In this study, we show for the first time that co-infection of P. caprae, a mosquito transmitted pathogen, with T. ovis and A. ovis. Analysis of goat blood samples revealed a positive correlation between A. ovis and T. ovis. Moreover, a negative correlation was seen between P. caprae, a mosquito transmitted pathogen, and the tick-borne pathogens T. ovis or A. ovis.

epidemiology↗