Photo-Induced Cell Damage Analysis for Single- and Multifocus Coherent Anti-Stokes Raman Scattering Microscopy

In this study, we investigated photo-induced damage to living cells during single-and multifocus excitations for coherent anti-Stokes Raman scattering (CARS) imaging. A near-infrared pulsed laser (709 nm) was used to induce cell damage. We compared the photo-induced cell damage in the single- and the multifocus excitation schemes with the condition to obtain the same CARS signal in the same frame rate. For the evaluation of cell viability, we employed 4', 6-diamidino-2-phenylindole (DAPI) fluorophores that predominantly stained the damaged cells. One-and two-photon fluorescence of DAPI fluorophores were, respectively, excited by an ultraviolet light source and the same near-infrared light source and were monitored to evaluate the cell viability during near-infrared pulsed laser irradiation. We found lower uptake of DAPI fluorophores into HeLa cells during the multifocus excitation compared with the single- focus excitation scheme in both the one- and the two-photon fluorescence examinations. This indicates a reduction of photo-induced cell damage in the multifocus excitation. Our findings suggested that the multifocus excitation scheme is expected to be suitable for CARS microscopy in terms of minimal invasiveness

Photo-Induced Cell Damage Analysis for Single- and Multifocus Coherent Anti-Stokes Raman Scattering Microscopy

In this study, we investigated photo-induced damage to living cells during single- and multifocus excitations for coherent anti-Stokes Raman scattering (CARS) imaging. A near-infrared pulsed laser (709 nm) was used to induce cell damage. We compared the photo-induced cell damage in the single- and the multifocus excitation schemes with the condition to obtain the same CARS signal in the same frame rate. For the evaluation of cell viability, we employed 4',6-diamidino-2-phenylindole (DAPI) fluorophores that predominantly stained the damaged cells. One- and two-photon fluorescence of DAPI fluorophores were, respectively, excited by an ultraviolet light source and the same near-infrared light source and were monitored to evaluate the cell viability during near-infrared pulsed laser irradiation. We found lower uptake of DAPI fluorophores into HeLa cells during the multifocus excitation compared with the single-focus excitation scheme in both the one- and the two-photon fluorescence examinations. This indicates a reduction of photo-induced cell damage in the multifocus excitation. Our findings suggested that the multifocus excitation scheme is expected to be suitable for CARS microscopy in terms of minimal invasiveness

A non-carboxylating pentose bisphosphate pathway in halophilic archaea

Bacteria and Eucarya utilize the non-oxidative pentose phosphate pathway to direct the ribose moieties of nucleosides to central carbon metabolism. Many archaea do not possess this pathway, and instead, Thermococcales utilize a pentose bisphosphate pathway involving ribose-1, 5-bisphosphate (R15P) isomerase and ribulose-1, 5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco). Intriguingly, multiple genomes from halophilic archaea seem only to harbor R15P isomerase, and do not harbor Rubisco. In this study, we identify a previously unrecognized nucleoside degradation pathway in halophilic archaea, composed of guanosine phosphorylase, ATP-dependent ribose-1-phosphate kinase, R15P isomerase, RuBP phosphatase, ribulose-1-phosphate aldolase, and glycolaldehyde reductase. The pathway converts the ribose moiety of guanosine to dihydroxyacetone phosphate and ethylene glycol. Although the metabolic route from guanosine to RuBP via R15P is similar to that of the pentose bisphosphate pathway in Thermococcales, the downstream route does not utilize Rubisco and is unique to halophilic archaea

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Seismic Exploration Using Active Sources at Kuchierabujima Volcano, Southwest Japan

Seismic exploration using artificial sources was conducted at Kuchierabujima volcano, southwest Japan in November 2004 by 40 participants from 9 national universities andJapan Meteorological Agency to investigate the subsurface seismic structure. The exploration was the 11th joint experiment under the National Project for Prediction of Volcanic Eruptions. A total of 183 temporal stations equippedwith a 2 Hz vertical component seismometer (including 75 3component seismometers) and a portable data logger were deployed on Kuchierabu Island. Dynamite shots with charges of 10-115 kg were detonated at 19 locations, and seismic signals were successfully recorded. To reveal the P-wave velocity structure, 2955 arrival times of the first motion were picked from the seismograms, and 2187 were classified into ranks A and B. From the record sections and the arrival time data, characteristics reflecting the geological structure were identified. Refracted waves of 5 km/s were observed at stations＞5km from the shot points. Apparent velocities near the shot points depend on the surface geology around the shots. P-wave arrived earlier at stations near the summits. Strongly scattered waves were observed similarly near the summits

Invited Article: Label-free nerve imaging with a coherent anti-Stokes Raman scattering rigid endoscope using two optical fibers for laser delivery

A coherent anti-Stokes Raman scattering (CARS) rigid endoscope using two optical fibers to deliver excitation beams individually is developed. The use of two optical fibers allows the correction of longitudinal chromatic aberration and enhances the CARS signal by a factor of 2.59. The endoscope is used to image rat sciatic nerves with an imaging time of 10 s. Imaging of the rabbit prostatic fascia without sample slicing is also demonstrated, which reveals the potential for the application of the CARS endoscope to robot-assisted surgery

<研究論文>Developing Teacher Educators and School Teachers through Collaborative School-based Action Research

[The World Association of Lesson Studies International Conference 2017] 24-27 November 2017, Nagoya University (http://www.walsnet.org/2017/

Hepatocyte Growth Factor Activator: A Proteinase Linking Tissue Injury with Repair

Hepatocyte growth factor (HGF) promotes pleiotropic signaling through its specific receptor tyrosine kinase, MET. As such, it has important roles in the regeneration of injured tissues. Since HGF is produced mainly by mesenchymal cells and MET is expressed in most epithelial, endothelial and somatic stem cells, HGF functions as a typical paracrine growth factor. HGF is secreted as an inactive precursor (proHGF) and requires proteolytic activation to initiate HGF-induced MET signaling. HGF activator (HGFAC) is a serum activator of proHGF and produces robust HGF activities in injured tissues. HGFAC is a coagulation factor XII-like serine endopeptidase that circulates in the plasma as a zymogen (proHGFAC). Thrombin, kallikrein-related peptidase (KLK)-4 or KLK-5 efficiently activates proHGFAC. The activated HGFAC cleaves proHGF at Arg494-Val495, resulting in the formation of the active disulfide-linked heterodimer HGF. Macrophage stimulating protein, a ligand of RON, is also activated by HGFAC in vivo. Although HGFAC functions primarily at the site of damaged tissue, a recent report has suggested that activated HGFAC relays a signal to stem cells in non-injured tissues via proHGF activation in the stem cell niche. This review focuses on current knowledge regarding HGFAC-mediated proHGF activation and its roles in tissue regeneration and repair