Methods in molecular biology
-
Fighting infections and developing novel drugs and vaccines requires advanced knowledge of pathogen's biology. Readily accessible genomic, functional genomic, and population data aids biological and translational discovery. ⋯ EuPathDB integrates preanalyzed data with advanced search capabilities, data visualization, analysis tools and a comprehensive record system in a graphical interface that does not require prior computational skills. This chapter describes guiding concepts common across EuPathDB sites and illustrates the powerful data mining capabilities of some of the available tools and features.
-
Advances in molecular biology and genetics have been used to elucidate the fundamental genetic mechanisms underlying central nervous system (CNS) diseases, yet disease-modifying therapies are currently unavailable for most CNS conditions. Antisense oligonucleotides (ASOs) are synthetic single stranded chains of nucleic acids that bind to a specific sequence on ribonucleic acid (RNA) and regulate posttranscriptional gene expression. Decreased gene expression with ASOs might be able to reduce production of the disease-causing protein underlying dominantly inherited neurodegenerative disorders. ⋯ A deep and wide-ranging understanding of the basic, preclinical, clinical, and epidemiologic components of drug development will improve the likelihood of success. This includes characterizing the natural history of the disease, including evolution of biomarkers indexing the underlying pathology; using predictive preclinical models to assess the putative gain-of-function of mutant Htt protein and any loss-of-function of the wild-type protein; characterizing toxicokinetic and pharmacodynamic effects of ASOs in predictive animal models; developing sensitive and reliable biomarkers to monitor target engagement and effects on pathology that translate from animal models to patients with HD; establishing a drug delivery method that ensures reliable distribution to relevant CNS tissue; and designing clinical trials that move expeditiously from proof of concept to proof of efficacy. This review focuses on the translational science techniques that allow for efficient and informed development of an ASO for the treatment of HD.
-
Reactive oxygen species (ROS) are involved in both physiological and pathological processes. This widely accepted concept is based more on the effects of antioxidant interventions than on reliable assessments of rates and sites of intracellular ROS formation. This argument applies also to mitochondria that are generally considered the major site for ROS formation, especially in skeletal and cardiac myocytes. ⋯ Thus, this approach can be used to characterize ROS formation in both isolated mitochondria and mitochondria within intact cells. This chapter describes three major examples of the use of fluorescent probes for monitoring mitochondrial ROS formation. Detailed methods description is accompanied by a critical analysis of the limitations of each technique, highlighting the possible sources of errors in performing the assay and results interpretation.
-
Recent advances in the CRISPR/Cas9 system have dramatically facilitated genome engineering in various cell systems. Among the protocols, the direct delivery of the Cas9-sgRNA ribonucleoprotein (RNP) complex into cells is an efficient approach to increase genome editing efficiency. ⋯ Here, we describe our routine methods for RNP complex-mediated gene deletion including the protocols to prepare the purified Cas9 protein and the in vitro transcribed sgRNA. Subsequently, we also describe a protocol to confirm the edited genomic positions using the T7E1 enzymatic assay and next-generation sequencing.
-
The analysis of genome-wide epigenomic alterations including DNA methylation has become a subject of intensive research for many complex diseases. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies can be considered the gold standard for a comprehensive and quantitative analysis of cytosine methylation throughout the genome. Several approaches including tagmentation- and post bisulfite adaptor tagging (PBAT)-based WGBS have been devised. ⋯ Spike-in of unmethylated DNA allows for the precise estimation of bisulfite conversion rates. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human samples as well as DNA extracted from plant tissues and yields robust and reproducible results.