Methods in molecular biology
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With the advent of high-throughput genomic and proteomic techniques, there is a massive amount of multidimensional data being generated and has increased several orders of magnitude. But the amount of data that is cataloged in the central repositories and shared publicly with the scientific community does not correlate the same rate at which the data is generated. Here, in this chapter, we discuss various proteomics data repositories that are freely accessible to the researchers for further downstream meta-analysis.
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Glycosylation of membrane proteins plays a crucial role in various physiological events, including intercellular recognition and intermolecular interactions on the cell surface (Gornik et al., Biochim Biophys Acta 1820:1318-1326, 2012). To study composition and function of N-glycans on membrane proteins one has to have an efficient and reproducible analytical method, which includes protein extraction and analysis of glycans. In this chapter we provide an analytical approach that includes cloud-point extraction (CPE) of total membrane proteins with the non-ionic detergent Triton X-114 and subsequent analysis of their N-glycans using hydrophilic interaction liquid chromatography (HILIC)-UPLC/HPLC. The protocol presented here can be used for parallel analysis of both membrane and intracellular proteins.
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Interstitial lung disease (ILD) comprises a large number of chronic lung disease characterized by varying degrees of inflammation and fibrosis. Mostly they are idiopathic including idiopathic pulmonary fibrosis (IPF), which is a specific disorder characterized by progressive fibrosis leading commonly to end-stage lung disease, respiratory failure, and fatal outcome. IPF and many of these fibrotic ILDs lack effective therapy despite recent approval of two drugs to slow progression in certain IPF patients. ⋯ Historically, among the first to be developed and used widely is the bleomycin model, which is the best-characterized and currently most extensively used animal model due to its ability to reproduce many aspects of IPF and other fibrotic ILDs, good reproducibility, and ease of induction. Studies using the bleomycin model have identified many of the cellular and molecular mechanisms now recognized as being important in pathogenesis of IPF and other fibrotic ILDs, as well as novel therapies for these diseases, including two recent drugs approved for treatment of IPF. This chapter will describe commonly used techniques for induction of the model by endotracheal administration of bleomycin through surgical and nonsurgical (transoral instillation).
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Allergic asthma is a heterogeneous inflammatory lung disease affecting millions of people worldwide and with a steadily increasing incidence. Mouse models have been of utmost importance in uncovering key inflammatory cell types, cytokines, and pathways in the development and maintenance of allergic asthma. ⋯ Here, we describe an often used and relevant HDM-based protocol to establish acute allergic asthma, and the methods we have developed to rapidly analyze inflammatory cell infiltration in the bronchalveolar lavage fluid by flow cytometry. Moreover, we explain the methods to restimulate T cells from lung-draining mediastinal lymph nodes with HDM to allow the measurement of cytokine secretion profiles of allergen reactive T cells.
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A high number of infectious diseases affecting livestock and companion animals are caused by pathogens of viral etiology. Ensuring the maximum standards of quality and welfare in animal production requires developing effective tools to halt and prevent the spread of those infectious diseases affecting animal husbandry. ⋯ One step ahead is needed to improve and adapt vaccine manufacturing to the use of new generation vaccine technologies already tested in experimental settings. Here we present in the context of animal viral diseases of veterinary interest, an overview of some current vaccine technologies that can be approached for virus pathogens with a brief insight in the type of immunity elicited.