Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse

ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CLT (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its Vmax⁡/mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dose-dependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3

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

Dose-Dependent Change in Elimination Kinetics of Ethanol due to Shift of Dominant Metabolizing Enzyme from ADH 1 (Class I) to ADH 3 (Class III) in Mouse

ADH 1 and ADH 3 are major two ADH isozymes in the liver, which participate in systemic alcohol metabolism, mainly distributing in parenchymal and in sinusoidal endothelial cells of the liver, respectively. We investigated how these two ADHs contribute to the elimination kinetics of blood ethanol by administering ethanol to mice at various doses, and by measuring liver ADH activity and liver contents of both ADHs. The normalized AUC (AUC/dose) showed a concave increase with an increase in ethanol dose, inversely correlating with β. CL T (dose/AUC) linearly correlated with liver ADH activity and also with both the ADH-1 and -3 contents (mg/kg B.W.). When ADH-1 activity was calculated by multiplying ADH-1 content by its V max /mg (4.0) and normalized by the ratio of liver ADH activity of each ethanol dose to that of the control, the theoretical ADH-1 activity decreased dosedependently, correlating with β. On the other hand, the theoretical ADH-3 activity, which was calculated by subtracting ADH-1 activity from liver ADH activity and normalized, increased dose-dependently, correlating with the normalized AUC. These results suggested that the elimination kinetics of blood ethanol in mice was dose-dependently changed, accompanied by a shift of the dominant metabolizing enzyme from ADH 1 to ADH 3

Comparing methods of detection and quantitation of RNA editing of rat glycine receptor alpha3P185L

<p><b>Background:</b> Recently, it has become evident that RNA editing-related changes are important in the modulation of neuronal information processing. Alternatively edited transcripts, when meagerly present, are hard to detect. Significant functional consequences may result, however, from small differences in editing efficiency. Moreover, it is difficult to evaluate the ratio of edited transcripts. The glycine receptor alpha3 subunit (GlyR alpha3) is expressed in the spinal cord, and transcripts of GlyR alpha3 are susceptible to RNA editing. The physiological role of this editing is still unclear. To analyze changes in RNA editing in various animal models, we need reliable and practical ways to detect and quantitate GlyR alpha3 RNA editing.</p> <p><b>Results:</b> We identified and assessed different ways of detecting edited RNA transcripts, including direct sequencing, denaturing high performance chromatography (DHPLC), allele-specific real-time PCR with TaqMan probes, and PCR with allele-specific primers. Using PCR with allele-specific primers on standard PCR products for edited and nonedited GlyR alpha3, we were able to detect as little as a 0.5% incidence of edited transcripts. We were able to detect a 5% incidence of RNA editing using direct sequencing and 2% using DHPLC. We could accurately determine the ratio of edited to non-edited RNA using DHPLC, direct sequencing, and allele-specific real-time PCR with TaqMan probes.</p> <p><b>Conclusion: </b>We demonstrated exact and sensitive methods of detecting RNA editing. In prepared samples, we showed means of quantitating the incidence of editing of a particular site. The demonstrated methodologies should be very useful when extended to the evaluation of other types of RNA editing and single base mutations.</p