Sevoflurane induces cardioprotection through reactive oxygen species-mediated upregulation of autophagy in isolated guinea pig hearts.

PURPOSE: Sevoflurane increases reactive oxygen species (ROS), which mediate cardioprotection against myocardial ischemia-reperfusion injury. Emerging evidence suggests that autophagy is involved in cardioprotection. We examined whether reactive oxygen species mediate sevoflurane preconditioning through autophagy. METHODS: Isolated guinea pigs hearts were subjected to 30 min ischemia followed by 120 min reperfusion (control). Anesthetic preconditioning was elicited with 2 % sevoflurane for 10 min before ischemia (SEVO). The ROS-scavenger, N-(2-mercaptopropionyl) glycine (MPG, 1 mmol/l), was administered starting 30 min before ischemia to sevoflurane-treated (SEVO + MPG) or non-sevoflurane-treated (MPG) hearts. Infarct size was determined by triphenyltetrazolium chloride stain. Tissue samples were obtained after reperfusion to determine autophagy-related protein (microtubule-associated protein light chain I and II: LC3-I, -II) and 5\u27 AMP-activated protein kinase (AMPK) expression using Western blot analysis. Electron microscopy was used to detect autophagosomes. RESULTS: Infarct size was significantly reduced and there were more abundant autophagosomes in SEVO compared with control. Western blot analysis revealed that the ratio of LC3-II/I and phosphorylation of AMPK were significantly increased in SEVO. These effects were abolished by MPG. CONCLUSIONS: Sevoflurane induces cardioprotection through ROS-mediated upregulation of autophagy

Sevoflurane confers additive cardioprotection to ethanol preconditioning associated with enhanced phosphorylation of glycogen synthase kinase-3β and inhibition of mitochondrial permeability transition pore opening.

OBJECTIVE: The purposes of this study were to investigate whether sevoflurane (SEVO) enhances moderate-dose ethanol (EtOH) preconditioning and whether this additional cardioprotection is associated with glycogen synthase kinase-3β (GSK-3β), protein kinase B (Akt), mammalian target of rapamycin (mTOR), 70-kDa ribosomal s6 kinase-1 (p70s6K), and/or mitochondrial permeability transition pore (MPTP) opening. DESIGN: In vitro study using an isolated heart Langendorff preparation. SETTING: University research laboratory. PARTICIPANTS: Male guinea pigs (n = 170). INTERVENTIONS: Isolated perfused guinea pig hearts underwent 30-minute ischemia and 120-minute reperfusion (control). The EtOH group received 5% EtOH in the drinking water for 8 weeks. Anesthetic preconditioning was elicited by a 10-minute exposure to 2% SEVO in EtOH (EtOH + SEVO group) or non-EtOH (SEVO group) hearts. The inhibition of GSK-3β phosphorylation and mTOR was achieved with LY294002 and rapamycin, respectively. GSK-3β, Akt, mTOR, and p70s6K expressions were determined by western blot. Calcium-induced MPTP opening was assessed in isolated calcein-loaded mitochondria. MEASUREMENTS AND MAIN RESULTS: After ischemia-reperfusion, the EtOH, SEVO, and EtOH + SEVO groups had higher left ventricular developed pressure recovery and lower end-diastolic pressure versus the control group. Infarct size was smaller in the EtOH and SEVO groups versus control and even smaller in the EtOH + SEVO group. Phosphorylation of GSK-3β and Akt, but not mTOR and p70s6K, was increased in the EtOH and SEVO groups. Phosphorylation of GSK-3β, but not mTOR and p70s6K, was further increased in the EtOH + SEVO group. The EtOH and SEVO groups exhibited a smaller calcium-induced MPTP opening, and the EtOH + SEVO presented an even smaller MPTP opening. CONCLUSIONS: SEVO and chronic EtOH preconditioning offer additive cardioprotection. This effect is associated with an increased GSK-3β phosphorylation and an inhibition of MPTP opening

Induction of autophagy restores the loss of sevoflurane cardiac preconditioning seen with prolonged ischemic insult.

Sevoflurane preconditioning against myocardial ischemia-reperfusion injury is lost if the ischemic insult is too long. Emerging evidence suggests that induction of autophagy may also confer cardioprotection against ischemia-reperfusion injury. We examined whether induction of autophagy prolongs sevoflurane preconditioning protection during a longer ischemic insult. Isolated guinea pigs hearts were subjected to 30 or 45 min ischemia, followed by 120 min reperfusion (control). Anesthetic preconditioning was elicited with 2% sevoflurane for 10 min prior to ischemia (SEVO-30, SEVO-45). Chloramphenicol (autophagy upregulator, 300 µM) was administered starting 20 min before ischemia and throughout reperfusion in SEVO-45 (SEVO-45+CAP). To inhibit autophagy, 3-methyladenine (10 μM) was administered during sevoflurane administration in SEVO-45+CAP. Infarct size was determined by triphenyltetrazolium chloride stain. Tissue samples were obtained before ischemia to determine autophagy-related protein (microtubule-associated protein light chain I and II: LC3-I, II), Akt and glycogen synthase kinase 3β (GSK3β) expression using Western blot analysis. The effect of autophagy on calcium-induced mitochondrial permeability transition pore (MPTP) opening in isolated calcein-loaded mitochondria was assessed. Electron microscopy was used to detect autophagosomes. Infarct size was significantly reduced in SEVO-30, but not in SEVO-45. Chloramphenicol restored sevoflurane preconditioning lost by 45 min ischemia. There were more abundant autophagozomes and LC3-II expression was significantly increased in SEVO-45+CAP. Induction of autophagy before ischemia enhanced GSK3β phosphorylation and inhibition of calcium-induced MPTP opening. These effects were abolished by 3-methyladenine. Pre-ischemic induction of autophagy restores sevoflurane preconditioning lost by longer ischemic insult. This effect is associated with enhanced inhibition of MPTP by autophagy

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

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

Carbon-ion Radiotherapy for Hepatocellular Carcinoma

AbstractClinical trials of carbon ion radiotherapy (C-ion RT) for hepatocellular carcinoma (HCC) were conducted at the National Institute of Radiological Sciences (NIRS) in Japan between April 1995 and August 2005. In four clinical studies, we tried to determine the optimal dose and to shorten the treatment duration using five different fractionation schedules comprising 15, 12, eight, four and two fractions. There have been no treatment-related deaths and no severe adverse events. As a result of these studies, the duration of treatment could be reduced from five weeks to two days. Two-fraction therapy is currently ongoing, licensed as a Highly Advanced Medical Technology. Between April 2003 and August 2012, 133 patients with HCC underwent two fraction C-ion RT. There were no cases of grade 4 toxicity. The local control rates were 98% and 83% at one and three years, respectively, with a total dose of 45.0 GyE. Because of the low toxicity and good local control rate, C-ion RT is a promising radical and minimally invasive therapeutic option for HCC

In-gel multiple displacement amplification of long DNA fragments diluted to the single molecule level

The isolation and multiple genotyping of long individual DNA fragments are needed to obtain haplotype information for diploid organisms. Limiting dilution of sample DNA followed by multiple displacement amplification is a useful technique but is restricted to short (<5 kb) DNA fragments. In the current study, a novel modification was applied to overcome these problems. A limited amount of cellular DNA was carefully released from intact cells into a mildly heated alkaline agarose solution and mixed thoroughly. The solution was then gently aliquoted and allowed to solidify while maintaining the integrity of the diluted DNA. Exogenously provided Phi29 DNA polymerase was used to perform consistent genomic amplification with random hexameric oligonucleotides within the agarose gels. Simple heat melting of the gel allowed recovery of the amplified materials in a solution of the polymerase chain reaction (PCR)-ready form. The haplotypes of seven SNPs spanning 240 kb of the DNA surrounding the human ATM gene region on chromosome 11 were determined for 10 individuals, demonstrating the feasibility of this new method

Solid-phase multiple displacement amplification for multi-locus genotyping of single chromosome molecules

(1) Background: Despite recent innovations in high throughput shotgun sequencing technologies, complex rearrangements in addition to the original dizygotic existence of homologous chromosomes complicate cancer genome sequencing. Thus there is an urgent need for establishing a convenient method to manipulate long stretches of individual chromosomes for analyzing sequence information.(2) Materials and Methods: A novel methodology has been developed to amplify single chromosomes for genotyping. A key feature of this methodology is a solid-phase multiple displacement amplification, that is an enzymatic reaction of Phi29 DNA polymerase, within a solidified agarose gel. It consists of the following seven steps. (I) Lysis of a limited number of cultured cells within a heated agarose gel solution to release chromosome molecules. (II) Careful aliquoting of small volumes of gel solutions containing limited number of chromosome molecules. (III) Solidification of the gel on ice. (IV) Solid-phase multiple displacement amplification of the gel-immobilized individual chromosome molecules. (V) Recovery of the amplification products by heating. (VI) Screening of target chromosomes by real-time QPCR. (VII) Multi-loci SNP typing using newly developed on-plastic chip allele-specific primer extension method (Michikawa et al., Anal. Sci., 2006, 22, 1537-1545). (3) Results: Utilization of agarose gel as a reaction matrix enabled reliable amplification-ready limited dilution of DNA to the level that homologous chromosomes rarely co-localize. Aggregation of chromosomes during the dilution step was dramatically reduced by incubating the gel solution at alkaline pH and at high temperature. Separation of homologous chromosomes by this method provided reliable determination of multi-loci genotypes on each homologous chromosome. Using this methodology, we have successfully determined haplotype of multiple SNPs in human ATM region that span 200 kilobase pairs. (4) Conclusions: The methodology developed in this study is effective for genotyping individual homologous chromosomes. Since amplified materials are easily recovered in a solution as PCR-ready form, this methodology is not restricted for genotyping. Further applications, such as chromosome-wide sequencing, are considerable.The 14th European Cancer Conference, ESTRO26 meeting, European Society for therapeutic radiology and oncolog