1,721,088 research outputs found
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
CRISPR-Cas9 recognition of enzymatically synthesized base-modified nucleic acids
Full text linkAn enzymatic method has been successfully established enabling the generation of partially base-modified RNA (previously named RZA) constructs, in which all G residues were replaced by isomorphic fluorescent thienoguanosine (thG) analogs, as well as fully modified RZA featuring thG, 5-bromocytosine, 7-deazaadenine and 5-chlorouracil. The transcriptional efficiency of emissive fully modified RZA was found to benefit from the use of various T7 RNA polymerase variants. Moreover, dthG could be incorporated into PCR products by Taq DNA polymerase together with the other three base-modified nucleotides. Notably, the obtained RNA products containing thG as well as thG together with 5-bromocytosine could function as effectively as natural sgRNAs in an in vitro CRISPR-Cas9 cleavage assay. N1-Methylpseudouridine was also demonstrated to be a faithful non-canonical substitute of uridine to direct Cas9 nuclease cleavage when incorporated in sgRNA. The Cas9 inactivation by 7-deazapurines indicated the importance of the 7-nitrogen atom of purines in both sgRNA and PAM site for achieving efficient Cas9 cleavage. Additional aspects of this study are discussed in relation to the significance of sgRNA-protein and PAM--protein interactions that were not highlighted by the Cas9-sgRNA-DNA complex crystal structure. These findings could expand the impact and therapeutic value of CRISPR-Cas9 and other RNA-based technologies.sponsorship: China Scholarship Council|G085321N], Fonds Wetenschappelijk Onderzoek|12Q8619N, Fonds Wetenschappelijk Onderzoek|1509920N, China Scholarship Council|201707040069 to H.Y., European Union’s Horizon 2020 research and innovation program|965135];status: Publishe
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
Ontwikkeling van antivirale geneesmiddelen gericht tegen hetinfluenzavirus PA endonuclease: een veelzijdige uitdaging.
No full textInfluenza viruses cause seasonal epidemics as well as pandemic outbreaks, associated with huge economic costs and significant morbidity and mortality, particularly in elderly or frail individuals. The widely recommended influenza vaccines require annual updates and provide inadequate protection, especially in immunocompromised and elderly persons, which are both steadily growing populations. In addition, vaccination cannot counteract the threat of a suddenly emerging influenza pandemic. Hence, antiviral drugs are an indispensable component of the broad approach to treat and prevent influenza infections, including for pandemic preparedness planning and response. Mutant viruses that are resistant to currently available anti-influenza drugs are widely distributed, even among untreated patients, and hence more potent drugs with a different and directly suppressive mode of action are urgently required. The influenza polymerase complex is widely recognized as a key drug target, given its critical role in virus replication and high degree of conservation among influenza A (of human or zoonotic origin) and B viruses. Its PA (polymerase acidic protein) subunit performs the ‘capsnatching’ endonuclease reaction in which cellular pre-mRNAs are cleaved to yield capped primers for transcription of viral RNA to mRNA. This PA endonuclease activity and the inhibition hereof by PA inhibitors (PAIs) to halt virus replication, represent the subject of this thesis project.
The foundations of our project were laid when we investigated the binding mode of one of the lead PAIs (L-742,001) in the PA active site. This β-diketo acid (DKA) compound was already discovered by researchers at Merck two decades ago, but at that time, the complete lack of knowledge regarding its target protein substantially hampered further PAI development. The crystal structure of the endonuclease catalytic site in the N-terminal part of PA (PA-Nter) was revealed in 2009, and this enabled us to perform computer-assisted docking of L-742,001 in the PA active site. A comprehensive mutational analysis was performed to reveal the binding mode of L-742,001 and determine which amino acid changes within the catalytic centre of PA or its surrounding hydrophobic pockets, alter the antiviral sensitivity to L-742,001 in cell culture. We explicitly chose to use cell culture-based methods, to obtain an accurate insight into the compound’s binding mode in a relevant virus infection model, instead of a system using isolated PA enzyme. A high level of resistance (up to 20-fold) to L-742,001 was noted for mutations I120T and H41A within the catalytic core of PA-Nter, consistent with the notion that the metalchelating effect of the bidentate DKA moiety is a crucial factor in endonuclease inhibition by L-742,001. Our observation that a virus carrying the H41A mutation in PA was able to replicate was totally unexpected, since it contradicts the assumption (which was based on PA crystal structures and enzymatic assays with isolated polymerases) that His41 is indispensable for metal-chelation and integrity of PA, and even of the entire polymerase complex. Mutations of the Gly81 residue (to Val, Phe or Thr) resulted in at least 9-fold resistance, suggesting that Gly81 may form a hydrophobic interaction with the benzyl moiety of L-742,001 or may be critical to shape the binding pocket for L-742,001. For PA mutant viruses and viral ribonucleoproteins (vRNPs) bearing mutations within the predicted hydrophobic binding pockets for L-742,001, only a modest (maximum 5-fold) level of resistance to L-742,001 was observed, which may be explained by suboptimal fitting of the compound’s aromatic wings in the proposed binding cavities, thus lacking tight binding interactions. Furthermore, our resistance data implicated a role for Arg124, Val122 and Tyr130, which surround the proposed RNA substrate binding site in PA; this was not acknowledged in previous cocrystal experiments performed by others.
In addition to the binding mode of L-742,001, our mutational analysis provided important information concerning potential resistance sites, of high relevance to anticipate on possible resistance development upon future clinical application of PAIs. The changes which totally destroyed the activity of the reconstituted vRNPs are all located in the catalytic centre of PA. This heavily compromised polymerase activity implies that it is highly unlikely that prolonged exposure to L-742,001 or another PAI would select viruses carrying these single mutations in their PA protein. On the other hand, several mutations situated at non-catalytic sites in PA had no or only marginal impact on the enzymatic functionality of viral ribonucleoprotein complexes, consistent with the less conserved nature of these PA residues. Thus, an endonuclease inhibitor optimized for tight binding to any of these residues might select for escape mutants at the corresponding sites. It is impossible to speculate on the consequences for the antiviral efficacy of such an inhibitor. Nevertheless, our biological data demonstrate that structure-based design of PA inhibitors should be accompanied by cell culture evaluation against specific PA mutant viruses, to verify the proposed mode of action and anticipate on any potential resistance sites that might be encountered during future clinical use.
Next, we focused on a series of DKA-based compounds encompassing different scaffolds and analysed their structure-activity relationship to build a plausible five-points 3D pharmacophore model, defining strict structural requirements along with chemical features. Several DKA derivatives were found to cause potent inhibition of the PA-Nter enzyme with IC50 values comparable to that of the prototype L-742,001. Three compounds (DKA-10, with a pyrrole scaffold, and DKA-40 and DKA-41, with an indole scaffold) exhibited moderate antiviral activity in cell culture, and were proven to affect viral RNA synthesis. In addition, we developed a novel real-time enzymatic assay based on a molecular beacon (MB) substrate, which is amenable to high-throughput screening and allowed to measure the enzyme kinetics of PA-Nter. Using this MB assay, we convincingly demonstrated that the setup of the enzymatic assay (i.e. substrate, metal cofactor and type of readout) should be carefully chosen during PAI evaluation. Whereas most enzymatic studies with isolated PA-Nter have indicated that the enzyme is considerably more active in the presence of Mn2+ compared to Mg2+, our MB assay works equally well with Mg2+ as with Mn2+. Since the intracellular concentration of free Mg2+ is at least 1000-fold higher than that of Mn2+, magnesium may be more biologically relevant, and evaluation of potential PAIs against both metals (as possible with our MB assay) seems recommended. For most of the DKA inhibitors tested here, the inhibitory activity against PA-Nter was far less with Mg2+ than with Mn2+, yet the lead compound L-742,001 appeared unique in having equal activity against either Mg2+ or Mn2+. This may implicate that PA-Nter assays using Mg2+ are more stringent for evaluating potential PAIs. Furthermore, while our enzymatic studies identified several PA inhibitors, only a few of these molecules were subsequently proven to have activity in influenza virus-infected cell cultures, which emphasizes the importance of proof-of-concept cell culture evaluation during early hit discovery of potential PAIs. Hence, we propose a comprehensive approach to guide PAI development, by integrating complementary enzymatic, cellular and mechanistic assays.
In the last part of our project, we aimed at implementing our gathered experience and assessed diverse scaffolds of potential metal-chelating PAIs, namely 2-hydroxybenzamides, thiosemicarbazones, N-acylhydrazones and dihydroxyindole-2-carboxamides. We combined our enzymatic and cellular assays to evaluate their inhibitory potency, and several compounds were found to produce marked inhibition in PA enzymatic assays (IC50 values < 10 μM), with promising antiviral activity in cell culture and favourable selectivity. However, although conceived as PAIs, for most of these inhibitors, the antiviral target in cell culture was unrelated to PA, but rather associated with an early or late event in the viral life cycle. We embarked on the further unravelling of their mechanism of action with time-of-addition studies and confocal microscopy, and identified at least one original compound which most likely blocks viral replication by interfering with PA endonuclease activity. In conclusion, we made an important contribution to the development and optimization of potential PAIs and achieved unique insights concerning the precise conformation and metal requirements of the PA enzyme. In addition, we revealed pitfalls in currently used enzymatic assays and convincingly demonstrated that, in order to be successful, the hit-to-lead process on novel PAIs requires rapid progression of potential hit compounds to relevant cell-based mechanistic assays.status: Publishe
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
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