Methods in molecular biology
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Clinical epidemiology is the science of human disease investigation with a focus on diagnosis, prognosis, and treatment. The generation of a reasonable question requires definition of patients, interventions, controls, and outcomes. ⋯ The hierarchy of evidence for clinical decision-making places randomized controlled trials (RCT) or systematic review of good quality RCTs at the top of the evidence pyramid. Prognostic and etiologic questions are best addressed with longitudinal cohort studies.
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cAMP-dependent protein kinase (PKA) is tethered at different subcellular locations by A-kinase anchoring proteins (AKAPs). AKAPs present amphipathic helices that bind to the docking and dimerization (D/D) domain of PKA regulatory subunits. ⋯ Accordingly, here we describe a bacteriophage screening procedure for identifying variants of PKA regulatory subunit D/D domains that bind selectively to individual AKAPs. This procedure can be adapted for engineering specificity into other shared protein interfaces.
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Pain assessment in animal models of osteoarthritis is integral to interpretation of a model's utility in representing the clinical condition, and enabling accurate translational medicine. Here we describe two methods for behavioral pain assessments available for use in animal models of experimental osteoarthritic pain: Von Frey filaments and spontaneous activity monitoring.
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Case Reports
Clinical diagnosis of sepsis and the combined use of biomarkers and culture- and non-culture-based assays.
Sepsis is among the most common causes of death in hospitalized patients, and early recognition followed by immediate initiation of therapy is an important concept to improve survival in these patients. According to the definition of sepsis, diagnosis of sepsis requires the recognition of the systemic inflammatory response syndrome (SIRS) caused by infection as well as recognition of possible infection-related organ dysfunctions for diagnosis of severe sepsis or septic shock. Both SIRS and organ dysfunctions may occur frequently in hospitalized patients for various reasons. ⋯ PCR-based pathogen detection can theoretically shorten the recognition of the underlying pathogen to about 8 h. However, this technique is expensive and requires additional staff in the laboratory; controlled prospective studies are missing. Although current studies suggest that PCR-based pathogen detection may be useful to shorten time to adequate antimicrobial therapy and diagnose invasive Candida infections, no general recommendations about the application of PCR for the diagnosis of sepsis can be given.
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Visualization of protein-protein interactions in vivo offers a powerful tool to resolve spatial and temporal aspects of cellular functions. The bimolecular fluorescence complementation (BiFC) makes use of nonfluorescent fragments of green fluorescent protein or its variants that are added as "tags" to target proteins under study. Only upon target protein interaction is a fluorescent protein complex assembled, and the site of interaction can be monitored by microscopy. In this chapter, we describe the method and tools for the use of BiFC in the yeast Saccharomyces cerevisiae and in mammalian cells.