Methods in molecular biology
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Luminescence exerts an ideal optical readout for imaging living subjects including no external light source, whereas the dim luminescence and poor color pallet should be addressed for the better utilities. To address the demerits and to prevail the advantages, we developed a bright luminescent protein, named yellow Nano-lantern, exhibiting about 10-20 times brighter than wild-type RLuc. In this chapter, we demonstrate two luminescence-based protocols in detail: i.e., (a) multicolor visualization of Ca(2+) dynamics in different cellular compartments in a single cell using Ca(2+) indicators based on cyan- and orange-Nano-lanterns and (b) video-rate tumor detection in a freely moving mouse using yellow Nano-lantern.
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The development and screening of pharmacological modulators of histone deacetylases (HDACs), and particularly sirtuins, is a promising field for the identification of new drugs susceptible to be used for treatment strategies in a large array of welfare-associated, autoimmune and oncologic diseases. Here we describe a comprehensive protocol to evaluate the impact of sirtuin-targeting drugs on inflammatory and innate immune responses in vitro and in a preclinical mouse model of endotoxemia. We first provide an overview on strategies to design in vitro experiments, then focus on the analysis of cytokine production by primary macrophages and RAW 267.7 macrophages at the mRNA and protein levels, and finally describe the setup and follow-up of a mouse model of inflammation-driven endotoxic shock.
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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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Traumatic brain injury (TBI) is the leading cause of death in young adults in industrialized nations and in the developing world the WHO considers TBI a silent epidemic caused by an increasing number of traffic accidents. Despite the major improvement of TBI outcome in the acute setting in the past 20 years, the assessment, therapeutic interventions, and prevention of long-term complications remain a challenge. ⋯ In addition, limitations and advantages of each TBI model are mentioned. This will hopefully give an insight into the possibilities of each model and be of value in choosing one when designing a study.