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

The genus Flavivirus within the family Flaviviridae includes many viral species of medical importance, such as yellow fever virus (YFV), Zika virus (ZIKV), and dengue virus (DENV), among others. Presently, the identification of flavivirus-infected cells is based on either the immunolabeling of viral proteins, the application of recombinant reporter replicons and viral genomes, or the use of cell-based molecular reporters of the flaviviral protease NS2B-NS3 activity. Among the latter, our flavivirus-activatable GFP and mNeptune reporters contain a quenching peptide (QP) joined to the fluorescent protein by a linker consisting of a cleavage site for the flavivirus NS2B-NS3 proteases (AAQRRGRIG). When the viral protease cleaves the linker, the quenching peptide is removed, and the

Connexins are membrane bound proteins that facilitate direct and local paracrine mediated cell-to-cell communication through their ability to oligomerise into hexameric hemichannels. When neighbouring channels align, they form gap-junctions that provide a direct route for information transfer between cells. In contrast to intact gap junctions, which typically open under physiological conditions, undocked hemichannels have a low open probability and mainly open in response to injury. Hemichannels permit the release of small molecules and ions (approximately 1kDa) into the local intercellular environment, and excessive expression/activity has been linked to a number of disease conditions. Carboxyfluorescein dye uptake measures functional expression of hemichannels, where increased ... [摘要]  [摘要]连接蛋白是有助于直接和局部旁分泌通过自己的能力介导的细胞-细胞通信膜结合蛋白寡聚成六聚半通道。当相邻通道对齐时，它们形成间隙连接，为单元之间的信息传输提供直接路径。与通常在生理条件下打开的完整的间隙连接相反，未对接的半通道的打开概率较低，主要是在受伤时打开。半通道允许小分子和离子（约1kDa）释放到局部细胞间环境中，并且过度表达/活性 与许多疾病状况有关。羧基荧光素染料的摄取量度了半通道的功能性表达，其中增加的半通道活性/功能反映了增加的负荷。该技术依赖于通过开放性半通道对不可渗透膜的荧光示踪剂的吸收，可用于比较在不同条件下（例如对照与疾病）培养的细胞单层之间的通道活性。其他技术（例如生物素化和电生理学）可以分别测量细胞表面表达和半通道开放可能性，但是，羧基荧光素的摄取提供了一种简单，快速且经济高效的方法来测定多种细胞类型的体外半通道活性。


图形摘要：

使用染料吸收量度半通道活性

[背景]连接蛋白（CX ）是一体的跨膜蛋白，其寡聚到连接子在细胞表面。连接子与相似的六聚体蛋白复合物停靠在相邻细胞上，形成用于间隙连接细胞间通讯的双向导管（GJIC；Bosco等人，2011 ...