| Fluorescent Detection of Intracellular Nitric Oxide in Staphylococcus aureus
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Author:
Date:
2016-07-20
[Abstract] Nitric Oxide (NO) is a highly-reactive radical gas that can modify a variety of cellular targets in both eukaryotes and bacteria. NO is produced endogenously by a wide variety of organisms: For example, as a cell-signaling molecule in mammals and bacteria via nitric oxide synthase (NOS) enzymes, and as a product of denitrification. As such, it is of great benefit to NO researchers to be able to sensitively detect intracellular NO and stable reactive nitrogen species (RNS) derived from NO. To this end, a protocol for fluorescent detection of intracellular NO/RNS in biofilm cultures of the Gram-positive pathogen Staphylococcus aureus has been optimized using the commercially-available cell-permeable fluorescent stain 4-Amino-5-Methylamino-2’,7’-Difluorofluorescein Diacetate (DAF-FM ...
[摘要] 一氧化氮（NO）是一种高反应性的自由基气体，其可以修饰真核生物和细菌中的多种细胞靶标。 NO由多种生物体内源性产生：例如，作为哺乳动物和细菌中的细胞信号分子，通过一氧化氮合酶（NOS）酶，以及作为脱氮的产物。因此，NO研究人员能够敏感地检测来自NO的细胞内NO和稳定的活性氮物质（RNS）是非常有益的。为此，已经使用商业上可获得的细胞可渗透的荧光染料4-氨基-5来优化用于荧光检测革兰氏阳性病原体金黄色葡萄球菌的生物膜培养物中的细胞内NO/RNS的方案 - 甲基氨基-2'，7'-二氟荧光素二乙酸酯（DAF-FM二乙酸酯）。该化合物扩散到细胞中并且通过酯酶的细胞内裂解释放弱荧光DAF-FM，其与NO或其它特异性RNS反应以变成高度荧光的（Kojima等人，1999）。虽然使用荧光板读数器进行荧光的定量，但设想该方案可适用于S的细胞内NO/RNS成像。 aureus biofilms by confocal microscopy。同样，这种技术可以被优化用于检测其它生长条件（即浮游生物培养物）和/或其它细菌/古细菌中的细胞内NO/RNS。
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| Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma
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Author:
Date:
2015-05-05
[Abstract] Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal ...
[摘要] 多种病毒可以共感染生殖道，修改免疫学和病毒学环境，并可能在病毒传播和发病中起作用。 我们的研究的目的是了解HIV-1 RNA和已知在HIV感染的男性生殖道中经常复制的多种人疱疹病毒之间的复杂关系（即巨细胞病毒[CMV]，Epstein 条形病毒[EBV]，单纯疱疹病毒[HSV] 1型和2型，以及人疱疹病毒[HHV] 6,7和8）（Gianella等人，2013a; Gianella等人 。，2013b; Gianella 等人，2013c; Gianella ，2014）。 该方案设计用于收集和处理男性生殖器分泌（GS），并使用定量实时PCR技术从精浆中分离并进一步定量7个HHV的HIV RNA和DNA。
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| Library Construction for Genome-wide Bisulfite Sequencing in Plants
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Author:
Date:
2013-12-20
[Abstract] DNA methylation is the most studied epigenetic modification, which involves the addition of a methyl group to the carbon-5 position of cytosine residues in DNA. DNA methylation is important for the regulation of gene expression. Bisulfite sequencing is the gold standard technique for determining genome-wide DNA methylation profiles in eukaryotes. This protocol describes how to prepare libraries of genomic DNA for whole-genome bisulfite sequencing in Arabidopsis, which could be adapted for use in other plant species.
[摘要] DNA甲基化是研究最多的表观遗传修饰，其涉及将甲基添加到DNA中胞嘧啶残基的碳-5位置。 DNA甲基化对于基因表达的调节是重要的。 亚硫酸氢盐测序是确定真核生物全基因组DNA甲基化谱的金标准技术。 该协议描述了如何在拟南芥中制备用于全基因组亚硫酸氢盐测序的基因组DNA文库，其可以适用于其他植物物种。
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