Methods in molecular biology
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In the past decades, a variety of publicly available data repositories and resources have been developed to support protein related information management, data-driven hypothesis generation and biological knowledge discovery. However, there is also an increasing confusion for the researchers who are trying to quickly find the appropriate resources to help them solve their problems. In this chapter, we present a comprehensive review (with categorization and description) of major protein bioinformatics databases and resources that are relevant to comparative proteomics research. We conclude the chapter by discussing the challenges and opportunities for developing new protein bioinformatics databases.
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Phosphoproteomics, the systematic study of protein phosphorylation events and cell signaling networks in cells and tissues, is a rapidly evolving branch of functional proteomics. Current phosphoproteomics research provides a large toolbox of strategies and protocols that may assist researchers to reveal key regulatory events and phosphorylation-mediated processes in the cell and in whole organisms. ⋯ We then discuss various tandem mass spectrometry approaches for phosphopeptide sequencing and quantification, and we consider aspects of phosphoproteome data analysis and interpretation. Efficient integration of these stages of phosphoproteome analysis is highly important to ensure a successful outcome of large-scale experiments for studies of phosphorylation-mediated protein regulation.
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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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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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Stem cell culture systems that rely on undefined animal-derived components introduce variability to the cultures and complicate their therapeutic use. The derivation of human embryonic stem cells and the development of methods to produce induced pluripotent stem cells combined with their potential to treat human diseases have accelerated the drive to develop xenogenic-free, chemically defined culture systems that support pluripotent self-renewal and directed differentiation. In this chapter, we describe four xeno-free culture systems that have been successful in supporting undifferentiated growth of hPSCs as well as methods for xeno-free subculture and cryopreservation of hPSCs. Each culture system consists of a xeno-free growth medium and xeno-free substratum: (1) TeSR2™ with human recombinant laminin (LN-511); (2) NutriStem™ with LN-511; (3) RegES™ with human foreskin fibroblasts (hFFs); (4) KO-SR Xeno-Free™/GF cocktail with CELLstart™ matrix.