Methods in molecular biology
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The smallpox vaccine based on the vaccinia virus was successfully used to eradicate smallpox, but although very effective, it was a very reactogenic vaccine and responsible for the deaths of one to two people per million vaccinated. Modified Vaccinia virus Ankara (MVA) is an attenuated derivative, also used in the smallpox eradication campaign and now being developed as a recombinant viral vector to produce vaccines against infectious diseases and cancer. MVA can encode one or more foreign antigens and thus can function as a multivalent vaccine. ⋯ Many clinical trials of these new vaccines have been conducted, and the safety of MVA is now well documented. Immunogenicity is influenced by the dose and vaccination regimen, and information on the efficacy of MVA-vectored vaccines is now beginning to accumulate. In this chapter, we provide protocols for generation, isolation, amplification, and purification of recombinant MVA for preclinical and clinical evaluation.
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Protein post-translational modifications (PTMs) are crucial for signal transduction in cells. In order to understand key cell signaling events, identification of functionally important PTMs, which are more likely to be evolutionarily conserved, is necessary. In recent times, high-throughput mass spectrometry (MS) has made quantitative datasets in diverse species readily available, which has led to a growing need for tools to facilitate cross-species comparison of PTM data. ⋯ Here, we describe an automated web-based tool, PhosphOrtholog, that accurately maps annotated and novel orthologous PTM sites from high-throughput MS-based experimental data obtained from different species without relying on existing PTM databases. Identification of conserved PTMs across species from large-scale experimental data increases our knowledgebase of evolutionarily conserved and functional PTM sites that influence most biological processes. In this Chapter, we illustrate with examples how to use PhosphOrtholog to map novel PTM sites from cross-species MS-based phosphoproteomics data.
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Post-translational modifications (PTMs) are an important source of protein regulation; they fine-tune the function, localization, and interaction with other molecules of the majority of proteins and are partially responsible for their multifunctionality. Usually, proteins have several potential modification sites, and their patterns of occupancy are associated with certain functional states. These patterns imply cross talk among PTMs within and between proteins, the majority of which are still to be discovered. Several methods detect associations between PTMs; these have recently combined into a global resource, the PTMcode database, which contains already known and predicted functional associations between pairs of PTMs from more than 45,000 proteins in 19 eukaryotic species.
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Allergic asthma, caused by inhaled allergens such as house dust mite or grass pollen, is characterized by reversible airway obstruction, associated with an eosinophilic inflammation of the airways, as well as airway hyper responsiveness and remodeling. The inhaled allergens trigger a type-2 inflammatory response with involvement of innate lymphoid cells (ILC2) and Th2 cells, resulting in high production of immunoglobulin E (IgE) antibodies. Consequently, renewed allergen exposure results in a classic allergic response with a distinct early and late phase, both resulting in bronchoconstriction and shortness of breath. ⋯ Finally, mice are challenged by three intranasal allergen administrations. We will describe the protocols as well as the most important read-out parameters including measurement of invasive lung function measurements, serum immunoglobulin levels, isolation of broncho-alveolar lavage fluid (BALF), and preparation of cytospins. Moreover, we describe how to restimulate lung single cell suspensions, perform flow cytometry measurements to identify populations of relevant immune cells, and perform ELISAs and Luminex assays to measure the cytokine concentrations in BALF and lung tissue.
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Ebolaviruses cause severe, often fatal hemorrhagic fever in Central, East, and West Africa. Until recently, they have been viewed as rare but highly pathogenic infections with regional, but limited, global public health impact. ⋯ We also describe the current animal models used in ebolavirus research, detailing each model's unique strengths and weaknesses. We focus on Ebola virus representing the type species Zaire ebolavirus of the genus Ebolavirus, as most work relates to this pathogen.