1,720,954 research outputs found
Genome - Wide Regulatory Principles of Essential Transcription Factors from Mycobacterium Tuberculosis
No full textAll life requires the expression of the genetic information stored in DNA into RNA, a process known as transcription. Hence, DNA-dependent RNA polymerases (RNAPs) perform the first step of gene expression, thereby dictating the amounts of gene products that proceed to downstream cellular reactions. The activity of RNAP can be modulated by numerous molecules including protein transcription factors (TFs), ligands, and antibiotics. Since bacteria only contain a single RNAP to express all genes, it is a prime therapeutic target. Most notably, the antibiotic rifampicin targets RNAP in the bacterial pathogen Mycobacterium tuberculosis (Mtb) and has therefore been a cornerstone of the frontline therapeutic regimen to treat tuberculosis (TB) since the 1960s. However, the rise of rifampicin-resistance necessitates new strategies to target RNAP and combat TB, which remains the leading cause of death from infectious disease worldwide. Efficient Mtb transcription requires multiple TFs that are essential for bacterial viability, pointing to specific steps of the transcription process that may be useful to target therapeutically. Yet, the direct genomic targets for most Mtb TFs are not known, limiting our understanding of Mtb gene expression. Obstacles include the lack of easily-predicted binding motifs, the degree of motif degeneracy tolerated, and the compensatory regulatory cascades triggered by perturbation of TFs in cells. In addition, the lack of tools to study non-model TFs, particularly those from difficult-to-culture microbes like Mtb, exacerbates these challenges. Specifically, we found that a major hindrance to identifying direct TF targets was the methodological gap between genomics and biochemistry. In cellulo genomics (e.g., ChIP-seq, RNA-seq) can provide genome-wide information, but pleiotropic indirect effects frequently obscure primary TF effects in cells, particularly when a TF is essential for viability or a global regulator. Conversely, in vitro transcription assays using purified RNAP can measure direct TF effects on RNA synthesis for a single gene, but these assays are too low-throughput for genome-scale transcription measurements and discovery of general principles. Therefore, the central aim of this thesis was the development of a novel cell-free genomics (CFG) method to bridge biochemistry and genomics. We reconstitute genome-wide transcription in vitro using purified components. We then quantify the output RNA using single-nucleotide-resolution RNA-seq and robust statistical analysis to count RNA 5\u27 ends (to study transcription initiation) or RNA 3\u27 ends (to study transcription elongation/termination) in the presence versus absence of essential TFs from tMb. CFG thus permits us to identify promoters and terminators whose expression is directly affected by TFs. We first validated CFG by counting the 5\u27 ends of transcripts in the presence versus absence of the cyclic AMP receptor protein (CRP), the archetypal TF that originated the study of transcription regulation in 1970. CFG revealed 90 promoters where Mtb CRP alone is sufficient to modulate transcription initiation levels. From these direct promoter targets, we re-discover the known Mtb CRP binding motif and reveal that the predicted strength of CRP binding to its consensus site is a quantitative predictor of its effect size on a given promoter. We also identify known target genes found in cellulo. Integration of the CFG-derived sufficiency regulon of CRP with the necessity regulon previously determined using RNA-seq and ChIP-seq in exponentially growing Mtb cells revealed where CRP can act autonomously; where it requires other cellular regulators to modulate transcription; and where it exerts indirect transcriptional effects. Our interdisciplinary synthesis thus provides a roadmap to gain unprecedented resolution of transcription regulatory networks. We next applied CFG to identify promoters regulated by the actinobacteria-specific transcription initiation factor holo-WhiB1 in Mtb. Holo-WhiB1 was previously intractable by other approaches, and thus its direct genomic targets were unknown in any species. We first performed a whiB1 knockdown RNA-seq time course in Mtb cells, demonstrating its essentiality and revealing its global effects on the Mtb transcriptome. We next used CFG to identify the direct effects of holo-WhiB1 on transcription initiation genome-wide. Integration of in cellulo and cell-free hits permitted the identification of promoters that are directly regulated by holo-WhiB1 in exponentially growing Mtb cells, revealing that holo-WhiB1 activates numerous genes involved in translation and fatty acid biosynthesis. CFG revealed that unlike CRP, holo-WhiB1 does not appear to bind to a consensus motif at the position where it contacts promoter DNA (directly upstream of the housekeeping –35 promoter element). Rather, holo-WhiB1 activates transcription of promoters that have suboptimal –35 elements and represses transcription from promoters that have strong –35 contacts. We validate direct holo-WhiB1 activation and repression using in vitro transcription initiation assays with minimal promoter DNA constructs, allowing further mechanistic dissection of holo-WhiB1\u27s effects using single-particle cryo-EM and functional mutagenesis. Lastly, we apply CFG to quantify the 3\u27 ends of transcripts, permitting the first genome scale in vitro quantification of transcription termination in a cell-free system. We chose to study the essential pro-termination TFs Mtb NusA (conserved in all bacteria and archaea) and Mtb NusG (the only transcription factor conserved in all three domains of life), alone and in combination. Their mechanisms have remained elusive in part because neither has a predicted nucleic acid binding motif. We validate new NusA and NusG terminator targets in the Mtb genome using gold standard in vitro transcription termination assays and reveal a novel selectivity mechanism for how NusA and NusG regulate some terminators but not others. Specifically, we find distinct contacts between NusA versus NusG on pre-termination complexes likely favor distinct mechanisms of RNA release from the RNAP active site. NusG contacts both RNAP and DNA, but not RNA; therefore, NusG fails to stimulate terminators that have AT-rich downstream DNA and thus likely favor forward translocation of RNAP to stimulate RNA release. In contrast, NusA contacts both RNA and RNAP, but not DNA; therefore, NusA fails to stimulate terminators with RNA terminator hairpins that are predicted to invade the RNA–DNA hybrid and thus likely require a concomitant rotational wrenching of the hairpin away from RNAP to stimulate RNA release. Since these NusA/G contacts with transcription complexes are highly conserved, aspects of our model may generalize across cellular life. In sum, we propose that cell-free genomics comprehensively fills a critical methodological gap in the field of gene expression. CFG facilitates the study of TFs with various modes of action, including those intractable to study by other approaches, those lacking clear bioinformatic predictors like binding motifs, or both. CFG also holds promise for broader applications, including higher-order experimental designs, other transcriptional perturbations (e.g. ligands, antibiotics), and diverse species. By complementing existing approaches, CFG brings biochemistry and enzymology into the genomics era to transform our understanding of fundamental transcriptional regulation
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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