Methods in molecular biology
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This chapter will outline strategies and ideas for the commercialization a promising wound healing technology discovered in an academic setting. This would include, but not limited to addressing topics such as intellectual property protection, funding, technology development, and regulatory aspects (i.e., navigating through the FDA).
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Human and mouse alkaline phosphatases (AP) are encoded by a multigene family expressed ubiquitously in multiple tissues. Gene knockout (KO) findings have helped define some of the precise exocytic functions of individual isozymes in bone, teeth, the central nervous system, and in the gut. For instance, deficiency in tissue-nonspecific alkaline phosphatase (TNAP) in mice (Alpl (-/-) mice) and humans leads to hypophosphatasia (HPP), an inborn error of metabolism characterized by epileptic seizures in the most severe cases, caused by abnormal metabolism of pyridoxal-5'-phosphate (the predominant form of vitamin B6) and by hypomineralization of the skeleton and teeth featuring rickets and early loss of teeth in children or osteomalacia and dental problems in adults caused by accumulation of inorganic pyrophosphate (PPi). ⋯ Analogous to the role of IAP in the gut, TNAP expression in the liver may have a proactive role from bacterial endotoxin insult. Finally, more recent studies suggest that neuronal death in Alzheimer's disease may also be associated with TNAP function on certain brain-specific phosphoproteins. This review recounts the established roles of TNAP and IAP and briefly discusses new areas of investigation related to multisystemic functions of these isozymes.
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By the combinations of high-throughput analytical technologies in the fields of transcriptomics, proteomics, and metabolomics, we are now able to gain comprehensive and quantitative snapshots of the intracellular processes. Dynamic intracellular activities and their regulations can be elucidated by systematic observation of these multi-omics data. ⋯ Moreover, interpretation of such multitude of data requires an intuitive pathway context. Here we describe such statistical methods for the integration and comparison of multi-omics data, as well as the computational methods for pathway reconstruction, ID conversion, mapping, and visualization that play key roles for the efficient study of multi-omics information.
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Promoter deletion analysis is a useful tool for identifying important regulatory regions involved in transcriptional control of gene expression. In this approach, a series of promoter deletion fragments are fused to a reporter gene, such as chloramphenicol acetyltransferase or luciferase gene in a vector, and then transfected into cells for induction. Screening the expression level of the reporter gene using either a qualitative or a quantitative assay, allows to identify the regulatory regions of interest (e.g., cis-acting elements or enhancer) in the promoter. ⋯ Therefore, the enzymatic activities of firefly and Renilla luciferases can be sequentially measured in a single sample by controlling reaction conditions. Here, we describe a dual-luciferase reporter assay, where the promoter of interest is fused to a firefly luciferase reporter and is co-transfected into cells with an internal control vector (pRL-CMV) to express Renilla luciferase. Both the Firefly and Renilla luciferases are measured using a dual-luciferase reporter assay system which improves experimental accuracy.
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Studies of DNA methylation in Arabidopsis have rapidly advanced from the analysis of a single reference accession to investigations of large populations. The goal of emerging population studies is to detect differentially methylated regions (DMRs) at the genome-wide scale, and to relate this variation to gene expression and phenotypic diversity. Whole-genome bisulfite sequencing (WGBS-seq) has established itself as a gold standard in DNA methylation analysis due to its high accuracy and single cytosine measurement resolution. ⋯ However, detection can be susceptible to strong signal distortions resulting from a combination of dye bias and the CG content of effectively unmethylated genomic regions. We show that these issues can be easily bypassed by taking appropriate data preparation steps and applying suitable analysis tools. We conclude that MeDIP-chip is a reasonable alternative to WGBS-seq in emerging Arabidopsis population epigenetic studies.