1,720,960 research outputs found
Towards the identification of small molecules inhibiting he dimerization of HCMV DNA polymerase processivity factor UL44
Full text linkHuman cytomegalovirus (HCMV) is a leading cause of congenital defects in humans. Currently available antivirals utilized for the therapy against HCMV infections have a series of contraindications due to their high cost, low bioavailability, high toxicity and the uprising of resistant viral strains. Furthermore no vaccine is available and no drugs are approved to prevent vertical transmission during pregnancy, therefore new, effective antiviral are highly needed.
HCMV DNA polymerase accessory protein UL44 plays an essential role in viral replication, conferring processivity to the DNA polymerase catalytic subunit UL54 by tethering it to the DNA. Binding of UL44 to dsDNA occurs in the absence of ATP and clamp loaders, and depends on UL44 homodimerization. Indeed, our research group recently demonstrated that the protein can dimerize in cells and point mutations disrupting protein self-interaction also prevent DNA binding and abolish viral oriLyt-dependent DNA replication in transient transcomplementation assays. Therefore, disruption of UL44 homodimerization represents an attractive target for the development of new anti-virals. Based on these observations, using the recently published crystal structure of UL44 homodimers our research group previously performed a virtual screening with the Glide software in combination with a library of 1.3 x 10^6 small molecules (SMs) to identify SMs potentially interfering with UL44 homodimerization. After three rounds of screening (HTVS: high-throughput virtual screening, SP: standard precision, XP: extra precision), followed by an in depth analysis of compounds chemical properties, 18 SMs were selected for further analysis. Selected compounds were obtained from a commercial supplier, to be tested in a variety of assays for their ability to inhibit UL44 homodimerization both in cells and in vitro, as well as on HCMV replication.
To this end, we applied a number of in cells and in vitro assays to monitor the effect of the SMs on UL44 dimerization. In cells methods included Fluorescent Resonant Energy Transfer (FRET) and Bioluminescence Resonant Energy Transfer (BRET), while as GST pull-down assays was chosen as the in vitro method.
FRET acceptor photobleaching was capable of detecting both UL44 homodimerization and UL44 binding to the catalytic subunit C-terminal domain (residues 1125-1242). Such interactions were sensitive to point mutations specifically impairing the two processes, highlighting the specificity of this technique. Therefore, we were able to confirm that UL44 forms dimers in cells. However, data acquisition and analysis proved quite time consuming and dependent on high fusion proteins expression levels.
BRET assays allowed to quickly and precisely quantitatively monitor UL44 self-interaction and binding to UL54 in living cells, and through saturation experiments allowed to precisely calculate Bmax and B50 values, relative to homodimerization or binding to UL54 for a number of single amino acids substitution derivatives of UL44 impaired for dimerization, binding to UL54 or to dsDNA, as well as for nuclear targeting. These data allowed gaining insights relative to the formation of HMCV DNA polymerase holoenzyme, suggesting conformational changes within UL44 upon DNA binding in complex to UL54. Furthermore, calculation of Bmax and B50 values was used to establish three different cellular systems expressing ideal amount of UL44 for screening purposes, in that they generated a BRET ratio similar to half of the Bmax. These included a fully stable expression system, whereby both RLuc-UL44 and YFP-UL44 are stably expressed in HEKA derived cells, a stable/transient system whereby YFP-UL44 is stably expressed and RLuc-UL44 is transiently expressed, or a fully transient system whereby both fusion proteins are transiently expressed. Competition assays performed overexpressing increasing amounts of FLAG-tagged UL44 as a competitor revealed that no inhibition could be obtained for the fully stably expressing system. This result highlighted the difficulty in disrupting a pre-existing protein complex and suggested to focus our attention on transient systems.
Keeping this in mind, a first small-scale screening was performed to study the impact of 18 SMs on UL44 dimerization by BRET using the stable/transient system. The 18 selected SMs were resuspended in DMSO and their toxicity was evaluated in cell culture, before being added at sub-toxic concentration to the YFP-UL44 cell line, six hours post-transfection with RLuc-UL44. Our analysis identified only compounds slightly inhibiting the BRET ratio relative to UL44 homodimerization.
Based on these disappointing results, we revaluated the BRET assays setup, by decreasing the time of SM incubation before assaying their effect from 42 to 18h, by switching to a completely transient system, and by decreasing the amount of expressed proteins. The last modification required a change in the substrate used for generation of bioluminescent signal from RLuc-fusion proteins from CTZ to hCTZ.
A new screening was performed, which resulted in very similar results to those obtained using the original setup. Furthermore when hit candidates were re-evaluated for their effect using a negative control, their effect on BRET ratio of UL44 proved unspecific. Additionally, BRET - similarly to FRET - failed to detect a specific effect on the UL54/UL44 interaction by a SM known to disrupt such interaction. Therefore we conclude that, with our current settings, BRET and FRET are not the ideal techniques to search for SM inhibitors of protein-protein interactions
We then focused on in vitro methods, starting with a GST pull-down assay, which was performed using UL44 C-terminal domain (residues 1-290), either fused to GST of a 6His-tag. Our results indicated that GST pull-down was capable of detecting differences in binding between wild-type UL44 and a dimerization-impaired mutant, suggesting a possible application for the screening of SMs. GST pull-down is currently being implemented for this purpose, and preliminary data suggest that a number of tested SMs could impair UL44 dimerization.
In summary, we have developed a number of assays to monitor UL44 dimerization. Whereas in cells RET based assays confirmed that UL44 form dimers in living cells, they proved suboptimal for screening proposes. On the other hand, in vitro methods such as GST pull-down might prove more sensitive and are currently being implemented for the identification of SMs inhibitors of UL44 dimerizatio
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
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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