Methods in molecular biology
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Kinases play a pivotal role in propagating the phosphorylation-mediated signaling networks in living cells. With the overwhelming quantities of phosphoproteomics data being generated, the number of identified phosphorylation sites (phosphosites) is ever increasing. Often, proteomics investigations aim to understand the global signaling modulation that takes place in different biological conditions investigated. ⋯ These algorithms employ different approaches to predict kinase consensus sequence motifs, mostly based on large scale in vivo and in vitro experiments. The context of the kinase and the phosphorylated proteins in a biological system is equally important for predicting association between the enzymes and substrates, an aspect that is also being tackled with available bioinformatics tools. This chapter summarizes the use of the larger phosphorylation databases, and approaches that can be applied to predict kinases that phosphorylate individual sites or that are globally modulated in phosphoproteomics datasets.
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Controlled cortical impact (CCI) is a commonly used and highly regarded model of brain trauma that uses a pneumatically or electromagnetically controlled piston to induce reproducible and well-controlled injury. The CCI model was originally used in ferrets and it has since been scaled for use in many other species. This chapter will describe the historical development of the CCI model, compare and contrast the pneumatic and electromagnetic models, and summarize key short- and long-term consequences of TBI that have been gleaned using this model. In accordance with the recent efforts to promote high-quality evidence through the reporting of common data elements (CDEs), relevant study details-that should be reported in CCI studies-will be noted.
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The goals of this chapter are to provide an introduction into the variety of animal models available for studying traumatic brain injury (TBI) and to provide a concise systematic review of the general materials and methods involved in each model. Materials and methods were obtained from a literature search of relevant peer-reviewed articles. Strengths and weaknesses of each animal choice were presented to include relative cost, anatomical and physiological features, and mechanism of injury desired. ⋯ Therefore, this chapter reflects a representative sampling of the TBI animal models available and is not an exhaustive comparison of every possible model and associated parameters. Throughout this chapter, special considerations for animal choice and TBI animal model classification are discussed. Criteria central to choosing appropriate animal models of TBI include ethics, funding, complexity (ease of use, safety, and controlled access requirements), type of model, model characteristics, and range of control (scope).
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Global phosphoproteomics investigations yield overwhelming datasets with up to tens of thousands of quantified phosphosites. The main challenge after acquiring such large-scale data is to extract the biological meaning and relate this to the experimental question at hand. ⋯ The use of these tools requires careful consideration with regard to the input data, and the interpretation demands a critical approach. This chapter provides a summary of the most appropriate tools for systems analysis of phosphoproteomics datasets, and the considerations that are critical for acquiring meaningful output.
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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.