1,721,122 research outputs found
Expression of the Bacterial Enzyme IdeS Using a GFP Fusion in the Yeast Saccharomyces cerevisiae
No full textBacterial proteases are important enzymes used in several technical applications where controlled cleavage of proteins is needed. They are challenging enzymes to express recombinantly as parts of the proteome can be hydrolyzed by their activity. The eukaryotic model organism Saccharomyces cerevisiae is potentially a good expression host as it tolerates several stress conditions and is known to better express insoluble proteins compared to bacterial systems. In this chapter we describe how the protease IdeS from Streptococcus pyogenes can be expressed in S. cerevisiae. The expression of IdeS was followed by constructing a fused protein with GFP and measuring the fluorescence with flow cytometry. The protease presence was confirmed with a Western blot assay and activity was measured with an in vitro assay. To reduce potentially toxic effect on the host cell, the growth and production phases were separated by using the inducible promoter GAL1p to control recombinant gene expression. The protocol provided may be adopted for other bacterial proteases through minor modifications of the fused protein
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
IgG glycan hydrolysis by EndoS inhibits experimental autoimmune encephalomyelitis
Get PDFAbstract Studies in experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis, have shown that B cells markedly influence the course of the disease, although whether their effects are protective or pathological is a matter of debate. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS has a protective effect in myelin oligodendrocyte glycoprotein peptide amino acid 35–55 (MOG35-55)-induced EAE, a chronic neuroinflammatory demyelinating disorder of the central nervous system (CNS) in which humoral immune responses are thought to play only a minor role. EndoS treatment in chronic MOG35-55-EAE did not impair encephalitogenic T cell priming and recruitment into the CNS of mice, consistent with a primary role of EndoS in controlling IgG effector functions. In contrast, reduced EAE severity coincided with poor serum complement activation and deposition within the spinal cord, suggesting that EndoS treatment impairs B cell effector function. These results identify EndoS as a potential therapeutic agent against antibody-mediated CNS autoimmune disorders.</p
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Isolation and Purification of Mycobacterial Extracellular Vesicles
No full textBacterial extracellular vesicles (EVs) contain numerous active substances that mediate bacterial interactions with their host and with other microbes. Best defined are the EVs from Gram-negative bacteria that have been shown to deliver virulence factors, modulate the immune responses, mediate antibiotic resistance, and also inhibit competitive microbes. Due to the complex cell wall structures of Gram-positive bacteria and mycobacteria, EVs from these bacteria were only recently reported. This protocol describes the isolation of EVs from mycobacteria
A Murine Mycobacterium marinum Infection Model for Longitudinal Analyses of Disease Development and the Inflammatory Response
No full textMycobacterial infections, including tuberculosis, are a major health problem globally. Prevention and treatments of tuberculosis are challenging due to the poor efficacy of the current vaccine and the emergence of drug-resistant strains. Therefore, it is critical to increase our basic understanding of mycobacterial virulence strategies as well as the host immune response during infection in the complex in vivo setting. While existing infection models provide valuable tools for investigating mycobacterial pathogenesis, they also exhibit limitations that can be addressed by the development of complementary models. Here we describe recent advances to the murine Mycobacterium marinum infection model, in which the bacteria produce a local infection restricted to the tail tissue. The M. marinum model has the advantage of mimicking some of the key hallmarks of human tuberculosis not replicated in the conventional murine Mycobacterium tuberculosis model, such as the formation of granulomas with central caseating necrosis and the spontaneous development of a latency-like stage. Moreover, the model is non-lethal and enables longitudinal analysis of disease development in live animals. In this chapter, we report protocols to prepare infected tissue samples for detailed and quantitative analysis of the immune response by flow cytometry, immunofluorescence microscopy, RT-qPCR, ELISA, and Western blot, as well as for the analysis of bacterial load and localization
Affinity-Purification Combined with Crosslinking Mass Spectrometry for Identification and Structural Modeling of Host-Pathogen Protein-Protein Complexes
No full textHost-pathogen protein-protein interactions are highly complex and dynamic and mediate key steps in pathogen adhesion to host, host invasion, and colonization as well as immune evasion. In bacteria, these interactions most often involve specialized virulence factors or effector proteins that specifically target central host proteins. Here, I present a mass spectrometry-based proteomics approach starting with the identification of host-pathogen interactions by affinity-purification followed by mapping the specific host-pathogen protein-protein interaction interfaces by crosslinking mass spectrometry and structural modeling of the complexes
Measurement of Antibody Binding Affinity on Bacterial Surfaces Using Flow Cytometry
No full textAntibody binding to bacterial surfaces plays a crucial role in immunity, and a key characteristic of this protein-protein interaction is the binding affinity. Determining the affinity of an antibody binding to its antigen is the first step in predicting the function in a physiological environment where other competing protein interactions may be present. Antibody-antigen affinity is often evaluated with isolated proteins. It is informative to also be able to assess antibody binding to a bacterial surface where many antigens might be present, including multiple copies of the specific antigen the antibody recognizes, and in a context where the antigen might be in a more natural conformation. In this chapter, we present a flow cytometry-based assay to measure and calculate the cell surface binding affinity or avidity of any mono- or polyclonal antibody solution
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