Methods in molecular biology
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RNA interference (RNAi) has become a powerful tool for modulating gene expression. While delivery of small interfering RNAs (siRNAs) has achieved silencing of pain-related genes in various animal models of nociception, delivery of short-hairpin RNA (shRNA) or artificial miRNA (miRNA) to dorsal root ganglia (DRG) has proven particularly challenging. This chapter describes a highly efficient method for in vivo gene silencing in sensory neurons using replication-defective vectors based on herpes simplex virus (HSV). This method can be utilised to obtain a better understanding of gene function, validate novel gene targets in drug discovery and potentially develop new RNAi-mediated approaches to achieve analgesia.
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Protein kinases (PKs) are widely recognized as valuable targets for disease diagnosis and drug discovery. For this reason, we have developed a sensitive peptide microarray for detecting intracellular PK activity. Peptides are immobilized on a glutaraldehyde-premodified high-amino terminal glass slide, by spotting 2 nL volumes of substrate peptide solutions with an automated microarray spotter. ⋯ The peptide microarray system involves simple peptide immobilization, requires low sample volumes and provides a high density array. Importantly, it provides high sensitivity for detecting PK activities in cell lysates. Thus, the peptide microarray system is expected to be useful for a high-throughput kinase assay to investigate intracellular kinase activity and has potential applications in disease diagnosis and drug discovery.
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Migraine is a high prevalence disorder which affects a significant proportion of the general population, especially women during their central and more productive time of the life, thus causing severe disability. The genetic basis of the disease is unknown and the mechanism is poorly understood. The possibility that following a perturbation in the central nervous system, and particularly in the brainstem, trigeminal neurons become hyperexcitable and produce an uncontrolled release of sensory neuropeptides which eventually results in arterial vasodilatation and neuronal sensitization, has been gaining credit from studies in experimental animals and humans. In particular, experimental and clinical data with antagonists of the calcitonin gene-related peptide (CGRP) propose this molecule and its receptor as a major target for migraine treatment.
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Amplifying and sequencing DNA after bisulfite treatment of genomic DNA reveals the methylation state of cytosine residues at the highest resolution possible. However, a thorough analysis is required for statistical evaluation of methylation at all sites in each genomic region. Several software tools were developed to assist in quantitative evaluation of bisulfite sequencing data from complex methylation patterns occurring in plants. ⋯ CyMATE is also able to perform a quality control of sequences and to detect redundancy among individual clones. The software is able to reveal methylation patterns on complementary strands by handling data from hairpin bisulfite sequencing. The tool is freely available for non-commercial use at http://www.cymate.org .
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The retinol carrier retinol-binding protein (RBP) forms in blood a complex with the thyroid hormone carrier transthyretin (TTR). The interactions of retinoid-RBP complexes, as well as of unliganded RBP, with TTR can be investigated by means of fluorescence anisotropy. RBP represents the prototypic lipocalin, in the internal cavity of which the retinol molecule is accommodated. ⋯ The fluorescence anisotropy technique is also suitable to study the interaction of TTR with apoRBP and RBP in complex with non-fluorescent retinoids. In the latter cases, the fluorescence signal is provided by a fluorescent probe covalently linked to TTR rather than by RBP-bound retinol. We report here on the preparation of recombinant human RBP and TTR, the covalent labeling of TTR with the fluorescent dansyl probe, and fluorescence anisotropy titrations for RBP and TTR.