Methods in molecular biology
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Patients with severe traumatic brain injury (TBI) frequently present with concomitant injuries that may cause secondary brain injury and impact outcomes. Animal models have been developed that combine contemporary models of TBI with a secondary neurologic insult such as hypoxia, shock, long bone fracture, and radiation exposure. ⋯ Here, we review these models and their collective contribution to the literature on TBI. In addition, we provide protocols and notes for two well-characterized models of TBI plus hemorrhagic shock.
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Protein kinases are widely considered to be invaluable target enzymes for drug discovery and for diagnosing diseases and assessing their prognosis. Effective analytical techniques for measuring the activities of cellular protein kinases are therefore required for studies in the field of phosphoproteomics. We have recently developed a highly sensitive microarray-based technique for tracing the activities of protein kinases. ⋯ In this chapter, we describe a standard protocol for detecting phosphopeptides by biotin-labeled Phos-tag. We also describe a microarray system for high-throughput profiling of intracellular protein kinase activities. The Phos-tag-based method is expected to be useful in the rapid detection of the complex range of phosphorylation reactions involved in cellular signaling events, and it has potential applications in high-throughput screening of kinase activators or inhibitors.
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Clostridium difficile is a challenging infection that can be difficult to treat with antibiotic therapy. This chapter outlines the processing material for fecal microbiota transplantation (FMT), also known as stool transplant. ⋯ FMT uses a stool sample collected from a healthy, screened donor to restore healthy microbiota in the colon of a patient with CDI for symptom resolution. Here, we describe a rapid method for FMT preparation that uses inexpensive and disposable materials.
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This chapter presents an introduction to data mining with machine learning. It gives an overview of various types of machine learning, along with some examples. It explains how to download, install, and run the WEKA data mining toolkit on a simple data set, then proceeds to explain how one might approach a bioinformatics problem. Finally, it includes a brief summary of machine learning algorithms for other types of data mining problems, and provides suggestions about where to find additional information.
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Obtaining high phosphoproteome coverage requires specific enrichment of phosphorylated peptides from the often extremely complex peptide mixtures generated by proteolytic digestion of biological samples, as well as extensive chromatographic fractionation prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Due to the sample loss resulting from fractionation, this procedure is mainly performed when large quantities of sample are available. To make large-scale phosphoproteomics applicable to smaller amounts of protein we have recently combined highly specific TiO2-based phosphopeptide enrichment with sequential elution from immobilized metal affinity chromatography (SIMAC) for fractionation of mono- and multi-phosphorylated peptides prior to capillary scale hydrophilic interaction liquid chromatography (HILIC) based fractionation of monophosphorylated peptides. In the following protocol we describe the procedure step by step to allow for comprehensive coverage of the phosphoproteome utilizing only a few hundred micrograms of protein.