Methods in molecular biology
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Mass spectrometry (MS) and nuclear magnetic resonance (NMR) have evolved as the most common techniques in metabolomics studies, and each brings its own advantages and limitations. Unlike MS spectrometry, NMR spectroscopy is quantitative and does not require extra steps for sample preparation, such as separation or derivatization. ⋯ Moreover, different MS approaches such as different ionization techniques and mass analyzer technology can be used in order to increase the number of metabolites that can be detected. In this chapter, the advantages, limitations, strengths, and weaknesses of NMR and MS as tools applicable to metabolomics research are highlighted.
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Tremor is the most common movement disorder. However; characterizing it in large populations is not easily accomplished since current methodologies are not adapted to large-scale field studies. To overcome this challenge, a smartphone application was developed as a stand-alone platform to assess tremor. ⋯ This allowed for the identification of the tremor properties that could reliably be characterized with the smartphone as well as the limits of the hardware. It also allowed for the identification of tasks that could be performed with the smartphone when tremor was being assessed. Finally, we confirmed the clinical relevance of the results provided by the smartphone application.
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Retinal cell apoptosis occurs in many eye conditions, including glaucoma, diabetic retinopathy and Alzheimer's disease. Real-time detection of retinal cell apoptosis has potential clinical value in early disease detection, as well as evaluating disease progression and treatment efficacy. Here, we describe our novel imaging technology DARC (Detection of Apoptosing Retinal Cells), which can be used to visualize single retinal neurons undergoing apoptosis in real time, by using fluorescently labeled Annexin A5 and confocal scanning laser ophthalmoscopy (cSLO ). Clinical trials of DARC in glaucoma patients are due to start shortly, but in this chapter, we describe this technique in experimental animal models.
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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.