1,720,954 research outputs found
Using GFP to investigate protein localisation, function and global cellular response
No full textIntegral membrane proteins (IMPs) make up 20-30 % of genes in all walks of life. They are major determinants of disease pathology, making them prime therapeutic targets for cancer, bacterial infection and genetic disorders. Despite this, they are underrepresented in the literature; this is often attributed to complications with IMP expression, purification and characterisation. This thesis aimed to tackle the difficulties with IMP characterisation. Exploitation of tagged versions of the IMPs has opened up new research fields, initially based on simple observations. The tag used is the well characterised GFP which has allowed for the simple optimisation of conditions for protein over-expression, determination of intracellular localisation of H. sapiens SWEET sugar transporter, development of a ligand binding method that does not rely on properties of the ligand and demonstrated that protein over-expression using the Escherichia coli pET system only occurs in mutant forms of this organism. The implication being that protein over-expression only occurs via genetically modified organisms. Ubiquitously expressed, the MFS is one of the largest protein superfamilies, with roles including metabolite and xenobiotic transport. They have been implicated in the development of antimicrobial resistance in E. coli, making them a clinically relevant target. An expression library of 63 GFP-tagged proteins was produced, before screening for optimal expression conditions. The larger amounts of transporter obtained via this approach enabled the implementation of a ligand-binding assay using the technique of thermophoresis. This approach produced novel binding substrates as well as identifying a new binding event for the well characterised drug efflux transporter mdfA. Significantly, the approach has shown that cyclic AMP also binds to mdfA and another MFS transporter kgtP, potentially identifying a novel role for these transporters in the catabolite repression process. The work presented in this thesis has shown the versatility of a reporter system like GFP to uncover fundamental properties at the cellular level (protein localisation experiments), the biochemical level (optimisation of protein over-expression and ligand binding studies) but also at the cellular level (E. coli’s use of mutants during protein over-expression). This research is a building block to identifying new drug targets to tackle the global problem of antimicrobial resistance
Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance
No full textBackground: the E. coli pET system is the most widely used protein over-expression system worldwide. It relies on the assumption that all cells produce target protein and it is generally believed that integral membrane protein (IMP) over-expression is more toxic than their soluble counterparts.Results: using GFP-tagged proteins, high level over-expression of either soluble or IMP targets results in > 99.9% cell loss with survival rate of only < 0.03%. Selective pressure generates three phenotypes: large green, large white and small colony variants. As a result, in overnight cultures, ~ 50% of the overall cell mass produces no protein. Genome sequencing of the phenotypes revealed genomic mutations that causes either the loss of T7 RNAP activity or its transcriptional downregulation. The over-expression process is bactericidal and is observed for both soluble and membrane proteins.Conclusions: we demonstrate that it is the act of high-level over-expression of exogenous proteins in E. coli that sets in motion a chain of events leading to > 99.9% cell death. These results redefine our understanding of protein over-production and link it to the adaptive survival response seen in the development of antimicrobial resistance.</p
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Variations on the Author
Full text link“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
Full text linkWe 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
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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