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Structure-Function Studies of Recombinant Apolipoproteins
No full textBioreactor-based foam fractionation is a method developed for the expression and purification of apolipoprotein alpha helix bundle domains for their use as nanoparticle scaffold protein components. Nanodisks (ND) are phospholipid nanoparticles comprised of a bilayer forming phospholipid and an amphipathic apolipoprotein scaffold, which circumscribes the exposed fatty acyl tails of the bilayer, conferring aqueous solubility to the complex. NDs comprised of foam fractionated apolipoproteins were used in a variety of studies to better understand the role of the amphipathic alpha helix bundle domain on cellular escape, foam fractionation potential, and ND stability. Recombinant apolipoproteins comprised entirely of an amphipathic alpha helix bundle such as apolipophorin III (apoLp-III) have previously been shown to escape their bacterial cell hosts when targeted to the periplasmic space using an N-terminal pelB leader signal sequence directly fused to the protein coding sequence. This characteristic was evaluated using other members of the exchangeable apolipoprotein family who contain an N-terminal alpha helix bundle domain in their tertiary protein structure. It was found that introducing C-terminal truncations into the protein coding sequence for recombinant apolipoprotein A-I (apoA-I (1-184)) and apolipoprotein E (apoE4-NT) resulted in cellular escape when directed to the periplasmic space. When conducting recombinant expression of these secreted apolipoproteins in a bioreactor setting, it was observed that formation of a thick foam appeared only after induction with isopropyl β-D-1-thiogalactopyranoside (IPTG). This phenomenon is specific to secreted apolipoprotein amphipathic alpha helix bundle proteins and is directly dependent upon a high concentration of secreted protein. Upon further investigation, I was concluded that foam produced during bioreactor-based expression contained the desired apolipoprotein as its sole major component. It was determined this foam could be fractionated into a separate collection vessel, and upon analysis, the protein produced in this manner contained no negative modifications as a result of the process. Ultimately, the novel foam fractionation purification method takes advantage of the natural biosurfactant like properties of alpha helix bundle apolipoproteins. The helix bundle is amphipathic in nature meaning the protein will preferentially bind free lipid in solution to stabilize its structure. It is believed that when the apolipoprotein is expressed in a bioreactor, the lipid-free protein found in the culture medium will bind microbubbles produced within the bioreactor chamber resulting in formation of a protein stabilized foam. This interaction is facilitated by the hydrophobic amino acid residues of the bundle core interacting with the gas component of the microbubble, and conversely, the hydrophilic amino acid residues interact with residual media forming a liquid interface between individual bubbles. Once a sufficient number of apolipoproteins have bound to the bubble surface, the stabilizing force produced is enough to prevent the bubble from bursting once it reaches the air/liquid interface of the culture medium and foam is produced. Foam fractionated protein was then used as a ND scaffold protein to investigate the lipid binding properties of the isolated protein. ND were formulated using a synthetic phospholipid, dimyristoylphosphatidylcholine (DMPC), and foam fractionated apolipoproteins (apoA-I (1-184), apoE4-NT, and apoLp-III). All three foam fractionated apolipoproteins were found to preferentially bind free DMPC in solution resulting in formation of stable NDs. Successful binding of an apolipoprotein scaffold to suitable lipid results in a dramatic clarification of the ND sample. This event also results in a molecular weight shift that can be visualized using size exclusion chromatography. Each of the apolipoproteins used for ND characterization studies had a characteristic elution peak of ~100-200 kDa, a drastic increase to the lipid-free apolipoprotein molecular weight and is indicative of successful ND formation. Further characterization of NDs constructed using foam fractionated scaffold protein was conducted using the cholesterol efflux capacity assay. This assay is a measure of total cholesterol efflux as a function of present lipid-free and lipid-bound apolipoproteins. It was found that lipid-free apoA-I (1-243; control), apoA-I (1-184), and apoE4-NT showed minimal cholesterol efflux when presented to differentiated THP-1 macrophage cells. Conversely, when incubating cells with NDs comprised of the same apolipoproteins, not only was the efflux value statistically higher than that of their lipid-free counterparts, but both C-terminal truncated variations (apoA-I (1-184) and apoE4-NT) had no statistical difference between particles comprised of a wild type apoA-I (1-243; control) protein. This is further evidence that the amphipathic alpha helix bundle is necessary for ND formation and NDs produced using these proteins exhibited definitive cholesterol efflux capacity. Characterized NDs were further evaluated as drug delivery vehicles for small hydrophobic molecules using the macrolide polyene antibacterial molecule, amphotericin-b (ampB). AmpB is a popular antifungal drug used to treat a variety of fungal infections in humans. The drawback of ampB treatment is the molecule becomes toxic at high concentrations over time and with the emergence of ampB resistant fungi on the rise, a new treatment regimen is required. NDs comprised of DMPC and foam fractionated apoE4-NT were investigated as a potential delivery vehicle of ampB. It was found that NDs enriched with ampB were not only able to drastically increase the solubility of the hydrophobic molecule, but also retained the molecules capacity to inhibit fungal growth. Fungal growth was measured using a yeast viability assay where the ampB NDs produced exhibited a strong growth inhibition with concentrations as low as 10 ng. The retention of biological activity suggests that foam fractionated apolipoprotein NDs represent a viable alternative to current drug delivery formulations. These data presented in this dissertation outline the practical use of foam fractionation for the production and purification of helix bundle apolipoproteins and present a possible alternative to conventional ND formulations for their utility as therapeutic nanoparticles
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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