Methods in molecular biology
-
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.
-
Many genetic mutations result in the disruption of (alternative) splicing. Prime examples are the SMN1 and SMN2 genes: a silent mutation in SMN2 leads to the skipping of the constitutive exon 7 in the majority of SMN2 transcripts, while this exon is generally included in SMN1 transcripts. ⋯ There are proteins and drugs that can chance alternative splicing events, e.g. increase the inclusion of exon 7 in SMN2. This chapter describes mini-genes and methods that can be employed to screen for candidate proteins and drugs.
-
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.
-
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.
-
Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. ⋯ The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.