| Whole-genome Identification of Transcriptional Start Sites by Differential RNA-seq in Bacteria
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Author:
Date:
2020-09-20
[Abstract] Gene transcription in bacteria often starts some nucleotides upstream of the start codon. Identifying the specific Transcriptional Start Site (TSS) is essential for genetic manipulation, as in many cases upstream of the start codon there are sequence elements that are involved in gene expression regulation. Taken into account the classical gene structure, we are able to identify two kinds of transcriptional start site: primary and secondary. A primary transcriptional start site is located some nucleotides upstream of the translational start site, while a secondary transcriptional start site is located within the gene encoding sequence.
Here, we present a step by step protocol for genome-wide transcriptional start sites determination by differential RNA-sequencing (dRNA-seq) ...
[摘要] [摘要] 细菌中的基因转录通常起始于起始密码子上游的一些核苷酸。识别SPE cific Ť ranscriptional 小号挞小号ITE （TSS）为遗传操作必需的，因为在许多情况下，起始密码子上游有中涉及的基因表达调控序列元件。考虑到经典的基因结构，我们能够鉴定出两种转录起始位点：一级和二级。主要转录起始位点位于翻译起始位点上游的一些核苷酸上，而次要转录起始位点位于基因编码序列内。
这里，我们提出一步步协议全基因组吨ranscriptional 小号馅饼小号ITES d etermination通过差RNA测序（DRNA 使用肠道病原体-SEQ）福氏痢疾杆菌血清型菌株5A作为M90T模型。但是，该方法可以用于选择的任何其他细菌物种。第一步，使用热酚法从细菌培养物中纯化总RNA。核糖体RNA（rRNA）是使用商业试剂盒通过杂交探针特异性去除的。然后准备一个富含5'- 一磷酸依赖性核酸外切酶（TEX）处理的，富含初级转录本的RNA文库，用于与未进行TEX处理的文库进行比较，然后连接已知序列的RNA接头衔接子，从而确定具有单核苷酸精度的TSS。最后，对RNA进行处理以制备Illumina测序文库，并按购买的服务进行测序。通过内部生物信息学分析鉴定TSS。
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| A Quantitative Single-cell Flow Cytometry Assay for Retrograde Membrane Trafficking Using Engineered Cholera Toxin
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Author:
Date:
2020-08-05
[Abstract] The organization and distribution of proteins, lipids, and nucleic acids in eukaryotic cells is an essential process for cell function. Retrograde trafficking from the plasma membrane to the Golgi and endoplasmic reticulum can greatly modify cell membrane composition and intracellular protein dynamics, and thus typifies a key sorting step. However, methods to efficiently quantify the extent or kinetics of these events are currently limited. Here, we describe a novel quantitative and effectively real-time single-cell flow cytometry assay to directly measure retrograde membrane transport. The assay takes advantage of the well-known retrograde trafficking of cholera toxin engineered with split-fluorescent proteins to generate novel tools for immediate monitoring of intracellular trafficking. ...
[摘要] [摘要]蛋白质、脂类和核酸在真核细胞中的组织和分布是细胞功能的重要过程。从质膜到高尔基体和内质网的逆向运输可以极大地改变细胞膜的组成和细胞内蛋白质的动态变化，因此是一个关键的分选步骤。然而，有效量化这些事件的程度或动力学的方法目前是有限的。在这里，我们描述了一种新的定量和有效的实时单细胞流式细胞术检测直接测量逆行膜转运。这项检测利用了众所周知的霍乱毒素逆行转移的特性，利用裂解荧光蛋白产生了新的工具，用于即时监测细胞内的转移。这种方法将大大扩展研究细胞内膜转运的生物学基础，以及细胞膜转运系统如何适应不同细胞类型和细胞状态的生理需要。
[背景]所有的真核细胞都依赖于它们动态地将分子分类和分离到膜结合的亚细胞器中的能力，以便组织和分配到细胞的特定区域。在这一过程中的一个重要步骤是早期分类内体和跨高尔基网络（TGN）。在从分选内体产生的其他贩运途径中，通过分泌途径到TGN的逆行贩运是一个关键的分选步骤（Johannes和Popoff，2008）。细胞膜蛋白和脂类发生逆向转运。从TGN到内质网（ER）的进一步逆行贩运可通过一些质膜脂质完成，并可巧妙地由几种细菌毒素和病毒协同作用而致病（Cho等人，2012年；Personnic等人，2016年；Williams和Tsai，2016年）。
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| In vivo Blood-brain Barrier Permeability Assays Using Clostridium perfringens Epsilon Toxin
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Author:
Date:
2020-08-05
[Abstract] In order for the brain to function properly, a carefully orchestrated homeostasis must be maintained. To help regulate this delicate balance, the brain has developed a highly selective blood-brain barrier (BBB). Under normal conditions, the BBB excludes harmful blood-borne material from the brain parenchyma. However, numerous neuropathological conditions can disrupt this barrier, causing BBB permeability and subsequent CNS dysfunction. Understanding the mechanisms involved in BBB permeability are essential to elucidating the pathology of various neurological disorders as well as identifying methods for drug delivery to the CNS. Here, we describe several in vivo methods to measure BBB permeability in mice using an array of diverse sized tracers including exogenous 376 Da ...
[摘要] [摘要]为了使大脑正常工作，必须维持精心安排的体内平衡。为了帮助调节这种微妙的平衡，大脑已经形成了一种高度选择性的血脑屏障（BBB）。在正常情况下，血脑屏障将有害的血液传播物质排除在脑实质之外。然而，许多神经病理学条件可以破坏这一屏障，导致BBB通透性和随后的CNS功能障碍。了解BBB通透性的机制对于阐明各种神经系统疾病的病理学以及确定药物输送到中枢神经系统的方法至关重要。在这里，我们描述了几种在体内使用不同大小的示踪剂（包括外源性376da荧光素盐、66.5kDa牛血清白蛋白、70kDa右旋糖酐以及内源性160kDa小鼠IgG）来测量BBB在小鼠体内的通透性。当静脉注射时，这些物质会被BBB排除在健康大脑之外。然而，BBB功能障碍可使这些示踪剂进入大脑，这种积聚可通过分光光度法、荧光显微镜和免疫组织化学进行测量。我们还描述了一种利用产气荚膜梭菌epsilon毒素诱导BBB通透性的方法。最后，我们将简要讨论每种方法的优缺点及其适当的下游应用。
[背景] ...
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