Neuromedin U-deficient rats do not lose body weight or food intake

Studies in genetically modified mice establish that essential roles of endogenous neuromedin U (NMU) are anorexigenic function and metabolic regulation, indicating that NMU is expected to be a potential target for anti-obesity agents. However, in central administration experiments in rats, inconsistent results have been obtained, and the essential role of NMU energy metabolism in rats remain unclear. This study aims to elucidate the role of endogenous NMU in rats. We generated NMU knockout (KO) rats that unexpectedly showed no difference in body weight, adiposity, circulating metabolic markers, body temperature, locomotor activity, and food consumption in both normal and high fat chow feeding. Furthermore, unlike reported in mice, expressions of Nmu and NMU receptor type 2 (Nmur2) mRNA were hardly detectable in the rat hypothalamic nuclei regulating feeding and energy metabolism, including the arcuate nucleus and paraventricular nucleus, while Nmu was expressed in pars tuberalis and Nmur2 was expressed in the ependymal cell layer of the third ventricle. These results indicate that the species-specific expression pattern of Nmu and Nmur2 may allow NMU to have distinct functions across species, and that endogenous NMU does not function as an anorexigenic hormone in rats

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

Successful production of genome-edited rats by the rGONAD method

Abstract Background Recent progress in development of the CRISPR/Cas9 system has been shown to be an efficient gene-editing technology in various organisms. We recently developed a novel method called Genome-editing via Oviductal Nucleic Acids Delivery (GONAD) in mice; a novel in vivo genome editing system that does not require ex vivo handling of embryos, and this technology is newly developed and renamed as “improved GONAD” (i-GONAD). However, this technology has been limited only to mice. Therefore in this study, we challenge to apply this technology to rats. Results Here, we determine the most suitable condition for in vivo gene delivery towards rat preimplantation embryos using tetramethylrhodamine-labelled dextran, termed as Rat improved GONAD (rGONAD). Then, to investigate whether this method is feasible to generate genome-edited rats by delivery of CRISPR/Cas9 components, the tyrosinase (Tyr) gene was used as a target. Some pups showed albino-colored coat, indicating disruption of wild-type Tyr gene allele. Furthermore, we confirm that rGONAD method can be used to introduce genetic changes in rat genome by the ssODN-based knock-in. Conclusions We first establish the rGONAD method for generating genome-edited rats. We demonstrate high efficiency of the rGONAD method to produce knock-out and knock-in rats, which will facilitate the production of rat genome engineering experiment. The rGONAD method can also be readily applicable in mammals such as guinea pig, hamster, cow, pig, and other mammals

Additional file 3: of Successful production of genome-edited rats by the rGONAD method

Table S1. Tyr-mediated mutations in F1 (DA male/WKY female) rat. Table S2. Tyr-mediated mutations in F1 (WKY male/DA female) rat. Table S3. Tyr-mediated mutations in F1 offspring. Table S4. Coat-color phenotypes recovered from albino in WKY rat. Table S5. CRISPR/Cas9 target sequence and ssODN used. (PDF 46 kb

The involvement of NF-κB activation in IL-1β-mediated-MMP-3 expression.

(a) The effect of IL-1β (100 pM, 24 h) on MMP-3 mRNA expression in melanoma cells with or without the NF-κB inhibitor, TPCA-1 (10 μM, 1 h). TBP was used as a reference, and the relative expression levels of MMP-3 mRNA in IL-1β-stimulated melanoma cells were compared with that of 0 h. The results are shown as mean ± standard error (SE) of biological triplicates. *PP<0.05.</p

The effect of IL-1β on intracellular pH (a) and the mRNA expression of NHE1 (b).

The cells were treated with (closed) or without (open) 100 pM IL-1β. IL-1β failed to induce the changes in intracellular pH (a, n = 20 cells, randomly selected ×20 fields from triplicate samples) and mRNA expression of NHE1 (b). Data are shown as the mean ± standard error of three independent experiments. (PDF)</p

Effect of the NF-κB inhibitor on the IL-1β-induced phosphorylation of p65/RelA and p105.

The cells were pretreated with the NF-κB inhibitor, TPCA-1 (10 μM, 1 h), followed by the stimulation of IL-1β (100 pM) for 15 min. Phosphorylated p65/RelA (p-p65) and p105 (p-p105), and total p65/RelA (t-p65) and p105 (t-p105) were detected by Western blotting. Representative images of the inhibitory effect of TPCA-1 on IL-1β-induced phosphorylation of p65/RelA (a) and p105 (c) are shown. The relative levels of [p-p65]/[t-p65] (b) and [p-p-105]/[t-105] (d) relative to levels without the inhibitor and IL-1β are illustrated. The results are represented as mean ± SE of biological triplicates. *P<0.05.</p

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Expression of MMP-3 and cellular migration in IL-1β-treated canine melanoma cells.

MMP-3 activity in the culture medium (a) and MMP-3 mRNA expression (c) of canine melanoma cells in the presence (closed circle) or absence (open circle) of canine recombinant IL-1β (100 pM). MMP-3 activity in the culture medium (b) and MMP-3 mRNA expression (d) in melanoma cells incubated with IL-1β (0 to 100 pM) for 24 h. TBP was used as a reference. Relative mRNA expression of MMP-3 in IL-1β-treated melanoma cells was compared with the expression at 0 h. (e, f) The cells were treated with (closed) or without (open) 100 pM IL-1β for 24 h in the presence (square) or absence (circle) of UK356618 (50 nM), an MMP-3 inhibitor, for 2 h. The representative data of cell migration (e) and wound area (f) values are shown. The data are represented as the mean ± standard error (SE) of three biological replicates. *P<0.05.</p