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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Clinical epidemiology is the science of human disease investigation with a focus on diagnosis, prognosis, and treatment. The generation of a reasonable question requires the definition of patients, interventions, controls, and outcomes. ⋯ The hierarchy of evidence for clinical decision making places randomized controlled trials (RCT) or systematic review of good quality RCTs at the top of the evidence pyramid. Prognostic and etiologic questions are best addressed with longitudinal cohort studies.
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This chapter posits that cancer is a complex and multifactorial process as demonstrated by the expression and production of key endocrine and steroid hormones that intermesh with lifestyle factors (physical activity, body size, and diet) in combination to heighten cancer risk. Excess weight has been associated with increased mortality from all cancers combined and for cancers of several specific sites. The prevalence of obesity has reached epidemic levels in many parts of the world; more than 1 billion adults are overweight with a body mass index (BMI) exceeding 25. ⋯ A reductionist approach is not sufficient for the basic biological mechanisms underlying the effect of diet and physical activity on cancer. The joint association between energy balance and cancer risk are hypothesized to share the same underlying mechanisms, the amplification of chemical mediators that modulate cancer risk depending on the responsiveness to those hormones to the target tissue of interest. Disentangling the connection between obesity, the insulin-IGF axis, endogenous hormones, inflammatory markers, and their molecular interaction is vital.