| Flow Cytometry Analysis and Fluorescence-activated Cell Sorting of Myeloid Cells from Lung and Bronchoalveolar Lavage Samples from Mycobacterium tuberculosis-infected Mice
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Author:
Date:
2020-05-20
[Abstract] Mycobacterium tuberculosis (Mtb) is transmitted by aerosol and can cause serious bacterial infection in the lung that can be fatal if left untreated. Mtb is now the leading cause of death worldwide by an infectious agent. Characterizing the early events of in vivo infection following aerosol challenge is critical for understanding how innate immune cells respond to infection but is technically challenging due to the small number of bacteria that initially infect the lung. Previous studies either evaluated Mtb-infected cells at later stages of infection when the number of bacteria in the lung is much higher or used in vitro model systems to assess the response of myeloid cells to Mtb. Here, we describe a method that uses fluorescent bacteria, a high-dose aerosol ...
[摘要] [摘要 ] 结核分枝杆菌（Mtb）通过气溶胶传播，可引起严重的肺部细菌感染，如果不及时治疗，可能致命。Mtb现在已成为全球传染病致死的主要原因。表征气溶胶激发后体内感染的早期事件对于了解先天免疫细胞如何对感染做出反应至关重要，但由于最初会感染肺的细菌数量少，因此在技术上具有挑战性。先前的研究或者在肺部细菌数量高得多时在感染后期评估Mtb感染的细胞，或者在体外使用 评估骨髓细胞对Mtb反应的模型系统。在这里，我们介绍一种使用荧光细菌，大剂量气溶胶感染模型和流式细胞术跟踪气溶胶感染和荧光激活细胞分选（FACS）之后立即分离肺中Mtb感染细胞的方法，以分离幼稚的旁观者，和Mtb感染的细胞用于下游应用，包括RNA测序。该协议提供了在肺环境中监视Mtb感染和细胞特异性反应的能力，已知该环境可调节常驻和募集人群的功能。使用此协议，我们发现肺泡巨噬细胞通过上调受转录因子Nrf2调节并有害于细菌早期控制的细胞保护性转录反应，在体内对Mtb感染作出反应。
[背景 ] 气溶胶传播是结核分枝杆菌（Mtb）感染自然周期的关键组成部分，有助于细菌的毒性并导致其在肺部的独特感染模式（North ，1995；Riley 等，1995）。 ; Pai et ...
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| Determination of Rifampicin-resistance Mutation Frequency and Analysis of Mutation Spectra in Mycobacteria
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Author:
Date:
2014-07-05
[Abstract] Understanding the genetic safeguarding mechanism of Mycobacterium tuberculosis (Mtb) may help us to explain i), how Mtb survive the genetic assaults elicited by both reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by host macrophages and ii), why some strains of Mtb, e.g., Mtb strains from East Asian lineage and Beijing sublineage, exhibit high mutation rate and are more likely to acquire drug resistant mutations (e.g., rifampicin-resistance mutation) during infection. Mutation frequency analysis is a basic methods to study the genetic safeguarding mechanism. Moreover, to study the molecular mechanism of mutation, it is necessary to analyse the mutation spectrum (For example, oxidized cytosine may induce CG to TA mutation.). This ...
[摘要] 了解结核分枝杆菌（Mtb）的遗传保护机制可以帮助我们解释i）如何Mtb存活由活性氧（ROS）和活性氮（RNS）产生的遗传攻击 宿主巨噬细胞和ii），为什么一些来自东亚谱系和北京亚系的Mtb菌株（例如eg）具有高突变率，并且更可能获得耐药突变（例如，利福平 - 抗性突变）。 突变频率分析是研究遗传保护机制的基本方法。 此外，为了研究突变的分子机制，有必要分析突变谱（例如，氧化的胞嘧啶可能诱导CG到TA突变）。 该协议描述了确定耻骨分枝杆菌（Msm）和Mtb中的突变频率和了解突变谱的方法。
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| Transport Assays in Aspergillus nidulans
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Author:
Date:
2013-11-20
[Abstract] Transport assays allow the direct kinetic analysis of a specific transporter by measuring apparent Km and Vmax values, and permit the characterization of substrate specificity profiles through competition assays. In this protocol, we describe a rapid and easy method for performing uptake assays in the model filamentous ascomycete Aspergillus nidulans. These assays make use of A. nidulans germinating conidiospores, thus avoiding technical difficulties associated with the use of mycelia. The ease of construction genetic null mutants in this model fungus permits the rigorous characterization of any transporter in the absence of similar transporters with overlapping specificities, a common problem in relevant studies.
[摘要] 运输测定允许通过测量表观上的最小值和最小值来测量特定转运蛋白的直接动力学分析，以及 允许通过竞争测定表征底物特异性谱。 在本协议中，我们描述了在模型丝状子囊菌实施吸收测定的快速和容易的方法 Aspergillus nidulans 。 这些测定利用了A。 构巢曲霉发芽分生孢子，从而避免与使用菌丝体相关的技术困难。 在该模型真菌中构建遗传无效突变体的容易性允许在不存在具有重叠特异性的类似转运蛋白的情况下严格表征任何转运蛋白，这是相关研究中的常见问题。
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