| A Quantitative Assay to Measure Stress Granule Association of Proteins and Peptides in Semi-permeabilized Human Cells
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Author:
Date:
2020-12-20
[Abstract] Stress granules (SGs) are membrane-less organelles that form in the cytoplasm through phase separation, in response to diverse stressors. SGs contain translationally stalled mRNAs, proteins involved in translation, and various RNA-binding proteins (RBPs). Due to the high local concentration of aggregation-prone RBPs, SGs might act as condensation sites for aberrant phase transitions of RBPs and could favor formation of solid protein aggregates underlying the pathological cytoplasmic inclusions found in numerous neurodegenerative diseases. Most assays aiming at studying the recruitment of RBPs into SGs are based on overexpression and SG recruitment of RBPs in intact cells. These approaches are, however, often limited by the predominantly nuclear localization of many RBPs, which precludes ...
[摘要] [摘要]圣RESS颗粒剂（SGS）是膜较少细胞器形式在通过相分离细胞质中，响应于不同的压力源。SG包含翻译停滞的mRNA，翻译中涉及的蛋白和各种RNA结合蛋白（RBP）。由于易于凝集的RBP的局部浓度很高，SG可能充当RBP异常相变的缩合位点，并且可能有助于形成在许多神经退行性疾病中发现的病理性细胞质内含物的固体蛋白质聚集体。大多数分析瞄准荷兰国际集团在研究限制性商业惯例的招募SG的基础上表达和SG招聘完整细胞的限制性商业惯例。但是，这些方法通常受到许多RBP的主要核定位的限制，这排除了足以进行SG定位的细胞质RBP浓度，并且没有解决独立于SG形成的RBP募集问题。在这里，我们提出了一个定量的方法来评估重组限制性商业惯例的募集至预先形成的SG，独立于RBP的核定位，用半透细胞和荧光显微镜。在该测定中，SG被第一LY由应激诱导的，然后第质膜ë应激的细胞随后被选择性地透化，以提供重组蛋白的访问的SG。通过用小麦胚芽凝集素阻塞核孔来阻止目的蛋白的核输入。这种测定方法使人们能够定量研究在没有核导入且在受控条件下将RBP募集到SG中的分子机制。该方法允许对野生型，突变体或直接比较翻译后修饰的限制性商业惯例，用于解决第其它蛋白质电子影响“预防或促进限制性商业惯例的SG关联，并且也适用于合成的肽。
图形摘要：
工作流概述在半SG招募重组蛋白质或肽的分析透化细胞 ...
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| Advances in Proximity Ligation in situ Hybridization (PLISH)
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Author:
Date:
2020-11-05
[Abstract] Understanding tissues in the context of development, maintenance and disease requires determining the molecular profiles of individual cells within their native in vivo spatial context. We developed a Proximity Ligation in situ Hybridization technology (PLISH) that enables quantitative measurement of single cell gene expression in intact tissues, which we have now updated. By recording spatial information for every profiled cell, PLISH enables retrospective mapping of distinct cell classes and inference of their in vivo interactions. PLISH has high sensitivity, specificity and signal to noise ratio. It is also rapid, scalable, and does not require expertise in molecular biology so it can be easily adopted by basic and clinical researchers.
[摘要] [摘要]在发育，维持和疾病的背景下了解组织需要确定单个细胞在其天然体内空间范围内的分子谱。我们开发了一种邻近连接原位杂交技术（PLISH），该技术能够定量测量完整组织中单细胞基因的表达，现已更新。通过记录每个分析细胞的空间信息，PLISH可以回顾性绘制不同细胞类别并推断其体内 互动。PLISH具有很高的灵敏度，特异性和信噪比。它也快速，可扩展，并且不需要分子生物学方面的专门知识，因此基础和临床研究人员可以轻松地采用它。
[背景技术]我们最近开发了一种复用原位称为PLISH（邻位连接杂交技术原位杂交）（Nagendran等人，2018）。PLISH与其他现有的空间转录组学技术不同，因为它结合了高性能，快速多路复用，低成本和技术简单性（Wilbrey -Clark等人，2020年）。可以通过自动计算完整的冷冻或石蜡包埋组织中单细胞表达图谱来分析PLISH结果，它与同时进行的免疫染色兼容。
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| Intestinal Enteroid Culture for Human Astroviruses
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Author:
Date:
2020-07-20
[Abstract] Human astroviruses (HAstV) are non-enveloped, positive-sense single stranded RNA viruses that typically cause gastroenteritis in children, the elderly and among immunocompromised individuals. Some HAstV species have also been implicated in neurological diseases. It is important to study these viruses to understand the pathogenesis and develop therapeutics. Here we describe HAstV infection in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. Although different HAstV clades have been propagated in transformed immortalized cell lines such as A549, Caco-2, HEK293T and Huh7.5, we chose HIE because they better mimic the human intestine and thus are more physiologically relevant. Additionally, HIE support the replication of all HAstV clades ...
[摘要] [摘要] 人类星状病毒（HAstV）是一种非包膜，正向单链RNA病毒，通常会导致儿童，老年人和免疫力低下的人群中的肠胃炎。一些HAstV物种也与神经系统疾病有关。研究这些病毒以了解其发病机理并开发治疗方法非常重要。在这里我们描述HAstV感染 从活检来源的肠隐窝中分离出的仅上皮的人肠小肠类固醇（HIE）。尽管已经在转化的永生化细胞系（例如A549，Caco-2，HEK293T和Huh7.5）中繁殖了不同的HAstV进化枝，但我们选择HIE是因为它们可以更好地模仿人的肠道，因此在生理上更相关。此外，HIE支持所有HAstV进化枝的复制，包括临床样品，因此使HIE成为研究HAstV生物学的有价值的潜在通用模型。
[背景] 人类星状病毒（HAstV）是高度流行的肠病毒，会引起从腹泻到脑炎的广泛感染。无症状，全身和肠道外感染很常见（Madeley和Cosgrove 1975；Bosch 等人，2014；Kolawole 等人，2019）。尽管具有医学重要性，但H AstV 是一些特征最弱的肠病毒。目前，已识别出三组人类AstV：经典HAstV和非经典HAstV-MLB（墨尔本），以及HAstV-VA / ...
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