Oat-enriched diet reduces inflammatory status assessed by circulating cell-derived microparticle concentrations in type 2 diabetes

This work was funded by the Chief Scientists Office of the Scottish Government by a joint grant to the University of the Highland and Islands, Grampian Health Board, Biomathematics and Statistics Scotland and the Rowett Institute of Nutrition and Health, University of Aberdeen. Additional support was provided by Provexis plc.Peer reviewedPublisher PD

Recovery and characterization of viral diversity from aquatic short- and long-read metagenomes

Viruses are the most abundant biological entities in marine ecosystems and play an essential role in global biogeochemical cycles. They have important ecological functions as drivers of bacterial populations through lytic infections and contribute to bacterial genetic diversification. Unfortunately, their study is severely limited by the difficulty to culture and isolate them in lab conditions. Culture-independent techniques such as metagenomics can complement culture-based approaches to capture more phage diversity. However, the vast majority of viral sequences recovered through these methods are uncharacterized and therefore do not provide any information about their interactions with the bacterial community, a phenomenon that has been named “viral dark matter”. In this thesis, several bioinformatic techniques are applied to both short- and long-read metagenomic datasets to recover biological information from marine viral sequences contained therein. A pipeline for recovering viral sequences based on a reference genome was developed and applied to the study of myophages infecting the alphaproteobacterial SAR11 clade, one of the most abundant bacterioplankton groups in surface marine and freshwater ecosystems. We were able to recover 22 new genomes which include the first genomes of myophages infecting LD12, the SAR11 freshwater clade. These sequences are underrepresented in datasets derived from the viral fraction, suggesting a bias of either technical or biological nature. Surprisingly, this family of phages code for an operon which resembles the secretion system type VIII operon in Escherichia coli. The function of this phage operon is still unknown. Next, a long-read dataset from the Mediterranean Sea was explored for viral contigs to contrast phage recovery between long- and short-read datasets. The analysis revealed that while long-read assemblies resulted in viral sequences of better quality, there was a sizable amount of intra-clade viral diversity that was not included in the assemblies. This viral diversity only found in long reads is even greater than previously thought. This untapped diversity could aid biotechnological efforts as evidenced by the discovery of new endolysins. Finally, a tool (Random Forest Assignment of Hosts, or RaFAH) for assigning hosts to phage sequences obtained from metagenomic datasets was created. The tool is based on a machine learning tool trained with phage protein clusters generated de novo. Benchmarking shows that RaFAH is on par with other state-of-the-art classifiers and is able to classify phage contigs at the level of Kingdom, which makes it the first classifier to accurately detect Archaea viruses from metagenomic samples. A feature importance analysis reveals that the protein clusters with the most predictive power are those involved in host recognition.Los bacteriófagos (”fagos”) son los organismos más abundantes en los ecosistemas marinos y tienen un papel esencial en los ciclos biogeoquímicos globales. Asimismo, influencian la evolución de las poblaciones bacterianas que infectan y contribuyen a la diversificación del acervo genético bacteriano. Desgraciadamente, su estudio se ve limitado por la dificultad de cultivar y aislar estos organismos en el laboratorio. El uso de técnicas que no requieren cultivo, como la metagenómica, pueden complementar el cultivo en laboratorio para recuperar una mayor diversidad de fagos. Sin embargo, la inmensa mayoría de secuencias virales recuperadas mediante metagenómica no pueden ser caracterizadas, por lo que no proporcionan ninguna información sobre sus interacciones con la comunidad bacteriana, un fenómeno que se ha nombrado “materia oscura viral”. En esta tesis se han utilizado múltiples procesos bioinformáticos en colecciones de metagenomas de lectura corta y larga para caracterizar las secuencias virales que contienen. Se ha desarrollado un procedimiento para recuperar secuencias virales a partir de un genoma de referencia y se ha aplicado al estudio de miofagos que infectan al clado SAR11 de las Alfaproteobacteria, uno de los grupos de bacterioplankton más abundantes en agua dulce y agua salada de superficie. Se consiguió recuperar 22 nuevos genomas que incluyen el primer genoma que infecta LD12, el subclado de SAR11 de agua dulce. Estos genomas están poco representados en colecciones obtenidas de la fracción viral, lo que sugiere que las afecta un sesgo técnico o biológico. Sorprendentemente, esta familia de fagos contiene un operón similar al sistema de secreción tipo VIII de Escherichia coli. La función de este operón es aún desconocida. Asimismo, se contrastó la recuperación de secuencias víricas entre colecciones de lectura corta y larga utilizando colecciones obtenidas en el mar Mediterráneo. Los resultados muestran que aunque los ensamblajes derivados de las lecturas largas producen secuencias virales de mejor calidad, en el proceso se pierde una gran cantidad de diversidad intraclado. Esta diversidad es mucho mayor de la recuperada con lecturas cortas, y podría explotarse para aplicaciones biotecnológicas, como el descubrimiento de nuevas endolisinas. Finalmente, se desarrolló un programa (Random Forest Assignment of Hosts, o RaFAH) para asignar hospedadores a secuencias virales obtenidas de colecciones metagenómicas. El programa se basa en el uso de algoritmos de machine learning entrenados con grupos de proteínas creados de novo. RaFAH muestra un rendimiento similar a otros clasificadores de secuencias y es capaz de clasificar secuencias víricas al nivel taxonómico de Reino, siendo así el primer clasificador capaz de detectar fagos que infectan arqueas con precisión. El análisis de importancia de rasgo revela que los grupos de proteínas con mayor poder predictivo son aquellos involucrados en el reconocimiento del hospedador

Structural studies of Pseudomonas and Chromobacterium ω-aminotransferases provide insights into their differing substrate specificity

Copyright © 2013 International Union of CrystallographyThe crystal structures and inhibitor complexes of two industrially important ω-aminotransferase enzymes from Pseudomonas aeruginosa and Chromobacterium violaceum have been determined in order to understand the differences in their substrate specificity. The two enzymes share 30% sequence identity and use the same amino acceptor, pyruvate; however, the Pseudomonas enzyme shows activity towards the amino donor β-alanine, whilst the Chromobacterium enzyme does not. Both enzymes show activity towards S-α-methylbenzylamine (MBA), with the Chromobacterium enzyme having a broader substrate range. The crystal structure of the P. aeruginosa enzyme has been solved in the holo form and with the inhibitor gabaculine bound. The C. violaceum enzyme has been solved in the apo and holo forms and with gabaculine bound. The structures of the holo forms of both enzymes are quite similar. There is little conformational difference observed between the inhibitor complex and the holoenzyme for the P. aeruginosa aminotransferase. In comparison, the crystal structure of the C. violaceum gabaculine complex shows significant structural rearrangements from the structures of both the apo and holo forms of the enzyme. It appears that the different rigidity of the protein scaffold contributes to the substrate specificity observed for the two ω-aminotransferases.University of Exeter - PhD GTA bursaryWellcome TrustBiotechnology and Biological Sciences Research Council (BBSRC)Engineering and Physical Sciences Research Council (EPSRC

Polytopic membrane protein folding at L17 in the ribosome tunnel initiates cyclical changes at the translocon

Interaction between L17 in the ribosome tunnel and folded nascent chain transmembrane segments during multi-spanning membrane protein synthesis triggers structural rearrangements in the ribosome that cause switching between cytosolic and ER lumenal targeting of the growing polypeptide

Exploitation of the Ligand-Binding Properties of the Mannose 6-Phosphate/Insulin-Like Growth Factor II (IGF-II) Receptor to Inhibit IGF-II-Dependent Growth of Cancer Cells

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is a multifunctional, type I transmembrane receptor that is a member of the P-type lectin family. A large, extracytoplasmic (EC) region of the M6P/IGF2R binds various ligands, allowing the receptor to regulate multiple biological functions, including the role as a tumor suppressor. Two major classes of ligands, M6P-glycosylated (i.e. any proteins that bear M6P due to post-translational modification in the trans-Golgi network (TGN)) and non-glycosylated (i.e., the mitogen insulin-like growth factor II (IGF-II)), bind within distinct regions of the EC of the receptor and are trafficked to the lysosome. The M6P/IGF2R as well as the cation-dependent mannose 6-phosphate receptor (CD-MPR) are mostly involved in lysosomal biogenesis, trafficking newly synthesized lysosomal enzymes from the TGN to the early endosomes, where the vesicles mature into lysosomes. The receptors undergo recycling during the late endosomal phase where they are retrograde transported back to the TGN for another round of trafficking. However, a fraction of the receptors is found on the cell surface, where the M6P/IGF2R, but not the CD-MPR, is able to bind extracellular ligands. Through this action, IGF-II can bind to the M6P/IGF2R and will be degraded in the lysosome, reducing the bioavailability of the growth factor for the mitogenic insulin-like growth factor I receptor (IGF1R); thus, the M6P/IGF2R is considered a clearance receptor and tumor suppressor. Due to its growth suppressive function, the M6P/IGF2R is believed to play a role in cancer biology. High-affinity, bivalent M6P-based ligands, such as lysosomal enzymes, bind and stabilize the dimeric M6P/IGF2R at the cell surface, leading to its internalization at a faster rate than when there is no M6P-based ligand bound. Therefore, the major goal of our work is to produce a panel of M6P-based ligands capable of bi- or multivalent binding to the M6P/IGF2R that could suppress IGF-II-dependent growth of cancer cells. Additionally, the M6P receptors (MPR) are well conserved through evolution, with the earliest form of “true” MPR known to date in the invertebrates such as mollusk. However, the social amoeba, D. discoideum, produces lysosomal enzymes that bind to the M6P/IGF2R, a discovery that predated identification of a receptor capable of transporting these acid hydrolases within this organism. We provide evidence of a putative MPR protein that retains all the necessary components of a M6P receptor homology domain that also binds M6P. The studies presented herein further our understanding of the origin of the M6P/IGFR as well as exploiting this receptor as a novel therapeutic target against IGF-II-dependent cancers