1,720,955 research outputs found
Crystal structure of Prp16 in complex with ADP
No full textDEAH-box helicases play a crucial role in pre-mRNA splicing as they are responsible for major rearrangements of the spliceosome and are involved in various quality-ensuring steps. Prp16 is the driving force during spliceosomal catalysis, remodeling the C state into the C* state. Here, the first crystal structure of Prp16 from Chaetomium thermophilum in complex with ADP is reported at 1.9 Å resolution. Comparison with the other spliceosomal DEAH-box helicases Prp2, Prp22 and Prp43 reveals an overall identical domain architecture. The β-hairpin, which is a structural element of the RecA2 domain, exhibits a unique position, punctuating its flexibility. Analysis of cryo-EM models of spliceosomal complexes containing Prp16 reveals that these models show Prp16 in its nucleotide-free state, rendering the model presented here the first structure of Prp16 in complex with a nucleotide
Ornithine is the central intermediate in the arginine degradative pathway and its regulation in Bacillus subtilis
No full textStructure-based Search for novel c-di-AMP Synthase Inhibiting Fragments
Full text linkOne of the major threats for the global health system is the increasing number of antibiotic resistant bacterial strains. The misuse of antibiotics during the Covid pandemic aggravates these problems. Therefore, the development of new antibiotics is mandatory to fight the rising threat of multi-resistant bacteria.
The essential second messenger c-di-AMP was discovered in 2008 and is mainly found in gram positive bacteria. It has been identified in several human pathogens like Listeria monocytogenes, Staphylococcus aureus or Enterococcus faecalis. It is a key player in the regulation of several pathways like DNA integrity scanning, cell wall metabolism or osmolyte homeostasis. Five different protein classes are able to synthesize c-di-AMP They all have the diadenylate cyclase domain (DAC) in common and need to dimerize to produce c-di-AMP in a metal-ion dependent manner from two ATP molecules. The protein class CdaA consists of three N-terminal transmembrane helices followed by the DAC domain and is often the sole diadenylate cyclase in several human pathogenic bacteria like Staphylococcus aureus, Streptococcus pneumoniae or Enterococcus faecium. The essentiality of c-di-AMP renders CdaA as potential target for the development novel antibiotic substances.
The major goal of this thesis was the establishment of a crystallization system of CdaA suitable for a fragment screening campaign in order to identify starting points for the development of inhibitors of CdaA. Therefore, the first part of this work is focused on the crystallization of CdaA from the soil bacterium Bacillus subtilis and the human pathogens Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae and Enterococcus faecium. To obtain constructs well suited for crystallography, the CdaAs of these organisms were N- and C-terminal truncated to solely consist of the DAC domain. The purity and homogeneity of every purified CdaA were verified via SDS-PAGE and DLS. To prove the enzymatic functionality of the purified proteins, the coralyne activity assay was applied with varying divalent cations. All purified CdaAs exhibit activity in the presence of manganese ions while their activity in presence of magnesium- or cobalt-ions differs. To explore these variations, crystallization trials of the different CdaAs in presence of their favorable metal-ion and ATP were carried out.
As a result, the crystal structure of Enterococcus faecium CdaA in complex with c-di-AMP and manganese as well as the crystal structure of Streptococcus pneumoniae CdaA in complex with c-di-AMP and magnesium were solved at a resolution of 2.1 Å (EfCdaA) and 2.2 Å (SpCdaA). Unfortunately, both models represent a post catalytic state where the metal ion is not coordinated in a catalytically active way, giving no further insights into the structural basis for the differing metal ion specificity. Nevertheless, if both obtained structures are compared to the Listeria monocytogenes CdaA structure in complex with c-di-AMPa conserved interaction pattern with c-di-AMP could be observed, rendering the interacting amino acids as possible targets for inhibitor design.
In order to identify potential fragments which can reduce the activity of CdaA, the CdaAs from all previously mentioned organisms were applied to crystallization trials in their APO state. For the CdaA of Enterococcus faecium and Bacillus subtilis these trials were successful. However, the crystallization system for Enterococcus faecium CdaA does not matches the requirements for a crystallographic fragment screening campaign as the crystals diffracted just to 2.4 Å and required several months to grow. In contrast, the Bacillus subtilis APO crystals diffracted up to 1.6 Å and were highly reproduceable. Further optimization of the crystallization condition led to a crystallization system which is greatly suitable for a fragment screening campaign. Through the following fragment campaign using the F2X-Entry screen, 32 unique fragments were identified to interact with BsCdaA. Mapping these fragments on a Consurf model of CdaA reduced the number to six different fragments which bound in the highly conserved active/dimerization site. These fragments were also biochemically characterized using the coralyne and malachite green assay. As none of the fragments reduced the cyclase activity of BsCdaA, an alternative approach was carried out to identify an inhibitory compound.
Here, the structure of BsCdaA in complex with AMP, which were determined in this work served as starting point for the identification of an inhibitory compound. By a computational defragmentation approach employing SeeSar, adenine was identified as fragment of AMP which is largely responsible for the biding capability of AMP. Combing the methods of computer aided drug design and structural information gained by X-ray crystallography, the Janus kinase inhibitor Ruxolitinib was identified as potential inhibitory compound for CdaA. The conserved binding mode of Ruxolitinib towards CdaA was proven as Ruxolitinib exhibits the same interaction pattern for CdaA from Bacillus subtilis and Listeria monocytogenes as it can be concluded by the X-ray structures obtained in this work as Ruxolitinib binds in the active site of the protein, it most likely acts as competitive inhibitor for CdaA. Moreover, the IC50 value of Ruxolitinib was determined as 2.7 µM for BsCdaA. Determining the cyclase activity of CdaA from Listeria monocytogenes, Staphylococcus aureus, Streptococcus pneumoniae or Enterococcus faecium in presence of Ruxolitinib also show a reduced activity, underlining the conservation of the protein-ligand interaction. Besides the in vitro studies, also in vivo experiments utilizing different Bacillus subtilis strains were carried out. The results from these experiments suggest a high specificity of Ruxolitinib towards Bacillus subtilis CdaA in vivo.2023-03-3
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
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