Methods in molecular biology
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Protocols for High-Resolution FluoRespirometry of intact cells, permeabilized cells, permeabilized muscle fibers, isolated mitochondria, and tissue homogenates offer sensitive diagnostic tests of integrated mitochondrial function using standard cell culture techniques, small needle biopsies of muscle, and mitochondrial preparation methods. Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols for analysis of oxidative phosphorylation (OXPHOS) improve our understanding of mitochondrial respiratory control and the pathophysiology of mitochondrial diseases. Respiratory states are defined in functional terms to account for the network of metabolic interactions in complex SUIT protocols with stepwise modulation of coupling control and electron transfer pathway states. ⋯ ET pathways with electron entry separately through NADH (pyruvate and malate or glutamate and malate) or succinate (succinate and rotenone) restrict ET capacity and artificially enhance flux control upstream of the Q-cycle, providing diagnostic information on specific ET-pathway branches. O2 concentration is maintained above air saturation in protocols with permeabilized muscle fibers to avoid experimental O2 limitation of respiration. Standardized two-point calibration of the polarographic oxygen sensor (static sensor calibration), calibration of the sensor response time (dynamic sensor calibration), and evaluation of instrumental background O2 flux (systemic flux compensation) provide the unique experimental basis for high accuracy of quantitative results and quality control in High-Resolution FluoRespirometry.
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Exon skipping therapy using synthetic DNA-like molecules called antisense oligonucleotides (ASOs) is a promising therapeutic candidate for overcoming the dystrophin mutation that causes Duchenne muscular dystrophy (DMD). This treatment involves splicing out the frame-disrupting segment of the dystrophin mRNA, which restores the reading frame and produces a truncated yet functional dystrophin protein. Phosphorodiamidate morpholino oligomer (PMO) is the safest ASO for patients among ASOs and has recently been approved under the accelerated approval pathway by the U. ⋯ Food and Drug Administration (FDA) as the first drug for DMD. Here, we describe the methodology and protocol of PMO transfection and evaluation of the exon skipping efficacy in the mdx52 mouse, an exon 52 deletion model of DMD produced by gene targeting. The mdx52 mouse model offers advantages over the mdx mouse, a spontaneous DMD model with a nonsense mutation in exon 23, in terms of the deletion in a hotspot of deletion mutations in DMD patients, the analysis of caveolae and also Dp140 and Dp260, shorter dystrophin isoforms.
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Superoxide and hydrogen peroxide produced by mitochondria play important roles in various physiological and pathological processes. This chapter describes a plate-based method to measure rates of superoxide and/or hydrogen peroxide production at specific sites in isolated mitochondria.
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Post-bisulfite adaptor tagging (PBAT) is a highly efficient procedure to construct libraries for whole-genome bisulfite sequencing (WGBS). PBAT attaches adaptors to bisulfite-converted genomic DNA to circumvent bisulfite-induced degradation of library DNA inherent to conventional WGBS protocols. Consequently, it enables PCR-free WGBS from nanogram quantities of mammalian DNA, thereby serving as an invaluable tool for methylomics.
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DNA methylation profiling in the epigenome of Arabidopsis thaliana (Arabidopsis) has provided great insights in the role of this epigenetic mark for the regulation of transcription in plants, and is often based on high-throughput sequencing. The analysis of these data involves a series of steps including quality checks, filtering, alignment, identification of methyl-cytosines, and the identification of differentially methylated regions. This chapter outlines the computational methodology required to profile genome-wide differential methylation patterns based on publicly available Arabidopsis base-resolution bisulfite sequencing data. The methylPipe Bioconductor package is adopted for the identification of the differentially methylated regions, and all the steps from the raw data to the required input are described in detail.