1,720,953 research outputs found
Exploring alternative expression systems for expression of heterologous proteins and metabolic pathways
No full textAugust 2016School of ScienceOur next goal was to use chromosomal integration for metabolic engineering. Multiple studies, from our laboratory and others, have found that modulatin of gene expression is key to pathway optimization. Generally, this has been achieved using promoters of varying strengths on plasmids of variable copy number. However, chromosomal integration offers advantages for metabolic engineering that could resilt in increased production of high-value chemical products. Here, we have shown, usnig a model five-gene pathway for the production of the purple pigment violacein from tryptophan, again that the location of the integration matters. We found that of the four genomic locations used, only one showed any level of violacein production, and it was surprisingly not the same location that resulted in increased levels of mCherry expression. Interestingly, when the one-gene TAL pathway, which converts phenylalanine to trans-cinnamic acid, was integrated into the same loci, very little difference in production was observed between the locations. These results indicate that optimization of production may not only be location dependent, but it may also depend on the genes being expressed.Next, we attempted to find an improved expression system for the heparin biosynthetic enzymes. To this end, two species of Bacillus and the yeast Pichia pastoris were tested for their ability to secrete the heparin biosynthetic enzymes. While intracellular expression of enzymatically active 3-OST-1 was achieved in the Bacillus species, no level of secretion was measured using a small library of signal peptides in Bacillus megaterium. Successful secretion of all of the sulfotransferases required for the chemoenzymatic synthesis of heparin was achieved in P. pastoris. Here, we hae shown that when expressed as glycoproteins in P. pastoris, these enzymes show favorable sulfotransferase activity compared to their E. coli-expressed counterparts. These results indicate that the post-translational modification of protein glycosylation plays an important role in the activity of the heparin biosynthetic enzymes and that the yeast P. pastoris may be a good host for the expression and secretion of the heparin biosynthetic enzymes.Heparin is the most widely used anticoagulant drug in the world. Current production of heparin from porchine intestinal tissue led to a contamination of the heparin supply in 2008 that resulted in over 100 deaths. This, coupled with the demand heparin puts on the worldwide pig population, has led to the need of non-animal sourced heparin. A chemeoenzymatic synthetic scheme, developed in our laboratory, has previously been used to successfully produce anticoagulant heparin. However, the enzymes for this synthesis are currently expressed and purified from Escherchia coli utilizing plasmid-based expression vectors. This Gram-negative organism is widely used for heterologous protein expression because of its fast growth and the vast molecular toolkit avilable. The work here aimed to alleviate problems with plasmid-based expression in E. coli.Heterologous protein expression has long been carried out using plasmids as expression vectors in E. coli. Problems with plasmid-based expression, primarily the necessity for selection using antibiotics that can be expensive and deleterious to the environment, led to the desire to integrate the genes for the chemoenzymatic synthesis of heparin on the genomic chromosome of E. coli. While the expression of these enzymes was unsuccessful from the genome, it became apparent that chromosomal integration had other uses. Here, we have used model reporter proteins and pathways to show that there is much to learn about chromosomal integration. By integrating the gene encoding the fluorescent reporter protein mCherry into various loci throughout the genome of E. coli, we found that expression level was location-dependent. Further, we found that integration of a single copy of the gene into one particular locus resulted in over 5-fold higher mCherry expression than a high-copy plasmid expressing the same gene. We had hypothesized that to achieve the same level of expression found in plasmid-based systems, we would need to increase the copy number of the gene. Surprisingly, we found that when we increased the copy number of the gene in a single genomic locus, there was actually a decrease in mCherry expression.Ph
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
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
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe 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
Author Under Sail The Imagination of Jack London, 1893-1902
No full textIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Intro -- Title Page -- Copyright Page -- Dedication -- Contents -- Acknowledgments -- Introduction -- 1. Spirit Truth -- 2. From Absorption to Theatricality and Back Again -- 3. "I Will Build a New Present" -- 4. Sons as Authors -- 5. Fathers as Publishers -- 6. The Daughter as Author -- 7. Lovers as Authors -- 8. At Sea with the Family -- 9. Yellow News, Yellow Stories -- 10. The Return Home -- Notes -- Bibliography -- Index -- About Jay WilliamsIn Author Under Sail, Jay Williams offers the first complete literary biography of Jack London as a professional writer engaged in the labor of writing. It examines the authorial imagination in London's work, the use of imagination in both his fiction and nonfiction, and the ways he defined imagination in the creative process in his business dealings with his publishers, editors, and agents. In this first volume of a two-volume biography, Williams traverses the years 1893 to 1902, from London's "Story of a Typhoon" to The People of the Abyss. The Jack London who emerges in the pages of Author Under Sail is a writer whose partnership with publishers, most notably his productive alliance with George Brett of Macmillan, was one of the most formative in American literary history. London pioneered many author models during the heyday of realism and naturalism, blurring the boundaries of these popular genres by focusing on absorption and theatricality and the representation of the seen and unseen. London created an impassioned, sincere, and extremely personal realism unlike that of other American writers of the time. Author Under Sail is a literary tour de force that reveals the full range of London as writer, creative citizen, and entrepreneur at the same time it sheds light on the maverick side of machine-age literature.Description based on publisher supplied metadata and other sources.Electronic reproduction. Ann Arbor, Michigan : ProQuest Ebook Central, YYYY. Available via World Wide Web. Access may be limited to ProQuest Ebook Central affiliated libraries
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