Methods in molecular biology
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Breakdown of the blood-brain barrier (BBB) is present in several neurological disorders such as stroke, brain tumors, and multiple sclerosis. Noninvasive evaluation of BBB breakdown is important for monitoring disease progression and evaluating therapeutic efficacy in such disorders. One of the few techniques available for noninvasively and repeatedly localizing and quantifying BBB damage is magnetic resonance imaging (MRI). ⋯ The accuracy and reliability of two of these multiparametric MRI measures, CBF by AST and DCE-MRI determined influx of Gd-DTPA, have been established by nearly congruent quantitative autoradiographic (QAR) studies with appropriate radiotracers. In addition, some of their linkages to local pathology have been shown via corresponding light microscopy and fluorescence imaging. This chapter describes: (1) multiparametric MRI techniques with emphasis on DCE-MRI and AST-MRI; (2) the measurement of the blood-to-brain influx rate constant and CBF; and (3) the role of each in determining BBB permeability.
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Lysine acetylation of histones is one of the major epigenetic regulators of chromatin conformation and gene expression. The dynamic nature of histone acetylation is determined by the counterbalancing activity of histone acetyltransferase and histone deacetylase (HDAC) enzymes. Acetylation of histones is generally associated with open and transcriptionally active chromatin, whereas the activity of HDACs leads to histone deacetylation, condensation of chromatin, and inhibition of transcription. ⋯ Abnormal activity of HDACs has been implicated in tumorigenesis and therefore considerable effort has been put into the development of HDAC inhibitors as a means of modifying histone acetylation status and reexpressing aberrantly silenced tumor suppressor genes. This has led to the generation of a number of structurally diverse compounds that can effectively inhibit HDAC activity, thus altering chromatin structure in cancer cells. This unit discusses the methods and recent technological developments with respect to the studies of HDAC inhibition in cancer.
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Tissue microarrays (TMAs) are produced by taking small punches from a series of paraffin-embedded (donor) tissue blocks and transferring these tissue cores into a positionally encoded array in a recipient paraffin block. Though TMAs are not used for clinical diagnosis, they have several advantages over using conventional whole histological sections for research. Tissue from multiple patients or blocks can be examined on the same slide, and only a very small amount of reagent is required to stain or label an entire array. ⋯ These advantages allow the use of TMAs in high-throughput procedures, such as screening antibodies for diagnostics and validating prognostic markers that are impractical using conventional whole tissue sections. TMAs can be used for immunohistochemistry, immunofluorescence, in situ hybridization, and conventional histochemical staining. Finally, several tissue cores may be taken without -consuming the tissue block, allowing the donor block to be returned to its archive for any additional studies.
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The primary function of skeletal muscle is to generate force. Muscle force production is compromised in various forms of acquired and/or inherited muscle diseases. An important goal of muscle gene therapy is to recover muscle strength. ⋯ These include ex vivo and in situ analysis of the contractile profile of a single intact limb muscle (the extensor digitorium longus for ex vivo assay and the tibialis anterior muscle for in situ assay), grip force analysis, and downhill treadmill exercise. Force measurement in a single muscle is extremely useful for pilot testing of new gene therapy protocols by local gene transfer. Grip force and treadmill assessments offer body-wide evaluation following systemic muscle gene therapy.
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Duchenne muscular dystrophy (DMD) is a severe muscle wasting X-linked genetic disease caused by dystrophin gene mutations. Gene replacement therapy aims to transfer a functional full-length dystrophin cDNA or a quasi micro/mini-gene into the muscle. ⋯ Further modification/optimization of these microgene vectors may improve the therapeutic potency. In this chapter, we describe a species-specific, codon optimization protocol to improve microdystrophin gene therapy in the mdx model.