The high-affinity binding of Clostridium botulinum type B neurotoxin to synaptotagmin II associated with gangliosides GT1b/GD1a

Abstract125I-labeled botulinum type B neurotoxin was shown to bind specifically to recombinant rat synaptotagmins I and II. Binding required reconstitution of the recombinant proteins with gangliosides GT1b/GD1a. Scatchard plot analyses revealed a single class of binding site with dissociation constants of 0.23 and 2.3 nM for synaptotagmin II and synaptotagmin I, respectively, values very similar to those of the high- (0.4 nM) and low-affinity (4.1 nM) binding sites in synaptosomes. The high-affinity binding of neurotoxin to synaptosomes was specifically inhibited by a monoclonal antibody recognizing with the amino-terminal region of synaptotagmin II. These results suggest that this region of synaptotagmin II participates in the formation of the high-affinity toxin binding site by associating with specific gangliosides

刺繍ミシンにおける縫糸切断機構

The mechanism of needle thread breakage during machine embroidering was investigated using a lotus pattern. Polyester embroidery thread with a higher twist is more difficult to break. Embroidery threads such as acrylics which break easier are made stronger through processing by which paraffin is added. When compared to lighter colored threads such as beige and white, darker colored threads such as black and purple showed a tendency to break more easily. The cause of thread breakage was investigated through analysis of needle thread tension wave profiles. There are two scenarios whereby a thread becomes broken. The first is estimated from the disorder of the needle thread tension wave profile. It is thought that first the thread becomes untwisted, allowing a portion of the needle thread to get caught and broken by the hook of the hook race which ultimately results in the entire thread becoming severed. The other thread breakage scenario is implicated by the disappearance of the peak tension from the needle thread tension wave profile. As the tip of the needle passes through fabric that has already been embroidered with multiple layers of thread, the needle thread is pulled taut and prevented from entering the fabric to cause the thread to break. In such cases, only a short amount of thread is left remaining on the underside of the fabric

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

7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway.

7-Ketocholesterol is a major dietary cholesterol oxidation product found in high concentrations in atherosclerotic plaques, which contribute to the development of atherosclerosis. This study aimed to investigate the effects of 7-ketocholesterol on endothelial inflammation, as well as the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 7-ketocholesterol significantly enhanced the total interactions between human monocytic cells (THP-1 cell line) and TNFα-activated HUVECs under physiological flow conditions, compared to pretreatment with cholesterol (TNFα+50 μM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 μM 7-ketocholesterol: 18.9 ± 0.35 cells/CPF, p < 0.01). 7-Ketocholesterol enhanced the expression of E-selectin, ICAM-1, and VCAM-1 proteins. It also activated p38 mitogen-activated protein kinase (MAPK), and treatment with a p38 MAPK inhibitor inhibited both E-selectin expression via ATF-2 activation and 7-ketocholesterol-induced THP-1 adhesion to HUVECs. These findings suggest that 7-ketocholesterol enhances leukocyte-endothelial interactions by upregulating the expression of adhesion molecules, presumably via the p38 MAPK-dependent pathway

Role of the high-affinity leukotriene B4 receptor signaling in fibrosis after unilateral ureteral obstruction in mice.

Leukotriene B4 (LTB4) is a lipid mediator that acts as a potent chemoattractant for inflammatory leukocytes. Kidney fibrosis is caused by migrating inflammatory cells and kidney-resident cells. Here, we examined the role of the high-affinity LTB4 receptor BLT1 during development of kidney fibrosis induced by unilateral ureteral obstruction (UUO) in wild-type (WT) mice and BLT1 knockout (BLT1-/-) mice. We found elevated expression of 5-lipoxygenase (5-LOX), which generates LTB4, in the renal tubules of UUO kidneys from WT mice and BLT1-/- mice. Accumulation of immunoreactive type I collagen in WT UUO kidneys increased over time; however, the increase was less prominent in BLT1-/- UUO kidneys. Accumulation of S100A4-positive fibroblasts increased temporally in WT UUO kidneys, but was again less pronounced in-BLT1-/- UUO kidneys. The same was true of mRNA encoding transforming growth factor-β (TGF)-β and fibroblast growth factor (FGF)-2. Finally, accumulation of F4/80-positive macrophages, which secrete TGF-β, increased temporally in WT UUO and BLT1-/- UUO kidneys, but to a lesser extent in the latter. Following LTB4 stimulation in vitro, macrophages showed increased expression of mRNA encoding TGF-β/FGF-2 and Col1a1, whereas L929 fibroblasts showed increased expression of mRNA encoding α smooth muscle actin (SMA). Bone marrow (BM) transplantation studies revealed that the area positive for type I collagen was significantly smaller in BLT1-/-BM→WT than in WT-BM→WT. Thus, LTB4-BLT1 signaling plays a critical role in fibrosis in UUO kidneys by increasing accumulation of macrophages and fibroblasts. Therefore, blocking BLT1 may prevent renal fibrosis

7-Ketocholesterol increases the expression of adhesion molecules and cytokines in human umbilical vascular endothelial cells (HUVECs).

<p>(A) HUVECs were pretreated with 50 μM 7-ketocholesterol or cholesterol or ethanol alone for 18 h, followed by stimulation with or without 0.1 ng/ml tumor necrosis factor (TNF)-α for an additional 4 h. The levels of E-selectin, ICAM-1, and VCAM-1 protein expression were analyzed by western blotting. Representative blots from three independent experiments are shown. (B) HUVECs were pretreated with 50 μM 7-ketocholesterol or cholesterol or ethanol alone for 18 h, followed by stimulation with or without 0.1 ng/ml TNF-α for an additional 2 h. IL-8 and MCP-1 mRNA levels were analyzed by RT-qPCR. Data are shown as mean ± standard errors of the means. *p < 0.05, **p < 0.01 by one-way analysis of variance followed by Tukey’s test.</p

Schematic representation of the signaling pathways involved in the 7-ketocholesterol-induced leukocyte-endothelial interactions.

<p>7-ketocholesterol induces E-selectin expression mediated by ATF-2 and involves the p38MAPK activation pathway, which together increase the number of leukocyte interaction to endothelial cells.</p

Effects of 7-ketocholesterol on tumor necrosis factor (TNF)-α-induced mitogen-activated protein kinase (MAPK) activity in human umbilical vascular endothelial cells (HUVECs).

<p>(A) HUVECs were treated with 50 μM cholesterol or 7-ketocholesterol for each indicated time interval, followed by stimulation with 0.1 ng/ml TNF-α for an additional 15 min. Western blotting was used to evaluate the levels of p38, phospho-p38, JNK and phospho-JNK proteins as described in the Materials and Methods. Representative blots from three independent experiments are shown. (B) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, followed by incubation with 5 μM p38MAPK inhibitor (SB203580) for 30 min and stimulation with TNF-α for an additional 4 h. A non-static adhesion assay was performed. Fluorescently labeled THP-1 cells were added to the HUVECs and allowed to adhere for 10 min under rotating conditions. Data are shown as means ± standard errors of the means (SEM). *p < 0.05 by a one-way analysis of variance followed by Tukey’s test. (C) HUVECs were pretreated with 50 μM of cholesterol or 7-ketocholesterol for 18 h, incubated with 5 μM p38 MAPK inhibitor (SB203580) for 30 min and stimulated with TNF-α for an additional 4 h. Western blotting was used to evaluate the expression of E-selectin, ICAM-1, and VCAM-1 proteins as described in the Materials and Methods. Representative blots from three independent experiments are shown.</p