Methods in molecular biology
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Although a number of animal models such as endotoxic shock and bacteremia have been used to study the pathogenesis of sepsis, cecal ligation and puncture (CLP) represents a peritonitis model with clinical features of polymicrobial infection comparable with those of peritonitis in humans. The CLP consists in the surgical perforation of the legated cecum of mice that results in immediate and constant drainage of cecal bacteria into the peritoneal cavity. The severity of the diseases depends on the diameter of the needle used for the perforation as well as on the number of cecal punctures. The CLP model of sepsis in mice is the most commonly used for studying the process of septic peritonitis and can be used as a preclinical model to test the efficacy of pharmacological agents for the treatment of sepsis.
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Protein functions rely on their ability to engage in specific protein-protein interactions and form complexes that are dynamically regulated by stimuli. Bioluminescence resonance energy transfer (BRET) is a highly sensitive technique, which allows monitoring of interaction between two proteins: one tagged with the luminescent donor Renilla luciferase, the other with a fluorescent acceptor such as YFP. ⋯ To this aim, we tag proteins of interest, transfect cells with these fusions, and use the high-sensitivity microscopy, combined with electron multiplying cooled charge-coupled device (EMCCD) cameras and improved bioluminescence probes. We thus achieve rapid acquisition of high-resolution BRET images and study the localization and dynamics of protein-protein interactions in individual live cells.
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Knowledge of novel antibiotic resistance genes aids in the understanding of how antibiotics function and how bacteria fight them. This knowledge also allows future generations of an antibiotic or antibiotic group to be altered to allow the greatest efficacy. The method described here is very simple in theory. ⋯ Any colony that grows will possess the antibiotic resistance gene and can be further examined. In actual practice, however, this technique can be complicated. The detailed protocol will need to be optimized for each bacterial strain, vector, and cell line chosen.
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Creating a robust and unbiased assay for the study of current and novel analgesics has been a daunting task. Traditional rodent models of pain and inflammation typically rely on a negative reaction to various forms of evoked stimuli to elicit a pain response and are subject to rater interpretation. ⋯ Rats, following prior administration of an activity-decreasing inflammatory insult, will positively increase spontaneous locomotor exploration when given single doses of known analgesics. The RSAA model capitalizes on a rat's spontaneous exploratory behavior in a novel environment with the aid of computer tracking software to quantify movement and eliminate rater bias.
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Human embryonic stem cells (hESCs) are pluripotent stem cells derived from the inner cell mass of human blastocysts. hESCs have become a great asset to studying human diseases and genetic functions of healthy organisms. The rate at which hESCs are being used in laboratories is exponentially increasing, and with that, the need for xeno-free hESCs is also increasing. ⋯ However, advances toward a xeno-free hESC environment are still being developed. Replacement of murine feeder layers with extracellular matrix proteins has advanced the research, and some advances toward a serum-free and feeder-free environment for hESCs are described in this chapter.