Biology subjects

Tabata, K. V.

Publications and source records attributed to Tabata, K. V..

3 recordsLinked to original sources

bioRxiv2024

High-speed Intracellular Temperature Mapping Reveals the Existence of Non-Conductive Dissipation of Energy by Heating

Recently, intracellular thermometry has revealed temperature variations within cells. Although the biological significance of intracellular temperature change is recognized, the physical principles of intracellular temperature change remain a mystery. Here, we investigate intracellular heat transfer through intracellular temperature mapping using a fluorescent polymeric thermometer and high-speed fluorescence lifetime imaging microscopy. Through infrared laser irradiation-assisted heating, we track changes in temperature distribution to examine the mechanism of intracellular heat dissipation in comparison with heat conduction. Continuous heating induces the significantly slower relaxation of the average temperature of single cells compared with that of liposomes containing homogeneous aqueous solutions of comparable size; to the scale of seconds. We additionally elucidate that these phenomena are impacted by intracellular structures and molecules. Finally, we discover that this slow intracellular temperature relaxation originates from non-diffusive heat dissipation distinct from the conventional heat conduction model. Our results provide insights into the mechanisms of temperature variation in cells that are unresolved based on our current understanding, establishing a framework for understanding intracellular thermodynamics under non-equilibrium conditions.

biophysics↗

bioRxiv2024

In vitro one-pot construction of influenza viral genomes for virus particle synthesis based on reverse genetics system

The reverse genetics system, which allows the generation of influenza viruses from plasmids encoding viral genome, is a powerful tool for basic research on viral infection mechanisms and application research such as vaccine development. However, conventional plasmid construction using Escherichia coli (E. coli) cloning is time-consuming and has difficulties handling DNA encoding genes toxic for E. coli or highly repeated sequences. These limitations hamper rapid virus synthesis. In this study, we establish a very rapid in vitro one-pot plasmid construction (IVOC) based virus synthesis. This method dramatically reduced the time for genome plasmid construction, which was used for virus synthesis, from several days or more to about 8 hours. Moreover, infectious viruses could be synthesized with a similar yield to the conventional E. coli cloning-based method with high accuracy. The applicability of this method was also demonstrated by the generation of recombinant viruses carrying reporter genes from the IVOC products. This method is expected to potentially advance further understanding of influenza viruses and apply to other RNA viruses.

synthetic biology↗

bioRxiv2020

Multi-dimensional digital bioassay platform based on an air-sealed femtoliter reactor array device

Single-molecule experiments have been helping us to get deeper inside biological phenomena by illuminating how individual molecules actually work. Digital bioassay, in which analyte molecules are individually confined in small compartments to be analyzed, is an emerging technology in single-molecule biology and applies to various biological entities (e.g., cells and virus particles). However, digital bioassay is not compatible with multi-conditional or multi-parametric assays, hindering understanding of analytes. This is because current digital bioassay lacks a repeatable solution-exchange system that keeps analytes inside compartments. To address this challenge, we developed a new digital bioassay platform with easy solution exchanges, called multi-dimensional (MD) digital bioassay, and tested its quantitativity and utility. We immobilized single analytes in arrayed femtoliter (10-15 L) reactors and sealed them with airflow. The solution in each reactor was stable and showed no cross-talk via solution leakage for more than 2 h, and over 30 rounds of perfect solution exchanges were successfully performed. To show the utility of our system, we investigated neuraminidase inhibitor (NAI) sensitivity on single influenza A virus (IAV) particles in a multi-conditional assay. We proved that IAV particles show a heterogeneous response to the NAI. Further, to demonstrate multi-parametric assays, we examined the sensitivity of individual IAV particles or model enzyme molecules to two different inhibitors. Our results support that MD digital bioassay is a versatile platform to unveil heterogeneities of biological entities in unprecedented resolution.

biophysics↗