Methods in molecular biology
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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.
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Mass spectrometry-based phosphoproteomics is an indispensible technique used in the discovery and quantification of phosphorylation events on proteins in biological samples. The application of this technique to tissue samples is especially useful for the discovery of biomarkers as well as biological studies. We herein describe the application of a large-scale phosphoproteome analysis and SRM/MRM-based quantitation to develop a strategy for the systematic discovery and validation of biomarkers using tissue samples.
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High-field asymmetric waveform ion mobility spectrometry (FAIMS) is a gas-phase separation technique which, when coupled with liquid chromatography tandem mass spectrometry, offers benefits for analysis of complex proteomics samples such as those encountered in phosphoproteomics experiments. Results from LC-FAIMS-MS/MS are typically complementary, in terms of proteome coverage and isomer identification, to those obtained by use of solution-phase separation methods, such as prefractionation with strong cation-exchange chromatography. Here, we describe the protocol for large-scale phosphorylation analysis by LC-FAIMS-MS/MS.
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Successful therapy for TBI disabilities awaits refinement in the understanding of TBI neurobiology, quantitative measurement of treatment-induced incremental changes in recovery trajectories, and effective translation to human TBI using quantitative methods and protocols that were effective to monitor recovery in preclinical models. Details of the specific neurobiology that underlies these injuries and effective quantitation of treatment-induced changes are beginning to emerge utilizing a variety of preclinical and clinical models (for reviews see (Morales et al., Neuroscience 136:971-989, 2005; Fujimoto et al., Neurosci Biobehav Rev 28:365-378, 2004; Cernak, NeuroRx 2:410-422, 2005; Smith et al., J Neurotrauma 22:1485-1502, 2005; Bose et al., J Neurotrauma 30:1177-1191, 2013; Xiong et al., Nat Rev Neurosci 14:128-142, 2013; Xiong et al., Expert Opin Emerg Drugs 14:67-84, 2009; Johnson et al., Handb Clin Neurol 127:115-128, 2015; Bose et al., Brain neurotrauma: molecular, neuropsychological, and rehabilitation aspects, CRC Press/Taylor & Francis, Boca Raton, 2015)). Preclinical models of TBI, essential for the efficient study of TBI neurobiology, benefit from the setting of controlled injury and optimal opportunities for biometric quantitation of injury and treatment-induced changes in the trajectories of disability. ⋯ Accordingly, use of this preclinical model offers an opportunity for (a) gaining a greater understanding of the relationships of TBI induced diffuse axonal injuries and associated long term disabilities, and (b) to provide a platform for quantitative assessment of treatment interactions upon the trajectories of TBI-induced disabilities. Using the impact acceleration closed head TBI model to induce mild/moderate injuries in the rat, we have observed and quantitated multiple morbidities commonly observed following TBI in humans (Bose et al., J Neurotrauma 30:1177-1191, 2013). This chapter describes methods and protocols used for TBI-induced multiple morbidity involving cognitive dysfunction, balance instability, spasticity and gait, and anxiety-like disorder.
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N-acylethanolamine-hydrolyzing acid amidase (NAAA) is a lysosomal hydrolase degrading various N-acylethanolamines at acidic pH. Since NAAA prefers anti-inflammatory and analgesic palmitoylethanolamide to other N-acylethanolamines as a substrate, its specific inhibitors are expected as a new class of anti-inflammatory and analgesic agents. Here, we introduce an NAAA assay system, using [(14)C]palmitoylethanolamide and thin-layer chromatography. The preparation of NAAA enzyme from native and recombinant sources as well as the chemical synthesis of N-[1'-(14)C]palmitoyl-ethanolamine is also described.