Methods in molecular biology
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The role of membrane proteins is critical for regulation of physiologic and pathologic cellular processes. Hence it is not surpassing that membrane proteins make ∼70% of contemporary drug targets. Quantitative profiling of membrane proteins using mass spectrometry (MS)-based proteomics is critical in a quest for disease biomarkers and novel cancer drugs. ⋯ After mixing, the differentially labeled peptides are fractionated using off-line strong cation exchange (SCX) followed by on-line reversed phase nanoflow reversed-phase liquid chromatography (nanoRPLC)-MS identification/quantiation of peptides/proteins. The use of methanol-based buffers in the context of the post-digestion (18)O exchange/labeling eliminates the need for detergents or chaotropes that interfere with LC separations and peptide ionization. Sample losses are minimized because solubilization, digestion, and stable isotope labeling are carried out in a single tube, avoiding any sample transfer or buffer exchange between these steps.
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Translational bioinformatics plays an indispensable role in transforming psychoneuroimmunology (PNI) into personalized medicine. It provides a powerful method to bridge the gaps between various knowledge domains in PNI and systems biology. Translational bioinformatics methods at various systems levels can facilitate pattern recognition, and expedite and validate the discovery of systemic biomarkers to allow their incorporation into clinical trials and outcome assessments. ⋯ Methods based on data integration, data mining, and knowledge representation are essential elements in building health information systems such as electronic health records and computerized decision support systems. Data integration of genes, pathophysiology, and behaviors are needed for a broad range of PNI studies. Knowledge discovery approaches such as network-based systems biology methods are valuable in studying the cross-talks among pathways in various brain regions involved in disorders such as Alzheimer's disease.
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Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. ⋯ However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene.
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A lumbosacral ventral root avulsion injury and repair model for studies of neuropathic pain in rats.
Neuropathic pain may develop after a variety of injuries to peripheral nerves and roots. Most injury models have included a direct injury to primary afferent fibers or neurons. ⋯ Interestingly, an acute replantation of the avulsed ventral roots into the spinal cord results in amelioration of the neuropathic pain. A detailed description of this injury and repair model is provided.
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After the publication of the First Edition of this book in the series of Methods in Molecular Medicine (volume 99 in the series) in 2004, pain research continues its rapid acceleration until 2009, during which it experienced a plateau of growth that likely resulted from the economic downturn started in 2008 (Fig. 1.1). This rapid growth in pain research could be the driving force for an impressive 66% increase in new randomized, double-blind, placebo-control trials for neuropathic pain medications in the past 5 years compared with the last four decades. Unfortunately, little improvement in pain medications has been obtained [1] due to primarily our limited understanding of mechanisms mediating different pain states, especially that for chronic pain. ⋯ It is estimated that the continuous increase in percentage of patients suffering from chronic pain (pain conditions lasting more than 6 months) arranges from 11 to 47% between 40 and 75 years of age [2], which will inevitably and continually increase the demand for better pain medications. Second, the cost of pain conditions to our society is high, estimated $55 billion per year in loss of productivity from full-time workers alone [3], so better pain management can significantly help economic growth and stability. Third, the swift advancement in technologies and our better understanding of sensory circuitries and pain pathways serves as a driving force for timely drug discovery research and development at an unprecedented pace to meet the demand for better pain medications.