| Adhesion Assay for Murine Bone Marrow Hematopoietic Stem Cells
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Author:
Date:
2017-02-20
[Abstract] Hematopoietic stem cells (HSCs) are defined by their functional abilities to self-renew and to give rise to all mature blood and immune cell types throughout life. Most HSCs are retained in a non-motile quiescent state within a specialized protective microenvironment in the bone marrow (BM) termed the niche. HSCs are typically distinguished from other adult stem cells by their motility capacity. Movement of HSCs across the physical barrier of the marrow extracellular matrix and blood vessel endothelial cells is facilitated by suppression of adhesion interactions, which are essential to preserve the stem cells retained within their BM niches. Importantly, homing of HSCs to the BM following clinical transplantation is a crucial first step for the repopulation of ablated BM as in the case of ...
[摘要] 造血干细胞（HSC）由其自我更新的功能定义，并在整个生命中产生所有成熟的血液和免疫细胞类型。大多数HSC在被称为利基的骨髓（BM）的专门的保护性微环境内保持在非运动性静止状态。 HSC通常通过其运动能力与其他成体干细胞区分开来。通过抑制粘附相互作用促进骨髓细胞外基质和血管内皮细胞的物理屏障的移动，这是保留在其BM细胞壁内保留的干细胞所必需的。重要的是，在临床移植后将HSC归巢到BM是重建消融BM的关键的第一步，就像血液恶性肿瘤治疗策略一样。归位过程结束于HSC的选择性访问和锚定到其在BM内的专门的位置。粘附分子是在干细胞移植的情况下增强归巢或减少BM保留以从匹配供体的血液中收集动员的HSC的靶标。在HSC上功能表达并参与其归巢和保留的主要粘附蛋白是整合素α4β1（非常晚的抗原-4; VLA4）。在该方案中，我们引入了针对表达VLA4的鼠骨髓干细胞优化的粘附测定。该测定法在分离表达VLA4的贴壁细胞后，通过流式细胞术与HSC富集细胞表面标记物定量粘附的HSC。
背景 HSCs主要保留在BM中，并通过与其微环境（niche）的粘合相互作用来调节。以这种方式，HSC保持在非运动性静止状态，保护它们免受DNA损伤代理（Boulais和Frenette，2015; Mendelson和Frenette，2014; ...
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| Design of a Transcription-based Secretion Activity Reporter (TSAR) for the Type III Secretion Apparatus of Shigella flexneri and Uses Thereof
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Author:
Date:
2014-10-20
[Abstract] Many gram-negative bacterial pathogens, including Shigella flexneri, are able to translocate bacterial proteins, dubbed effectors, across the host cell plasma membrane into the host cell cytosol using a syringe-like structure, the type three secretion apparatus (T3SA). While some bacteria use their T3SA to modulate their phagosomal environment (Salmonella spp.), establish pedestal structure to form microcolonies on the plasma membrane (Enteropathogenic Escherichi coli) or lyse their entry vacuole (Shigella spp.), they all have in common a tightly regulated activity of their T3SA. However, the tracking of the activity of the T3SA in infected cells and tissue has been difficult to perform. Using the property of MxiE-dependent promoters that are ...
[摘要] 包括灵芝氏菌在内的许多革兰氏阴性细菌病原体能够使用注射器样结构将类型三分泌物穿过宿主细胞质膜转运细菌蛋白质（配体效应子）到宿主细胞胞质溶胶中装置（T3SA）。虽然一些细菌使用它们的T3SA调节它们的吞噬体环境（沙门氏菌 spp 。），建立基质结构以在质膜上形成微集落（Enteropathogenic < em="">）或溶解它们的进入泡（志贺氏菌属。），它们都具有共同的严格调节的他们的T3SA的活性。然而，T3SA在感染的细胞和组织中的活性的跟踪一直难以进行。使用在T3SA是活性时上调的MxiE依赖性启动子的性质，我们最近设计了基于转录的分泌活性报道分子（TSAR），其允许以下的S的活性。使用快速成熟的GFP内在荧光，在组织培养细胞中实时地和体内实时分析灵敏度。在这里我们描述TSAR的设计及其应用于固定和活体样品的显微镜和流式细胞仪在结肠上皮细胞模型使用TC7组织培养细胞。
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| Detection of the Secreted and Cytoplasmic Fractions of IpaB, IpaC and IpaD by Lysozyme Permeabilization
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Author:
Date:
2014-10-20
[Abstract] Gram negative bacterial pathogens, such as Shigella flexneri, which possess a Type Three Secretion System (T3SS), are able to transfer bacterial proteins, dubbed translocators and effectors, from their cytoplasm into the cytoplasm of their host cells using a syringe like needle complex. For Shigella, it has been shown that during cellular invasion, the intrabacterial pool of translocators and effectors is completely depleted upon activation of the TTS Apparatus and is then progressively replenished while bacteria remain inside host cells. Replenishment of effectors allows for cell-to-cell spreading events, which also necessitate reactivation of the T3SA, and lead to another round of depletion of intrabacterial effector stores. To understand the state of individual ...
[摘要] 具有三型分泌系统（T3SS）的革兰氏阴性细菌病原体如灵芝氏菌能够将细菌蛋白质，配位的易位蛋白和效应子从其细胞质转移到其宿主细胞的细胞质中使用注射器如针复合物。对于志贺氏菌（Shigella），已经表明在细胞侵袭过程中，转位子和效应子的细菌池在TTS装置激活时完全耗尽，然后逐渐补充，同时细菌保留在宿主细胞内。补充效应物允许细胞间的扩散事件，这也需要T3SA的再活化，并导致细胞内效应子库的另一轮消耗。为了理解感染期间单个细胞内细菌的状态，因此有兴趣的是能够定位和评估细菌和分泌的易位蛋白和效应子池的相对数量。我们最近改编了基于EDTA和溶菌酶的方法以透化宿主细胞内存在的细菌的细胞壁，以标记尖端蛋白IpaD和易位蛋白IpaB和IpaC的细菌池。在这里，我们详细描述执行连续标记细菌和分泌池的协议。这种方法理论上可扩展到由其他分泌系统和其他细菌病原体分泌的毒力因子。
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