Biology subjects

Ohno, A.

Publications and source records attributed to Ohno, A..

4 recordsLinked to original sources

bioRxiv2025

Evaluation of graphene oxide-mediated NET formation using HL-60-derived neutrophil-like cells

Neutrophil extracellular traps (NETs) are chromatin-based structures released by activated neutrophils in response to pathogens or chemical stimuli, contributing to host defense but also implicated in autoimmune disease and inflammation. As NET formation gains attention as an endpoint in in vitro immunotoxicity screening, the lack of reproducible and scalable systems hampers its broader application. Here, we developed an in vitro assay using HL-60-derived neutrophil-like cells (dHL-60) differentiated with all-trans retinoic acid to evaluate NET-inducing activity in a standardized, non-animal model. Graphene oxide (GO), a model nanomaterial known to trigger NETs in primary neutrophils, induced concentration-dependent NET formation in dHL-60 cells, with maximal induction at intermediate doses and attenuation at higher concentrations, likely due to particle aggregation. NET formation was validated by extracellular DNA staining and scanning electron microscopy. GO also induced superoxide-mediated ROS production, as confirmed by electron spin resonance, consistent with canonical NETosis pathways. Furthermore, GO suppressed PMA-induced NET formation, suggesting a dose- and context-dependent immunomodulatory effect. Collectively, our results demonstrate that dHL-60 cells recapitulate key features of NETosis observed in primary neutrophils and provide a practical, reproducible model for investigating immune responses to nanomaterials. This system supports the development of non-animal approaches for assessing immunological effects of chemical substances under controlled in vitro conditions.

pharmacology and toxicology↗

bioRxiv2020

Rapid profiling of drug-resistant bacteria using DNA-binding dyes and a nanopore-based DNA sequencer

Spread of drug-resistant bacteria is a serious problem worldwide. We thus designed a new sequence-based protocol that can quickly identify bacterial compositions of clinical samples and their drug-resistance profiles simultaneously. Here we utilized propidium monoazide (PMA) that prohibits DNA amplifications from dead bacteria, and subjected the original and antibiotics-treated samples to 16S rRNA metagenome sequencing. We tested our protocol on bacterial mixtures, and observed that sequencing reads derived from drug-resistant bacteria were significantly increased compared with those from drug-sensitive bacteria when samples were treated by antibiotics. Our protocol is scalable and will be useful for quickly profiling drug-resistant bacteria.

microbiology↗

bioRxiv2020

Cystal structure of inhibitor-bound human MSPL/TMPRSS13 that can activate high pathogenic avian influenza

Infection of certain influenza viruses is triggered when its hemagglutinin (HA) is cleaved by host cell proteases such as proprotein convertases and type II transmembrane serine proteases (TTSP). HA with a monobasic motif is cleaved by trypsin-like proteases, including TMPRSS2 and HAT, while the multibasic motif found in high pathogenicity avian influenza HA is cleaved by furin, PC5/6, or MSPL. MSPL belongs to the TMPRSS family and preferentially cleaves [R/K]-K-K-R{downarrow} sequences. Here, we solved the crystal structure of the extracellular region of human MSPL in complex with an irreversible substrate-analog inhibitor. The structure revealed three domains clustered around the C-terminal -helix of the SPD. The inhibitor structure and its putative model show that the P1-Arg inserts into the S1 pocket, whereas the P2-Lys and P4-Arg interacts with the Asp/Glu-rich 99-loop that is unique to MSPL. Based on the structure of MSPL, we also constructed a homology model of TMPRSS2, which is essential for the activation of the SARS-CoV-2 spike protein and infection. The model may provide the structural insight for the drug development for COVID-19.

biophysics↗

bioRxiv2020

CO2 reduction driven by a pH gradient

All life on Earth is built of organic molecules, so the primordial sources of reduced carbon are a major open question in studies of the origin of life. A variant of the alkaline-vent theory suggests that organics could have been produced by the reduction of CO2 via H2 oxidation, facilitated by geologically sustained pH gradients. The process would be an abiotic analog--and proposed evolutionary predecessor--of the modern Wood-Ljungdahl acetyl-Co-A pathway of extant archaea and bacteria. The first energetic bottleneck of the pathway involves the endergonic reduction of CO2 with H2 to formate, which has proven elusive in low-temperature abiotic settings. Here we show the reduction of CO2 with H2 at moderate pressures (1.5 bar), driven by microfluidic pH gradients across inorganic Fe(Ni)S precipitates. Isotopic labelling with 13C confirmed production of formate. Separately, deuterium (2H) labelling indicated that electron transfer to CO2 did not occur via direct hydrogenation with H2. Instead, freshly deposited Fe(Ni)S precipitates appear to facilitate electron transfer in an electrochemical-cell mechanism with two distinct half-reactions. Decreasing the pH gradient significantly, or removing either H2 or the precipitate, yielded no detectable product. Our work demonstrates the feasibility of spatially separated, yet electrically coupled geochemical reactions as drivers of otherwise endergonic processes. Beyond corroborating the ability of early-Earth alkaline hydrothermal systems to couple carbon reduction to hydrogen oxidation through geologically plausible and biologically relevant mechanisms, these results may also be of significance for industrial and environmental applications, where other redox reactions could be facilitated using similarly mild approaches.

biochemistry↗