Methods in molecular biology
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Mitochondrial respiration is associated with the formation of reactive oxygen species, primarily in the form of superoxide (O2 (•-)) and particularly hydrogen peroxide (H2O2). Since H2O2 plays important roles in physiology and pathology, measurement of hydrogen peroxide has received considerable attention over many years. Here we describe how the well-established Amplex Red assay can be used to detect H2O2 production in combination with the simultaneous assessment of mitochondrial bioenergetics by high-resolution respirometry. ⋯ The sensitivity of the H2O2 assay was strongly influenced by compounds contained in different mitochondrial respiration media, which also exerted significant effects on chemical background fluorescence changes. Near linearity of the fluorescence signal was restricted to narrow ranges of accumulating resorufin concentrations independent of the nature of mitochondrial respiration media. Finally, we show an application example using isolated mouse brain mitochondria as an experimental model for the simultaneous measurement of mitochondrial respiration and H2O2 production in SUIT protocols.
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Bioluminescence resonance energy transfer (BRET) is a valuable tool to detect protein-protein interactions. BRET utilizes bioluminescent and fluorescent protein tags with compatible emission and excitation properties, making it possible to examine resonance energy transfer when the tags are in close proximity (<10 nm) as a typical result of protein-protein interactions. Here we describe a protocol for detecting BRET from two known protein binding partners (Gαi1 and RGS14) in HEK 293 cells using Renilla luciferase and yellow fluorescent protein tags. We discuss the calculation of the acceptor/donor ratio as well as net BRET and demonstrate that BRET can be used as a platform to investigate the regulation of protein-protein interactions in live cells in real time.
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Over the past 20 years, advances in high-throughput biological techniques and the availability of computational resources including fast Internet access have resulted in an explosion of large genome-scale data sets "big data." While such data are readily available for download and personal use and analysis from a variety of repositories, often such analysis requires access to seldom-available computational skills. As a result a number of databases have emerged to provide scientists with online tools enabling the interrogation of data without the need for sophisticated computational skills beyond basic knowledge of Internet browser utility. This chapter focuses on the Eukaryotic Pathogen Databases (EuPathDB: http://eupathdb.org) Bioinformatic Resource Center (BRC) and illustrates some of the available tools and methods.
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The diagnosis of sepsis, and especially its differentiation from sterile inflammation, may be challenging. TREM-1, the triggering receptor expressed on myeloid cells-1, is an amplifier of the innate immune response. ⋯ In this chapter, we review the numerous studies that have evaluated the usefulness of sTREM-1 concentration determination for the diagnosis and the prognosis evaluation of sepsis or localized infection. Nowadays, sandwich ELISA kits are available and the assay is described.
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The ability to inhibit gene expression via RNA interference (RNAi) has a broad therapeutic potential for various human diseases such as infections and cancers. Recent advances in mechanistic understanding of RNAi have improved the design of functional small interfering (si) RNAs with superior potency and specificity. With respect to delivery, new developments in delivery strategies have facilitated preclinical and clinical siRNA applications. This review provides valuable insights to guide the design and delivery of therapeutic siRNAs.