1,720,968 research outputs found
Novel Approaches to Fight Antimicrobial Resistance: a Structural Biology Perspective
No full textAntimicrobial resistance is emerging as one of the worst threats to public health of our century. The use (and often misuse) of antimicrobials challenges bacterial pathogens to evolve molecular mechanisms that counteract the effect of bactericidal and bacteriostatic drugs. The SOS response is a widely conserved bacterial pathway activated in reply to genotoxic stress and is recognized as one of the main drivers of stress-induced (hyper)mutation and consequent acquisition of antimicrobial resistance. Its protein actors (RecA and LexA) regulate a large and species-specific number of genes and cellular functions, ranging from DNA repair and recombination to horizontal gene transfer, expression of virulence factors, biofilm organization and differentiation of persister bacterial populations. Pharmacological suppression of the SOS response has been suggested as a useful approach for the development of new, unconventional weapons in antimicrobial warfare, with the aim of prolonging the usability of currently available antibiotics and re-sensitizing resistant microbes.
In this thesis, several efforts are presented to find inhibitors of the SOS response, screening large libraries of small drug-like molecules, cyclic peptides and antibody fragments (nanobodies). Deep biochemical and biophysical characterization of hit compounds is given in order to estimate binding affinities for the protein targets and inhibitory potency on isolated RecA and LexA, in vitro. Since anti-LexA nanobodies emerged as the most active SOS suppressors discovered so far, microbiological testing was focused on this class of molecular tools and revealed that they can significantly downregulate the expression of pro-mutagenic factors in bacterial cells undergoing to antibiotic-induced DNA stress. X-ray structures of LexA-nanobody complexes revealed a peculiar and previously unreported mechanism of inhibition on LexA and sustained the advancement of anti- LexA nanobodies (NbSOSs) by rational protein engineering. In particular, both the increase of NbSOSs affinity for Escherichia coli LexA and the tuning of their specificity towards Pseudomonas aeruginosa LexA have been investigated and preliminary results are presented. The latter task relied on the availability of structural information on P. aeruginosa LexA, whose atomic structure has been solved by x-ray crystallography and is described in detail. Towards a full comprehension of SOS response activation in P. aeruginosa, a high-resolution structure of P. aeruginosa RecA has also been obtained by cryoEM in this work.
Last, another strategy to counteract antibiotic resistance has been pursued by developing inhibitors of NDM-1 metallo-b-lactamase, a major factor of resistance to b-lactam antibiotics. Among the tested compounds, potent enzymatic inhibitors were discovered, and one was able to strongly increase the bactericidal effect of a b-lactam. Once again, structural investigations on NDM-1 inhibitors were carried out by x-ray crystallography and disclosed the main binding determinants.
Studies presented in this thesis pave the way for the development of novel classes of biopharmaceuticals that might reinforce the current arsenal of antimicrobial weapons. Moreover, evidence is given of the great impact that structural biology studies have in biotechnology and pharmaceutical chemistry, sustaining both the understanding of mechanisms underlying macromolecular machineries and the design of new functionalities.Antimicrobial resistance is emerging as one of the worst threats to public health of our century. The use (and often misuse) of antimicrobials challenges bacterial pathogens to evolve molecular mechanisms that counteract the effect of bactericidal and bacteriostatic drugs. The SOS response is a widely conserved bacterial pathway activated in reply to genotoxic stress and is recognized as one of the main drivers of stress-induced (hyper)mutation and consequent acquisition of antimicrobial resistance. Its protein actors (RecA and LexA) regulate a large and species-specific number of genes and cellular functions, ranging from DNA repair and recombination to horizontal gene transfer, expression of virulence factors, biofilm organization and differentiation of persister bacterial populations. Pharmacological suppression of the SOS response has been suggested as a useful approach for the development of new, unconventional weapons in antimicrobial warfare, with the aim of prolonging the usability of currently available antibiotics and re-sensitizing resistant microbes.
In this thesis, several efforts are presented to find inhibitors of the SOS response, screening large libraries of small drug-like molecules, cyclic peptides and antibody fragments (nanobodies). Deep biochemical and biophysical characterization of hit compounds is given in order to estimate binding affinities for the protein targets and inhibitory potency on isolated RecA and LexA, in vitro. Since anti-LexA nanobodies emerged as the most active SOS suppressors discovered so far, microbiological testing was focused on this class of molecular tools and revealed that they can significantly downregulate the expression of pro-mutagenic factors in bacterial cells undergoing to antibiotic-induced DNA stress. X-ray structures of LexA-nanobody complexes revealed a peculiar and previously unreported mechanism of inhibition on LexA and sustained the advancement of anti- LexA nanobodies (NbSOSs) by rational protein engineering. In particular, both the increase of NbSOSs affinity for Escherichia coli LexA and the tuning of their specificity towards Pseudomonas aeruginosa LexA have been investigated and preliminary results are presented. The latter task relied on the availability of structural information on P. aeruginosa LexA, whose atomic structure has been solved by x-ray crystallography and is described in detail. Towards a full comprehension of SOS response activation in P. aeruginosa, a high-resolution structure of P. aeruginosa RecA has also been obtained by cryoEM in this work.
Last, another strategy to counteract antibiotic resistance has been pursued by developing inhibitors of NDM-1 metallo-b-lactamase, a major factor of resistance to b-lactam antibiotics. Among the tested compounds, potent enzymatic inhibitors were discovered, and one was able to strongly increase the bactericidal effect of a b-lactam. Once again, structural investigations on NDM-1 inhibitors were carried out by x-ray crystallography and disclosed the main binding determinants.
Studies presented in this thesis pave the way for the development of novel classes of biopharmaceuticals that might reinforce the current arsenal of antimicrobial weapons. Moreover, evidence is given of the great impact that structural biology studies have in biotechnology and pharmaceutical chemistry, sustaining both the understanding of mechanisms underlying macromolecular machineries and the design of new functionalities
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
Protein Electrostatics: from Computational and Structural Analysis to Discovery of Functional Fingerprints and Biotechnological Design
Get PDFComputationally driven engineering of proteins aims to allow them to withstand an extended range of conditions and
to mediate modified or novel functions. Therefore, it is crucial to the biotechnological industry, to biomedicine and to
afford new challenges in environmental sciences, such as biocatalysis for green chemistry and bioremediation. In
order to achieve these goals, it is important to clarify molecular mechanisms underlying proteins stability and
modulating their interactions. So far, much attention has been given to hydrophobic and polar packing interactions
and stability of the protein core. In contrast, the role of electrostatics and, in particular, of surface interactions has
received less attention. However, electrostatics plays a pivotal role along the whole life cycle of a protein, since early
folding steps to maturation, and it is involved in the regulation of protein localization and interactions with other
cellular or artificial molecules. Short- and long-range electrostatic interactions, together with other forces, provide
essential guidance cues in molecular and macromolecular assembly. We report here on methods for computing
protein electrostatics and for individual or comparative analysis able to sort proteins by electrostatic similarity. Then,
we provide examples of electrostatic analysis and fingerprints in natural protein evolution and in biotechnological
design, in fields as diverse as biocatalysis, antibody and nanobody engineering, drug design and delivery, molecular
virology, nanotechnology and regenerative medicine
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
The Family Keeps on Growing: Four Novel Fungal OYEs Characterized
Get PDFAiming at expanding the portfolio of Old Yellow Enzymes (OYEs), which have been systematically studied to be employed in the chemical and pharmaceutical industries as useful biocatalysts, we decided to explore the immense reservoir of filamentous fungi. We drew from the genome of the two Ascomycetes Aspergillus niger and Botryotinia fuckeliana four new members of the OYE superfamily belonging to the classical and thermophilic-like subfamilies. The two BfOYEs show wider substrate spectra than the AnOYE homologues, which appear as more specialized biocatalysts. According to their mesophilic origins, the new enzymes neither show high thermostability nor extreme pH optimums. The crystal structures of BfOYE4 and AnOYE8 have been determined, revealing the conserved features of the thermophilic-like subclass as well as unique properties, such as a peculiar N-terminal loop involved in dimer surface interactions. For the classical representatives BfOYE1 and AnOYE2, model structures were built and analyzed, showing surprisingly wide open access to the active site cavities due to a shorter β6-loop and a disordered capping subdomain
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
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