Mutations in the intersubunit bridge regions of 16S rRNA affect decoding and subunit–subunit interactions on the 70S ribosome

The small and large subunits of the ribosome are held together by a series of bridges, involving RNA-RNA, RNA-protein and protein-protein interactions. Some 12 bridges have been described for the Escherichia coli 70S ribosome. In this work, we have targeted for mutagenesis, some of the 16S rRNA residues involved in the formation of intersubunit bridges B3, B5, B6, B7b and B8. In addition to effects on subunit association, the mutant ribosomes also affect the fidelity of translation; bridges B5, B6 and B8 increase decoding errors during elongation, while disruption of bridges B3 and B7b alters the stringency of start codon selection. Moreover, mutations in the bridge B5, B6 and B8 regions of 16S rRNA also correct the growth and decoding defects associated with alterations in ribosomal protein S12. These results link bridges B5, B6 and B8 with the decoding process and are consistent with the recently described location of translation factor EF-Tu on the ribosome and the proposed involvement of h14 in activating Guanosine-5'-triphosphate (GTP) hydrolysis by aminoacyl-tRNA center dot EF-Tu center dot GTP. These observations are consistent with a model in which bridges B5, B6 and B8 contribute to the fidelity of translation by modulating GTP hydrolysis by aminoacyl-tRNA center dot EF-Tu center dot GTP teRNAry complexes during the elongation phase of protein synthesis

Comunicación bacteriana: fundamentos y estrategias de interferencia

El conocimiento de la comunicación bacteriana, o quorum sensing (QS), es crucial para el desarrollo de nuevos agentes antimicrobianos. Esta tesis ofrece un enfoque novedoso para explicar la regulación del QS dependiente de proteínas LuxR bajo el marco de la teoría de la percolación. Además, propone nuevos mecanismos de inactivación de estos sistemas. En concreto, se centra sobre el diseño y optimización de nanopartículas a base de quitosano (CS NPs) para interferir con el QS. Un hallazgo clave de la tesis es que las CS NPs interaccionan con las bacterias e inhiben el QS en momentos específicos del crecimiento. El trabajo plasmado en esta tesis abre el horizonte para la aplicación de CS NPs al control de la virulencia de bacterias patógenas

Aprendizaxe baseada en problemas en Xenética Molecular

[Resumo] A aprendizaxe baseada en problemas (ABP) trátase dunha metodoloxía na que os alumnos, guiados polo profesor, deben resolver un problema. No proceso, os alumnos constrúen e aplican coñecementos en lugar de ser meros receptores. Neste traballo, propoñemos aplicar ABP na materia Xenética Molecular do Grao en Bioloxía e do Programa de Simultaneidade do Grao en Bioloxía e o Grao en Química, impartidos na Facultade de Ciencias da UDC. O obxectivo é espertar un maior interese pola Bioinformática, unha parte da materia considerada a miúdo ardua e tediosa. Tamén pretendemos incrementar a orientación da materia cara á práctica profesional. A actividade que se propón consta de tarefas prácticas estruturadas, idénticas ás que se realizan nun laboratorio típico de Xenética Molecular. Así, o problema a resolver enfronta aos alumnos a unha situación real que implica non só aplicar coñecementos senón tamén tomar decisións. Para resolver o problema, os alumnos recibirán unha explicación previa do profesor e disporán de orientación mediante titorías e un foro en Moodle. A actividade desenvolverase en grupos de 4-5 alumnos. A aprendizaxe avaliarase en base á exposición oral apoiada nunha presentación Power Point que realice un membro do grupo elixido polo profesor e aos comentarios críticos que realicen os demais membros do grupo. Para organizar as titorías de avaliación, usarase a ferramenta Doodle. A actividade de ABP que se presenta contribuirá a desenvolver competencias recollidas na guía docente da materia e debería axudar aos alumnos a ver a relevancia do traballo realizado en Xenética Molecula

Screening of Bacterial Quorum Sensing Inhibitors in a Vibrio fischeri LuxR-Based Synthetic Fluorescent E. coli Biosensor

A library of 23 pure compounds of varying structural and chemical characteristics was screened for their quorum sensing (QS) inhibition activity using a synthetic fluorescent Escherichia coli biosensor that incorporates a modified version of lux regulon of Vibrio fischeri. Four such compounds exhibited QS inhibition activity without compromising bacterial growth, namely, phenazine carboxylic acid (PCA), 2-heptyl-3-hydroxy-4-quinolone (PQS), 1H-2-methyl-4-quinolone (MOQ) and genipin. When applied at 50 µM, these compounds reduced the QS response of the biosensor to 33.7% ± 2.6%, 43.1% ± 2.7%, 62.2% ± 6.3% and 43.3% ± 1.2%, respectively. A series of compounds only showed activity when tested at higher concentrations. This was the case of caffeine, which, when applied at 1 mM, reduced the QS to 47% ± 4.2%. In turn, capsaicin, caffeic acid phenethyl ester (CAPE), furanone and polygodial exhibited antibacterial activity when applied at 1mM, and reduced the bacterial growth by 12.8% ± 10.1%, 24.4% ± 7.0%, 91.4% ± 7.4% and 97.5% ± 3.8%, respectively. Similarly, we confirmed that trans-cinnamaldehyde and vanillin, when tested at 1 mM, reduced the QS response to 68.3% ± 4.9% and 27.1% ± 7.4%, respectively, though at the expense of concomitantly reducing cell growth by 18.6% ± 2.5% and 16% ± 2.2%, respectively. Two QS natural compounds of Pseudomonas aeruginosa, namely PQS and PCA, and the related, synthetic compounds MOQ, 1H-3-hydroxyl-4-quinolone (HOQ) and 1H-2-methyl-3-hydroxyl-4-quinolone (MHOQ) were used in molecular docking studies with the binding domain of the QS receptor TraR as a target. We offer here a general interpretation of structure-function relationships in this class of compounds that underpins their potential application as alternatives to antibiotics in controlling bacterial virulence

Diversification linked to larval host plant in the butterfly Eumedonia eumedon

It is widely accepted that the relationship between phytophagous insects and their host plants influences insect diversification. However, studies addressed at documenting host-associated genetic differentiation (HAD) and the mechanisms that drive reproductive isolation in host-associated lineages (or host races) are still scarce relative to insect diversity. To uncover further evidence on the HAD processes in Lepidoptera, we investigated the genetic structure of the geranium argus butterfly (Eumedonia eumedon) and tested for isolation by ecology (IBE) vs. isolation by distance (IBD). Genomic data revealed an array of host races (three of them in the same mountain range, the Cantabrian Mountains, northern Iberia) at apparently distinct levels of reproductive isolation. We found a pattern of IBE mediated by HAD at both local and European scales, in which genetic differentiation between populations and individuals correlated significantly with the taxonomic relatedness of the host plants. IBD was significant only when considered at the wider European scale. We hypothesize that, locally, HAD between Geranium-feeding populations was caused (at least partially) by allochrony, that is via adaptation of adult flight time to the flowering period of each host plant species. Nevertheless, the potential reproductive isolation between populations using Erodium and populations using Geranium cannot be explained by allochrony or IBD, and other mechanisms are expected to be at play.Peer reviewe

Political economy of oil production from 1850s to 1974

A study of the oil industry in its modern development from the 1850s to 1973. During this period the industry underwent significant changes in terms of its productive expansion, the diversity of its products, its role in general production, its corporate organisation and in terms of its significance to the very reproduction of advanced societies. The examination of the oil industry focuses on a political economy of its historical expansion. The thesis uses a Marxist theoretical framework to examine issues related to oil production as well as synthesising the elemental features of oil production into a structured conceptual model of the oil industry. The thesis divides the analysis of oil between chapters dealing with economic and political concerns in the context of historic epochs. The economic components of the thesis deal with the capitalist development of oil, its relationship with other sectors of production and consumption and an assessment of its role in economic growth as a whole. This provides the basis for the subsequent politically focused analyses. The political chapters deal with two primary issues, including the state response to the monopolisation of the oil industry and the effect of the expanding importance of oil on political relations. The analysis of the monopolisation of the oil industry provides an opportunity to study the relationship between the state in a regulatory function and the subsequent constraint on oil industry autonomy. The study of interstate relations focuses in turn on the effect of expanding oil production on the economic interests of states, in their support for the reproduction of capital in their domains

Recruitment of RNA molecules by connexin RNA-binding motifs: Implication in RNA and DNA transport through microvesicles and exosomes

Connexins (Cxs) are integral membrane proteins that form high-conductance plasma membrane channels, allowing communication from cell to cell (via gap junctions) and from cells to the extracellular environment (via hemichannels). Initially described for their role in joining excitable cells (nerve and muscle), gap junctions (GJs) are found between virtually all cells in solid tissues and are essential for functional coordination by enabling the direct transfer of small signalling molecules, metabolites, ions, and electrical signals from cell to cell. Several studies have revealed diverse channel-independent functions of Cxs, which include the control of cell growth and tumourigenicity. Connexin43 (Cx43) is the most widespread Cx in the human body. The myriad roles of Cx43 and its implication in the development of disorders such as cancer, inflammation, osteoarthritis and Alzheimer's disease have given rise to many novel questions. Several RNA- and DNA-binding motifs were predicted in the Cx43 and Cx26 sequences using different computational methods. This review provides insights into new, ground-breaking functions of Cxs, highlighting important areas for future work such as transfer of genetic information through extracellular vesicles. We discuss the implication of potential RNA- and DNA-binding domains in the Cx43 and Cx26 sequences in the cellular communication and control of signalling pathwaysThis work was supported in part through funding from the Society for Research on Bone and Mineral Metabolism - Grant number FEIOMM2016 (to M.D.M.), by grant PRECIPITA-2015-000139 from the FECYT-Ministry of Economy and Competitiveness (to M.D.M), by grants PI13/00591 and PI16/00035 from the Health Institute “Carlos III” (ISCIII, Spain) and co-financed by the European Regional Development Fund, “A way of making Europe” from the European Union (to M.D.M.), by a grant from Xunta de Galicia (pre-doctoral fellowship) to M.V.-E., and by a grant from the Ministry of Education, Culture and Sports, Spain (FPU grant to M.R.-C.M.)S

An investigation of the interactions between an E. coli bacterial quorum sensing biosensor and chitosan-based nanocapsules

We examined the interaction between chitosan-based nanocapsules (NC), with average hydrodynamic diameter ∼114–155 nm, polydispersity ∼0.127, and ζ-potential ∼+50 mV, and an E. coli bacterial quorum sensing reporter strain. Dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) allowed full characterization and assessment of the absolute concentration of NC per unit volume in suspension. By centrifugation, DLS, and NTA, we determined experimentally a “stoichiometric” ratio of ∼80 NC/bacterium. By SEM it was possible to image the aggregation between NC and bacteria. Moreover, we developed a custom in silico platform to simulate the behavior of particles with diameters of 150 nm and ζ-potential of +50 mV on the bacterial surface. We computed the detailed force interactions between NC-NC and NC-bacteria and found that a maximum number of 145 particles might interact at the bacterial surface. Additionally, we found that the “stoichiometric” ratio of NC and bacteria has a strong influence on the bacterial behavior and influences the quorum sensing response, particularly due to the aggregation driven by NC

Structural analysis of mitochondrial rRNA gene variants identified in patients with deafness

The last few years have witnessed dramatic advances in our understanding of the structure and function of the mammalian mito-ribosome. At the same time, the first attempts to elucidate the effects of mito-ribosomal fidelity (decoding accuracy) in disease have been made. Hence, the time is right to push an important frontier in our understanding of mitochondrial genetics, that is, the elucidation of the phenotypic effects of mtDNA variants affecting the functioning of the mito-ribosome. Here, we have assessed the structural and functional role of 93 mitochondrial (mt-) rRNA variants thought to be associated with deafness, including those located at non-conserved positions. Our analysis has used the structural description of the human mito-ribosome of the highest quality currently available, together with a new understanding of the phenotypic manifestation of mito-ribosomal-associated variants. Basically, any base change capable of inducing a fidelity phenotype may be considered non-silent. Under this light, out of 92 previously reported mt-rRNA variants thought to be associated with deafness, we found that 49 were potentially non-silent. We also dismissed a large number of reportedly pathogenic mtDNA variants, 41, as polymorphisms. These results drastically update our view on the implication of the primary sequence of mt-rRNA in the etiology of deafness and mitochondrial disease in general. Our data sheds much-needed light on the question of how mt-rRNA variants located at non-conserved positions may lead to mitochondrial disease and, most notably, provide evidence of the effect of haplotype context in the manifestation of some mt-rRNA variants

L11 domain rearrangement upon binding to RNA and thiostrepton studied by NMR spectroscopy

Ribosomal proteins are assumed to stabilize specific RNA structures and promote compact folding of the large rRNA. The conformational dynamics of the protein between the bound and unbound state play an important role in the binding process. We have studied those dynamical changes in detail for the highly conserved complex between the ribosomal protein L11 and the GTPase region of 23S rRNA. The RNA domain is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of the L11 protein (L11(ctd)). In addition, the N-terminal domain of L11 (L11(ntd)) is implicated in the binding of the natural thiazole antibiotic thiostrepton, which disrupts the elongation factor function. We have studied the conformation of the ribosomal protein and its dynamics by NMR in the unbound state, the RNA bound state and in the ternary complex with the RNA and thiostrepton. Our data reveal a rearrangement of the L11(ntd), placing it closer to the RNA after binding of thiostrepton, which may prevent binding of elongation factors. We propose a model for the ternary L11–RNA–thiostrepton complex that is additionally based on interaction data and conformational information of the L11 protein. The model is consistent with earlier findings and provides an explanation for the role of L11(ntd) in elongation factor binding