Methods in molecular biology
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Animal models of traumatic brain injury (TBI) provide important tools for studying the pathobiology of brain trauma and for evaluating therapeutic or diagnostic targets. Incorporation of additional insults such as hemorrhagic shock (HS) and/or hypoxemia (HX) into these models more closely recreates clinical scenarios as TBI often occurs in conjunction with these systemic insults (i.e., polytrauma). ⋯ The physiological, histological, and behavioral aspects of this animal model have been characterized and have demonstrated exacerbating effects of systemic insults on penetrating TBI. As such, this model may facilitate the use of simultaneous assessments of multiple mechanisms and provide a platform for testing novel diagnostic and therapeutic targets.
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Protein phosphorylation, a process in which kinases modify serines, threonines, and tyrosines with phosphoryl groups is of major importance in eukaryotic biology. Protein phosphorylation events are key initiators of signaling responses which determine cellular outcomes after environmental and metabolic stimuli, and are thus highly regulated. ⋯ Peptides are separated on a C18 reversed-phase column under basic conditions and fractions collected in timed intervals followed by concatenation of the fractions. Each Fraction is subsequently enriched for phosphopeptides using TiO2 followed by LC/MS analysis.
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Immobilized metal affinity chromatography (IMAC) has been the method of choice for phosphopeptide enrichment prior to mass spectrometric analysis for many years and it is still used extensively in many laboratories. Using the affinity of negatively charged phosphate groups towards positively charged metal ions such as Fe(3+), Ga(3+), Al(3+), Zr(4+), and Ti(4+) has made it possible to enrich phosphorylated peptides from peptide samples. However, the selectivity of most of the metal ions is limited, when working with highly complex samples, e.g., whole-cell extracts, resulting in contamination from nonspecific binding of non-phosphorylated peptides. ⋯ After binding, the enriched phosphopeptides are released from the metal ions using alkaline buffers of pH 10-11, EDTA, or phosphate-containing buffers. Here we describe a protocol for IMAC using Fe(3+) for phosphopeptide enrichment. The principles are illustrated on a semi-complex peptide mixture.
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Physical trauma in the central nervous system (CNS) is usually the result of a number of forces in different directions and dimensions. A large number of experimental models have been developed to improve the possibilities to understand the outcome of CNS trauma. ⋯ Models can serve different needs, such as: to test new treatments for injuries, to reveal thresholds for injuries, to provide a better understanding of injury mechanisms, or to test tools and methods for translation between experiments and clinical data. In this chapter, we will discuss on the validation of models and translation between experimental and clinical studies.
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In patients with muscle injury or muscle disease, assessment of muscle damage is typically limited to clinical signs, such as tenderness, strength, range of motion, and more recently, imaging studies. Animal models provide unmitigated access to histological samples, which provide a "direct measure" of damage. However, even with unconstrained access to tissue morphology and biochemistry assays, the findings typically do not account for loss of muscle function. ⋯ The majority of animal models testing contractile force have been limited to the muscle groups moving the ankle, with advantages and disadvantages depending on the equipment. Here, we describe in vivo methods to measure torque, to produce a reliable muscle injury, and to follow muscle function within the same animal over time. We also describe in vivo methods to measure tension in the leg and thigh muscles.