Methods in molecular biology
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Mass spectrometry-based quantitative proteomics can identify and quantify thousands of proteins in complex biological samples. Improved instrumentation, quantification strategies and data analysis tools now enable protein analysis on a genome-wide scale. ⋯ The spectrum of applications ranges from bacteria and eukaryotic cell culture systems to multicellular organisms. Here, we provide a step-by-step protocol on how to plan and perform large-scale quantitative proteome analysis using SILAC, from sample preparation to final data analysis.
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For decades, researchers have used animal self-administration models to examine the effects drugs of abuse have on physiology and behavior. Sophisticated self-administration procedures have been developed to model many different aspects of drug addiction. ⋯ In this way, animals can hold the lever down for any duration of time thereby self-administering any dose on a continuous spectrum. This procedure eliminates some of the ambiguity in translating results from effects only observed at one unit dose and allows examination of which dose the animal "prefers" at different times.
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Mitochondria are considered as the main source of reactive oxygen species (ROS) in the cell. For this reason, they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids, and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. ⋯ In this chapter, we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depends on the metabolic state of this organelle.
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Psychoneuroimmunology (PNI) may provide the scientific basis for personalized and systems medicine. The exploration of the extensive interactions among psychological and behavioral factors, the nervous system, the immune system, and the endocrine system may help understand the mechanisms underlying health, wellness, and diseases. PNI theories based on systems biology methodologies may contribute to the identification of patient patterns for establishing psychological and physiological profiles for personalized medicine. ⋯ As inflammation is a critical connection among different diseases, the elucidation of the associations may contribute to the findings of systemic therapeutic targets. With the understanding of the translational implications of PNI, integrative interventions in multiple dimensions can be applied to modulate stress responses and promote healthier behaviors. These interventions include combination drug therapies, diets, nutritional supplements, meditation, and other behavioral and mind-body strategies.
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Astrocytes produce numerous mediators under conditions of inflammation in the central nervous system. One such mediator is nitric oxide (NO) derived from nitric oxide synthase-2 (NOS-2), the high output, inducible NOS isoform. Expression of NOS-2 and production of NO can be stimulated in astrocyte cultures by combinations of cytokines and lipopolysaccharide, a gram-negative bacterial endotoxin. This chapter details methods to induce and analyze NOS-2 expression and NO production in astrocyte cultures.