1,720,969 research outputs found
Conditional gene expression using ribozymes : Post-transcriptional control of amino acid identity in protein synthesis and temperature-dependent gene expression
No full textDuring the main research study we wanted to demonstrate that it is possible to control the amino acid identity in vivo on a post-transcriptional level by switching designer tRNAs via the ligand-dependent regulation of ribozyme activity.For this purpose we designed and generated various modular constructs that are composed of distinct parts: an aptamer, a catalytic ribozyme core (hammerhead ribozyme) and of course the appropriate amber suppressor tRNA. Our design included the connection of aptamer (theophylline or thiamine pyrophosphate) to ribozyme via a small connecting sequence, which was randomized. With the help of in vivo screening process we managed to identify functional riboswitches that possess a desired activity (ON or OFF). This activity resulted in the conditional release or not of a suppressor tRNA which in turn serves as tool used for decoding of the amber stop codon in the mRNA of our reporter gene, a fluorescent protein.We managed to obtain novel, individual tRNA riboswitches that we successfully combined in a dual fashion, in order to switch one or the other tRNA, therefore the protein synthesis of one or the other protein variant.Considering the fact that suppression efficiency is inherently low due to release factor competition to the generated suppressor tRNA, this methodology is not quantitative. However, we demonstrated that the protein of interest can be selectively generated in relatively high yields with the use of desired small molecule trigger.This idea can be further developed and exploited in ways that benefit the field of protein chemistry. Considering the high modularity of our constructs, one can envision the multitude of possibilities arising from substituting or expanding by adding one or more parts to the construct. For example, use of different aptamer sensor in combination with a tRNA will unlock the potential generation of more than two different protein variants, using all the while, the same message RNA containing one amber stop codon for which all suppressor tRNAs compete for. Of course optimization of the current system by utilizing more stringent expression control systems could improve the overall performance of the riboswitching system. Another approach which can enhance the performance of our current system is the use of release factor knockout strains which is documented to double the efficiency of suppression systems. Moreover, the applicability of our already versatile tool can be exponentially increased when combined with the fascinating and thriving field of unnatural amino acids. Considering the fact that the central dogma is highly conserved throughout all domains of life, our approach should be able to be realized in higher organisms as well.Finally, we demonstrated that it is possible to design and synthesize ribozyme-based gene regulation elements that are able to respond to temperature changes. This can be achieved with the aid of a thermosensing hairpin that melts with increased temperatures which in turn modulates the function of the hammerhead ribozyme. By replacing the ligand responsive aptamer in the hammerhead ribozyme construct with a Salmonella RNA thermometer we expanded the concept of hammerhead ribozyme regulated gene expression. In this new fusion construct the hammerhead ribozyme functions by exposing a ribosome-binding site upon self-cleavage, while an RNA hairpin undergoes a conformational change caused by an increase in temperature. After in vivo screening of several temperature-sensitive clones for gene regulation activity, we identified two of such thermozymes. The designed tool acts in reverse manner to the natural thermo-sensitive RNA hairpin, which is used by Salmonella to increase gene expression. New tools were developed in order to regulate gene expression in response to temperature changes.On the whole, we presented methodologies that include the use of RNA-only toolboxes which can be adapted, modified and applied in the field of protein chemistry and protein engineering. Further development of the existing tools, along with their potential application in higher organisms, is possible to shed light in investigation of protein synthesis pathways, mutant generation and their mechanisms and functions.publishe
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
Assay development for the discovery of small-molecule inhibitors of bacterial adhesion to collagen
No full textWe set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from Yersinia enterocolitica that binds to collagen.
Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance.
Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to control wells. Our assay is easy to scale up, and can be adopted to different ECM component/Adhesin combinations. Alternatively, bacterial pathogens (harboring deletion mutants of adhesins compared to wildtype) could be used directly in the same assay if they express GFP as a reporter at high levels
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
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