Biology subjects

Wada, J.

Publications and source records attributed to Wada, J..

7 recordsLinked to original sources

bioRxiv2026

Modeling Risk Group 4 virus infection and antiviral treatment in microfluidic lung organ-on-chips in maximum containment laboratories

Development of candidate countermeasures against human pathogens frequently includes nonhuman animal experimentation. Preclinical animal pathogen exposure studies are conducted to model diseases and accumulate preliminary and hypothetically translatable data to inform and justify the design of clinical trial evaluation of countermeasure safety and efficacy. In addition to frequent ethical critiques, challenges associated with animal experimentation include considerable resources needed to achieve statistical power and robustness, replicability and reproducibility concerns, potentially compromised objectivity through lack of blinding, fundamental species-specific biological differences, and risk of unpredictable pathogen adaptation to the experimental animal. Recent U.S. and U.K. government initiatives aim to reduce animal experimentation by complementing or potentially replacing them with new approach methodologies (NAMs), i.e., increasingly sophisticated in silico, in chemico, and in vitro approaches. We piloted development of one type of NAM, organ-on-chips (OOCs), in the highly challenging environment of a maximum (biosafety level 4) containment laboratory. Using a Risk Group 4 virus, Nipah virus (NiV), and two types of lung OOCs seeded with human or porcine cells, we demonstrated the recapitulation of key features of NiV lung infection, including viral infection, replication, and translocation, that are associated with proinflammatory cytokine secretion, immune cell recruitment, and disruption of the air-liquid interface barrier. We reproduced the known anti-NiV activity of remdesivir and evaluated that of another potential antiviral, zotatifin. Our results pave the way for similar applications of advanced microphysiological systems for modeling infections caused by high-consequence viruses.

microbiology↗

bioRxiv2025

Claudin-1 Inhibitor PDS-0330 Ameliorates Diabetic Kidney Disease by Suppressing Src/Akt/mTOR Signaling Pathway in Podocytes

IntroductionRecent studies have reported that claudin-1, which is reactively expressed in malignant tumors and inflammatory diseases, plays an important role in disease progression. In diabetic kidney disease (DKD), upregulated expression of claudin-1 in podocytes has also been shown to contribute to disease progression. However, it remains unclear whether claudin-1 itself could be a therapeutic target in DKD. In this study, we investigated whether the claudin-1 inhibitor PDS-0330 could ameliorate DKD. MethodsFive-week-old male C57BL/6J mice were fed either a high-fat diet (HFD) or a normal diet (ND). At 15 weeks of age, PDS-0330 (Inh) or vehicle as control (Ctrl) was administered orally once per week for 4 weeks. At the end of the treatment period, blood, urine, and kidney samples were collected. In in vitro study, immortalized podocytes were treated with Inh or Ctrl and cultured for 48 hours in either normal glucose medium (5.5 mM; NG) or high glucose medium (25 mM; HG), after which proteins were extracted for analysis. ResultsMice fed an HFD showed significant increases in body weight and serum glucose levels compared with ND mice, and these parameters were not significantly affected by Inh treatment. HFD mice treated with Ctrl (HFD-Ctrl) exhibited significantly higher urinary albumin excretion than ND mice treated with Ctrl (ND-Ctrl), whereas the Inh-treated HFD mice (HFD-Inh) showed a significant reduction. Transmission electron microscopy revealed foot process effacement in HFD-Ctrl podocytes, which was markedly improved in HFD-Inh mice. Immunofluorescence staining demonstrated claudin-1 expression in podocytes of HFD mice. Furthermore, phospho-mTOR staining in podocytes was significantly increased in HFD-Ctrl compared with ND-Ctrl and was significantly attenuated in HFD-Inh. Western blot analysis of kidney samples revealed activation of the Src/Akt/mTOR signaling pathway in HFD-Ctrl mice, which was significantly reduced in HFD-Inh mice. In in vitro study, Ctrl-treated podocytes cultured in HG (HG-Ctrl) showed significant activation of Src/Akt/mTOR signaling compared with those in NG (NG-Ctrl). This activation was significantly suppressed in Inh-treated podocytes (HG-Inh). ConclusionThese findings suggest that podocyte claudin-1 could be a potential therapeutic target in DKD.

physiology↗

bioRxiv2025

Weak SLP-76-PLC-γ1 interaction in the LAT-nucleated multi-protein complex fine-tunes TCR signal strength to optimize T cell responsiveness

Upon TCR engagement several protein tyrosine kinases are recruited and activated, and adapter proteins and enzymes are phosphorylated on tyrosine residues, leading to further events characterizing activated T cells. Phosphorylation of the LAT adapter protein enables binding of the enzyme PLC-{gamma}1 and of a dimer of two additional adapter proteins Gads and SLP-76, forming a tetrameric structure. Within this heterotetramer there is a weak interaction between SLP-76 and PLC-{gamma}1, and the relevant binding sites of SLP-76 and PLC-{gamma}1 are highly conserved in vertebrates. To address the biological relevance of this weak interaction, we introduced a mutation in the SLP-76 that enhanced its affinity for PLC-{gamma}1 and found that this mutation increased PLC-{gamma}1 activity and altered thymocyte development and peripheral T cell responses due to enhanced TCR signal strength. The conserved weak SLP-76-PLC-{gamma}1 interaction is critical for the controlled activation of PLC-{gamma}1, thus fine-tuning TCR signal strength to optimize T cell-mediated immunity.

immunology↗

bioRxiv2025

Discovery of Light-Powered Organic Ion Transport by a Natural Protein

Membrane transport proteins play vital roles in living cells by selectively transporting ions and molecules across biological membranes. Among these, microbial rhodopsins are unique in their unparalleled capacity to harness light to drive ion translocation. This distinctive feature has led to their widespread use as optogenetic tools in neuroscience, physiology, and biomed-ical applications. While not all microbial rhodopsins function as ion transporters, many ion-translocating variants have been discov-ered since the identification of the first member--a light-driven H+ pump--in the 1970s. These proteins share a compact structure composed of only seven transmembrane helices and have long been thought to specialize exclusively in transporting small inorganic ions such as H+, Cl-, and Na+. Here, we show that several anion-pumping microbial rhodopsins can also transport organic anions. In particular, a rhodopsin from cyanobacteria is capable of transporting bulky organic anions, including those containing benzene rings, with molecular volumes up to [~]120 [A]3--five times that of Cl-. These organic ions bind to the dark state and are translocated upon photoactivation, following a mechanism similar to that of inorganic anion transport. Mutational analysis indicates that both classes of substrates share a common binding site. Only anions with pKa values below 2 were transported, suggesting that a retained negative charge is essential for binding to the dark state--a prerequisite for transport. This study expands the known sub-strate repertoire of microbial rhodopsins and introduces new possibilities for optogenetic strategies based on light-driven delivery of bioactive organic molecules.

biophysics↗

bioRxiv2025

Development and Validation of a Stem Cell-Based Kidney Organoid Platform for Nephrotoxicity Assessment

Background and hypothesisRecent cases of acute kidney injury (AKI) in Japan have been linked to the Beni-koji CholesteHelp supplement. Renal biopsies from affected patients revealed tubular damage, and puberulic acid was subsequently identified as a potential nephrotoxic contaminant. Recognizing the urgent need for a reliable in vitro nephrotoxicity testing platform, we hypothesized that a kidney organoid-based system could replicate nephrotoxic injury and help identify novel nephrotoxicants. In this study, we developed a screening model using kidney organoids derived from adult rat kidney stem (KS) cells and applied it to evaluate the toxicity of puberulic acid. MethodsKidney organoids were generated from KS cells and exposed to established nephrotoxicants, including cisplatin and gentamicin, to validate the model. The nephrotoxicity of puberulic acid was evaluated using both KS cell-derived organoids and wild-type mice. Nephrotoxicity was assessed by morphological changes, Kim-1 mRNA expression, transmission electron microscopy (TEM), and analysis of markers related to mitochondrial injury, oxidative stress, and apoptosis. ResultsThe kidney organoids reproduced morphological features of injury induced by known nephrotoxins and showed significant upregulation of Kim-1 mRNA. Puberulic acid-treated organoids displayed ultrastructural features consistent with acute tubular necrosis (ATN) and increased Kim-1 expression. In vivo, puberulic acid-exposed mice exhibited impaired renal function and histological findings consistent with ATN. Both in vitro and in vivo models revealed mitochondrial structural abnormalities and reduced expression of cytochrome c oxidase subunit IV (COX-IV). Additionally, oxidative stress and apoptotic markers, including 8-hydroxy-2-deoxyguanosine (8-OHdG) and cleaved caspase-3, were significantly elevated, suggesting that puberulic acid induces mitochondrial dysfunction and oxidative stress, leading to tubular cell death. ConclusionPuberulic acid-induced nephrotoxicity was demonstrated using our kidney organoid model. KS cell-derived kidney organoids provide a simple, reproducible, and rapid platform for nephrotoxicity assessment, which contribute to a reduction of animal use in toxicology research.

pharmacology and toxicology↗

bioRxiv2020

Characteristic and quantifiable COVID-19-like abnormalities in CT- and PET/CT-imaged lungs of SARS-CoV-2-infected crab-eating macaques (Macaca fascicularis)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing an exponentially increasing number of coronavirus disease 19 (COVID-19) cases globally. Prioritization of medical countermeasures for evaluation in randomized clinical trials is critically hindered by the lack of COVID-19 animal models that enable accurate, quantifiable, and reproducible measurement of COVID-19 pulmonary disease free from observer bias. We first used serial computed tomography (CT) to demonstrate that bilateral intrabronchial instillation of SARS-CoV-2 into crab-eating macaques (Macaca fascicularis) results in mild-to-moderate lung abnormalities qualitatively characteristic of subclinical or mild-to-moderate COVID-19 (e.g., ground-glass opacities with or without reticulation, paving, or alveolar consolidation, peri-bronchial thickening, linear opacities) at typical locations (peripheral>central, posterior and dependent, bilateral, multi-lobar). We then used positron emission tomography (PET) analysis to demonstrate increased FDG uptake in the CT-defined lung abnormalities and regional lymph nodes. PET/CT imaging findings appeared in all macaques as early as 2 days post-exposure, variably progressed, and subsequently resolved by 6-12 days post-exposure. Finally, we applied operator-independent, semi-automatic quantification of the volume and radiodensity of CT abnormalities as a possible primary endpoint for immediate and objective efficacy testing of candidate medical countermeasures.

microbiology↗

bioRxiv2018

Human, Nonhuman Primate, and Bat Cells Are Broadly Susceptible to Tibrovirus Particle Cell Entry

In 2012, the genome of a novel rhabdovirus, Bas-Congo virus, was discovered in the acute-phase serum of a Congolese patient with presumed viral hemorrhagic fever. In the absence of a replicating virus isolate, fulfilling Kochs postulates to determine whether Bas-Congo virus is indeed a human virus and/or pathogen has been impossible. However, experiments with vesiculoviral particles pseudotyped with Bas-Congo glycoprotein suggested that Bas-Congo virus particles can enter cells from multiple animals, including humans. In 2015, genomes of two related viruses, Ekpoma virus 1 and Ekpoma virus 2, were detected in human sera in Nigeria. Isolates could not be obtained. Phylogenetic analyses led to the classification of Bas-Congo virus, Ekpoma virus 1, and Ekpoma virus 2 in the same genus, Tibrovirus, together with five biting midge-borne rhabdoviruses (i.e., Beatrice Hill virus, Bivens Arm virus, Coastal Plains virus, Sweetwater Branch virus, and Tibrogargan virus) not known to infect humans. Using individual recombinant vesiculoviruses expressing the glycoproteins of all eight known tibroviruses and more than 75 cell lines representing different animal species, we demonstrate that the glycoproteins of all tibroviruses can mediate vesiculovirus particle entry into human, bat, nonhuman primate, cotton rat, boa constrictor, and Asian tiger mosquito cells. Using four of five isolated authentic tibroviruses (i.e., Bivens Arm virus, Coastal Plains virus, Sweetwater Branch virus, and Tibrogargan virus), our experiments indicate that many cell types may be partially resistant to tibrovirus replication after virion cell entry. Consequently, experimental data solely obtained from experiments using tibrovirus surrogate systems (e.g., vesiculoviral pseudotypes, recombinant vesiculoviruses) cannot be used to predict whether Bas-Congo virus, or any other tibrovirus, infects humans.

microbiology↗