| Assay for Assessing Mucin Binding to Bacteria and Bacterial Proteins
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Author:
Date:
2021-03-05
[Abstract] Legionella pneumophila, a Gram-negative bacterium and the causative agent of Legionnaires’ disease, exports over 300 effector proteins/virulence factors, through its type II (T2SS) and type IV secretion systems (T4SS). One such T2SS virulence factor, ChiA, not only functions as a chitinase, but also as a novel mucinase, which we believe aids ChiA-dependent virulence during lung infection. Previously published protocols manipulated wild-type L. pneumophila strain 130b and its chiA mutant to express plasmid-encoded GFP. Similarly, earlier studies demonstrated that wheat germ agglutinin (WGA) can be fluorescently labeled and can bind to mucins. In the current protocol, GFP-labeled bacteria were incubated with type II and type III porcine stomach mucins, which were then labeled with ...
[摘要] [摘要]嗜肺军团杆菌是革兰氏阴性细菌，是军团菌病的病原体，通过其II型（T2SS）和IV型分泌系统（T4SS）出口了300多种效应蛋白/毒力因子。一种这样的T2SS毒力因子ChiA不仅起几丁质酶的作用，而且还起新型粘蛋白酶的作用，我们认为它可以在肺部感染期间帮助ChiA依赖性毒力。以前发表的协议操纵野生型肺炎嗜血杆菌130b菌株及其chiA突变体，以表达质粒编码的GFP。同样，较早的研究表明，小麦胚芽凝集素（WGA）可以进行荧光标记，并可以与粘蛋白结合。 在当前方案中，将GFP标记的细菌与II型和III型猪胃粘蛋白孵育，然后用TexasRed标记的WGA进行标记，并通过流式细胞术进行分析，以测量在有或没有HLA的情况下细菌与粘蛋白的结合。内源性ChiA。另外，我们分析了纯化的ChiA与II型和III型猪胃粘蛋白的结合。该方案将细菌和直接蛋白结合到粘蛋白上，并且是第一个使用WGA和流式细胞术分析革兰氏阴性细菌与粘蛋白结合的方法。
图形摘要：
自动生成手机说明的屏幕截图
评估细菌和蛋白质与粘蛋白结合的策略
[背景技术]嗜肺军团菌（LPN ），革兰氏阴性细菌，是军团病，肺炎的严重形式的病原体。L ...
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| Using RNA Sequencing and Spike-in RNAs to Measure Intracellular Abundance of lncRNAs and mRNAs
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Author:
Date:
2020-10-05
[Abstract] Long noncoding RNAs (lncRNAs) play essential roles in normal physiology and in disease but their mechanisms of action can be challenging to identify. For mechanistic studies, it is often useful to know a lncRNA’s intracellular abundance, i.e., approximately how many molecules of the lncRNA are present in a typical cell of a cell-type of interest. At least two approaches have been used to approximate lncRNA intracellular abundance: single-molecule sensitivity RNA fluorescence in situ hybridization (smFISH) and single-gene, calibrated reverse-transcription followed by quantitative PCR (RT-qPCR). However, like all experimental approaches, these methods have their limitations. smFISH, when analyzed using diffraction-limited microscopy, can underestimate intracellular ...
[摘要] [摘要]长非编码RNA（lncRNA）在正常生理和疾病中起着至关重要的作用，但其作用机理可能难以鉴定。对于机理研究，了解lncRNA的胞内丰度（即在目标细胞类型的典型细胞中大约存在多少个lncRNA分子）通常很有用。至少两种方法已用于估算lncRNA细胞内丰度：单分子敏感性RNA荧光原位杂交（smFISH ...
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| Alphavirus Purification Using Low-speed Spin Centrifugation
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Author:
Date:
2018-03-20
[Abstract] Chemical and sedimentation procedures are used to purify virus particles. While these approaches are successful for wild-type viruses, they are often not feasible for purifying mutant viruses with assembly defects. We combined two published methods (Atasheva et al., 2013; Moller-Tank et al., 2013), to generate a protocol that uses low-speed centrifugation to purify both wildtype and mutant enveloped virus particles at high yield with minimal handling steps. This protocol has successfully been used to purify alphavirus particles for imaging and structural studies (Wang et al., 2015; Ramsey et al., 2017).
[摘要] 化学和沉淀过程用于纯化病毒颗粒。 虽然这些方法对于野生型病毒是成功的，但它们通常不能用于纯化具有装配缺陷的突变病毒。 我们结合了两种公开的方法（Atasheva等人，2013; Moller-Tank等人，2013），以生成使用低速离心纯化两种方法的方案 野生型和突变体包膜病毒颗粒，产量高，处理步骤最少。 该方案已成功用于纯化甲病毒颗粒用于成像和结构研究（Wang等人，2015; Ramsey等人，2017）。
【背景】传统上，病毒纯化基于化学沉淀（例如PEG）或密度梯度离心。离心方案包括以高速（>100,000μgx g）或通过沉淀基质如铯，Nycodenz，碘克沙醇，蔗糖，甘油或酒石酸丸粒化颗粒。在梯度沉降之后，纯化的病毒样品通常需要额外的步骤来除去梯度基质，浓缩纯化的颗粒，并且缓冲液交换成用于下游应用的稳定缓冲液。这些方法包括透析，通过离心过滤器离心或PEG沉淀。虽然这些方法会产生纯化的颗粒，但有几个缺点：（1）总产量可能很低，（2）纯化所需的时间可能延长到一周，（3）形态上不均匀的颗粒不能以相同的效率纯化，（ 4）过程中可能会损坏颗粒，以及（5）组装突变体常常不能在净化过程中存活，使得某些下游分析具有挑战性。
这里描述的协议使用温和的方法来净化包膜病毒颗粒。我们使用最小的离心力来减少对粒子的损伤，这是由于负染透射电子显微镜（TEM）中增加的形态异质性或通过TEM或传染性测定法测定的易碎粒子的全部损失而观察到的。另外，我们希望减少纯化后纯化病毒体的操作。通过合并文献中的两个方案（Atasheva等人，2013; ...
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