Methods in molecular biology
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Studies of DNA methylation in Arabidopsis have rapidly advanced from the analysis of a single reference accession to investigations of large populations. The goal of emerging population studies is to detect differentially methylated regions (DMRs) at the genome-wide scale, and to relate this variation to gene expression and phenotypic diversity. Whole-genome bisulfite sequencing (WGBS-seq) has established itself as a gold standard in DNA methylation analysis due to its high accuracy and single cytosine measurement resolution. ⋯ However, detection can be susceptible to strong signal distortions resulting from a combination of dye bias and the CG content of effectively unmethylated genomic regions. We show that these issues can be easily bypassed by taking appropriate data preparation steps and applying suitable analysis tools. We conclude that MeDIP-chip is a reasonable alternative to WGBS-seq in emerging Arabidopsis population epigenetic studies.
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Peptide microarray technology can be used to identify substrates for recombinant kinases, to measure kinase activity and changes thereof in cell lysates and lysates from fresh frozen (tumor) tissue. The effect of kinase inhibitors on the kinase activities in relevant tissues can be investigated as well. The method for performing experiments on dynamic peptide microarrays with real-time readout is described, as well as the influence of assay parameters and suggestions for optimization of experiments.
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Macrophages play a key role in the innate immune response and help to direct the acquired immune response. Early in the innate immune response, they produce reactive oxygen species and pro-inflammatory cytokines and chemokines to drive inflammation and are referred to as "classically activated" or "killer" macrophages (M1). During the resolution phase of inflammation, they switch to what is known as an "alternatively activated" phenotype or "healer" macrophage (M2) and contribute to debris scavenging, angiogenesis, and wound healing. ⋯ M1 macrophages also produce relatively higher levels of pro-inflammatory IL-12 and lower levels of anti-inflammatory IL-10 relative to M2 macrophages. In this chapter, we describe in vitro derivation of polarized bone marrow macrophages and methods to analyze the resulting phenotype including Q-PCR, Western blotting, and enzyme assays to determine argI and iNOS expression and activity, as well as production of IL-12p40 and IL-10 and determination of IL-12/IL-10 ratios. Production of iNOS, NO, IL-12p40, and IL-10 are measured after treatment with LPS.
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The search for potential drugs to treat neurodegenerative diseases has been intense in the last two decades. Among many candidates, erythropoietin (EPO) was identified as a potent protectant of neurons suffering from various adverse conditions. A wide array of literature indicates that endogenous or exogenous recombinant human erythropoietin and its variants activate cell signaling that initiates survival-promoting events in neurons and neuronal cells. ⋯ The signaling pathways involved in EPO are multiple; some are well known whereas others are still under intense investigation and few are observed in very specific cell types. It is important to note that neuronal signaling events triggered by EPO are still incomplete and require further research. Therefore, excellent review articles that explore specific EPO-signaling events are referenced.
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The various biochemical cascades that follow primary brain injury result in secondary brain injury which can adversely affect the clinical outcome. Over the last few years it has been well established that molecules like erythropoietin (Epo) have a neuroprotective role in experimental traumatic brain injury (TBI). Epo is shown to produce this effect by modulating multiple cellular processes, including apoptosis, inflammation, and regulation of cerebral blood flow. ⋯ Peptides that mimic a portion of the Epo molecule, including Helix B surface peptide and Epotris, have also been developed to isolate the neuroprotective activities. The TBI model in rodents most commonly used to study the effect of Epo and these derivatives in TBI is controlled cortical impact injury, which is a model of focal contusion following a high velocity impact to the parietal cortex. Following TBI, rodents are given Epo or an Epo derivative vs. placebo and the outcome is evaluated in terms of physiological parameters (cerebral blood flow, intracranial pressure, cerebral perfusion pressure), behavioral parameters (motor and memory), and histological parameters (contusion volumes, hippocampus cell counts).