| A Workflow for Ultra-rapid Analysis of Histone Post-translational Modifications with Direct-injection Mass Spectrometry
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Author:
Date:
2020-09-20
[Abstract] Chromatin modifications, like histone post translational modifications (PTMs), are critical for tuning gene expression and many other aspects of cell phenotype. Liquid chromatography coupled to mass spectrometry (LC-MS) has become the most suitable method to analyze histones and histone PTMs in a large-scale manner. Selected histone PTMs have known functions, and their aberrant regulation is linked to a wide variety of diseases, including cancer. However, histone analysis is scarcely used in diagnostics, partially due to the limited throughput and not ideal reproducibility of LC-MS based analysis. We describe a workflow that allows for high-throughput sample preparation is less than a day using 96-well plates. Following preparation, samples are sprayed into MS without LC, using an ...
[摘要] [抽象]像组蛋白翻译后修饰（PTM）一样，染色质修饰对于调节基因表达和细胞表型的许多其他方面至关重要。液相色谱-质谱联用（LC-MS）已成为最适合大规模分析组蛋白和组蛋白PTM的方法。选定的组蛋白PTM具有已知功能，其异常调节与包括癌症在内的多种疾病有关。但是，组蛋白分析很少用于诊断中，部分是由于通量有限且基于LC-MS的分析的重现性不理想。我们描述了一种使用96孔板进行少于一天的高通量样品制备的工作流程。制备后，使用自动直接进样（DI-MS）方法将样品喷雾到无LC的MS中。每次分析都可以通过45个PTM（甲基化，乙酰化和磷酸化（共151个组蛋白标记）和16个未修饰的组蛋白肽进行组蛋白变体的相对定量。由于没有残留或基于LC的批处理效应，该工作流程允许MS运行少于1分钟，并具有更高的重现性和耐用性。最后，我们描述了一种工程化的肽序列，用于精确监控样品制备的效率，可以在DI-MS运行期间检测到该效率。
[背景] 组蛋白是具有球形头部和N末端尾巴的碱性蛋白质，富含精氨酸和赖氨酸残基。一对典型的组蛋白H2A，H2B，H3和H4（称为核心组蛋白）形成一个八聚体，其周围147 ...
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| Candida albicans Agar Invasion Assays
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Author:
Date:
2020-08-20
[Abstract] The ability of the human fungal pathogen Candida albicans to disseminate into tissues is promoted by a switch from budding to invasive hyphal growth. This morphological transition is stimulated by multiple environmental factors that can vary at different sites of infection. To identify genes that promote invasive growth, C. albicans mutants can be screened for defects in growing invasively into solid agar medium as a substitute for studying tissue invasion. This in vitro approach has advantages in that it permits the media conditions to be varied to mimic different host environments. In addition, the concentration of agar can be varied to determine the effects of altering the rigidity of the matrix into which the cells invade, as this provides a better indicator ...
[摘要] [摘要 ] 人类真菌病原体白色念珠菌传播到组织的能力是由从发芽到侵入性菌丝生长的转换而增强的。多种环境因素可能会刺激这种形态转变，这些环境因素可能在不同的感染部位有所不同。为了鉴定促进侵入growt基因小时，白色念珠菌突变体可以在侵入性生长成固体琼脂培养基作为研究组织侵袭的替代品进行筛选的缺陷。这个体外 该方法的优点在于，它允许改变媒体条件以模仿不同的主机环境。另外，可以改变琼脂的浓度以确定改变细胞侵袭的基质的刚度的作用，因为与液体培养中形成菌丝的能力相比，这提供了更好的侵袭性生长指标。在多种条件下的测试可以用来鉴定具有最强缺陷的突变细胞。因此，将描述用于分析白色念珠菌在不同条件下的侵袭性生长的方案和培养基，其适合于测试突变株白色念珠菌菌株的单个菌株或高通量分析。
[背景 ] 白色念珠菌是一种多形性真菌病原体，可以通过形成芽（小球形细胞），假菌丝细胞（细长细胞链）或菌丝细胞（具有平行平行线的长丝状细胞链）生长（图1）（贵族等人，2017）。已证明宿主中遇到的多种环境刺激可促进向侵袭性生长的转变，包括人体温度（37 °C ），碱性pH，CO 2 ，血清中的因子和糖GlcNAc（N-乙酰氨基葡萄糖） ）（Kornitzer，2019）。萌芽细胞被认为促进胃肠道的血流传播和定植（Pierce和Kumamoto ，2012 ；Witchley ...
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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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