Understanding nutrient transport across the outer membrane by members of the human gut microbiota

PhD ThesisThe human gut contains a dense group of microbes termed the microbiota, which has been shown to play a major role in health and disease. Despite significant diversity at species level, the microbiota is dominated by only two phyla, the Gram-negative Bacteroidetes and Gram-positive Firmicutes. The Bacteroidetes are able to use a wide range of different complex glycans from both dietary and host sources. Bacteroidetes express groups of co-regulated, cell envelope associated proteins termed polysaccharide utilisation loci (PULs). Each PUL is specific for a different glycan with some species of Bacteroides, one of the major genera of the gut, encoding >100 predicted PULs. PULs encodes enzymes, binding proteins, a regulator and a transporter which are localised to the outer-membrane or periplasm for the complete degradation and transport of the target glycan. Following initial, partial degradation of the target polysaccharide at the cell surface, the resulting oligosaccharides are transported into the periplasm. This process involves an outer membrane complex consisting of a substrate binding lipoprotein (SusD-like) and a β-barrel TonB dependent transporter (SusC-like). This SusCD transporter complex is vital to utilisation of glycans by Bacteroidetes as oligosaccharide breakdown to monosaccharides occurs in the periplasm. Despite the importance of this process to microbiota function, the mechanism of SusCD function is unclear. How extracellular substrate binding by the SusD-like protein is coupled to import by SusC is unknown as other classes of TonB dependent transporters do not have a partner lipoprotein. A SusCD complex with two auxiliary lipoproteins, BT2261-4, was expressed natively and purified directly from Bacteroides thetaiotaomicron. The X-ray crystal structure shows the SusC transporters form a homodimer with a SusD-like binding protein capping each barrel like a lid. The structure also shows a linear peptide bound at the interface of the SusC and SusD proteins via interactions with the peptide backbone. Expression levels of the BT2261-4 complex indicated that the proteins were required for growth under nutrient stress conditions which suggests a possible role in peptide scavenging. A classical glycan targeting SusCD complex, BT1762-3 from the Bacteroides thetaiotaomicron levan PUL, was targeted by adding a His-tag to the genomic copy of the SusD-like binding protein BT1762. The His-tag allowed purification of the BT1762-3 complex which led to two further SusCD structures; apo and with a levan oligosaccharide bound. BT1762-3 has the same overall conformation as the peptide importing SusCD suggesting the dimeric SusC transporter and SusD protein ‘cap’ general structure is conserved across SusCD complexes. MD simulations and electrophysiology experiments allowed us to propose a model for SusCD function where the SusD-like binding protein sits on top of the SusC-like transporter like a lid and is able to open like a pedal bin to allow oligosaccharide binding and uptake.Barbour Foundatio

Breaking the Code Archival Lighting Plan, 8th - 12th August, 2017

Lighting Plan for the 2017 Graduating Actor's Production of Breaking the Code by Hugh Whitemor

Electromagnetic Actuator for Camless Engines

This document summarizes the research, objectives, project plan, and design for developing an electromagnetic actuator valve for use in a camless internal combustion engine. While electromagnetic valve actuators have not been implemented into a working product to date, there have been many attempts to research and develop working prototypes. Similar products have been developed, but they do not use purely electromagnetic actuation. This research is significant because it shows the challenge that must be overcome and outlines potential design solutions to the problem. The objectives section highlights the problem statement and what is aimed to be achieved in this project. The concept design section outlines the chosen design direction and the methods used to arrive at this design. The final design section describes the finalized design decisions and details the analysis used to justify the design. The manufacturing section details the process for building the finalized design of the actuator and establishes the cost and timeline of development and testing as well as the required equipment and tools. The design verification section explains the testing required to verify if the design meets the intended specifications. The accomplished testing section provides the results of tests that we were able to perform under the circumstances of the COVID-19 crisis. The new project scope section highlights the revised goals of the project while under quarantine restrictions. Finally, the project management section outlines how the goals and project milestones will be achieved

Influence of Voice Intonation on Understanding Irony by Polish-Speaking Preschool Children

The main aim of the presented study was to investigate the influence of voice intonation on the comprehension of ironic utterances in 4- to 6-year-old Polish-speaking children. 83 preschool children were tested with the Irony Comprehension Task (Banasik & Bokus, 2012). In the Irony Comprehension Task, children are presented with stories in which ironic utterances were prerecorded and read by professional speakers using an ironic intonation. Half of the subjects performed the regular Irony Comprehension Task while the other half were given a modified version of the Irony Comprehension Task (ironic content was uttered using a non-ironic intonation). Results indicate that children from the ironic intonation group scored higher on the Irony Comprehension Task than children who heard ironic statements uttered using a neutral voice. Ironic voice intonation appeared to be a helpful cue to irony comprehension

The identification of intrinsic chloramphenicol and tetracycline resistance genes in members of the Bacillus cereus group (sensu lato)

Bacillus toyonensis strain BCT-7112T (NCIMB 14858T) has been widely used as an additive in animal nutrition for more than 30 years without reports of adverse toxigenic effects. However, this strain is resistant to chloramphenicol and tetracycline and it is generally considered inadvisable to introduce into the food chain resistance determinants capable of being transferred to other bacterial strains, thereby adding to the pool of such determinants in the gastro-enteric systems of livestock species. We therefore characterized the resistance phenotypes of this strain and its close relatives to determine whether they were of recent origin, and therefore likely to be transmissible. To this end we identified the genes responsible for chloramphenicol (catQ) and tetracycline (tetM) resistance and confirmed the presence of homologs in other members of the B. toyonensis taxonomic unit. Unexpectedly, closely related strains encoding these genes did not exhibit chloramphenicol and tetracycline resistance phenotypes. To understand the differences in the behaviors, we cloned and expressed the genes, together with their upstream regulatory regions, into Bacillus subtilis. The data showed that the genes encoded functional proteins, but were expressed inefficiently from their native promoters. B. toyonensis is a taxonomic unit member of the Bacillus cereus group (sensu lato). We therefore extended the analysis to determine the extent to which homologous chloramphenicol and tetracycline resistance genes were present in other species within this group. This analysis revealed that homologous genes were present in nearly all representative species within the B. cereus group (sensu lato). The absence of known transposition elements and the observations that they are found at the same genomic locations, indicates that these chloramphenicol and tetracycline resistance genes are of ancient origin and intrinsic to this taxonomic group, rather than recent acquisitions. In this context we discuss definitions of what are and are not intrinsic genes, an issue that is of fundamental importance to both Regulatory Authorities, and the animal feed and related industries

Study Protocol for RESORP – Resolution of Organ Injury in Acute Pancreatitis – an observational prospective cohort study

Introduction Survivors of acute pancreatitis (AP) have shorter overall survival and increased incidence of new-onset cardiovascular, respiratory, liver and renal disease, diabetes mellitus and cancer compared with the general population, but the mechanisms that explain this are yet to be elucidated. Our aim is to characterise the precise nature and extent of organ dysfunction following an episode of AP.Methods and analysis This is an observational prospective cohort study in a single centre comprising a University hospital with an acute and emergency receiving unit and clinical research facility. Participants will be adult patient admitted with AP. Participants will undergo assessment at recruitment, 3 months and 3 years. At each time point, multiple biochemical and/or physiological assessments to measure cardiovascular, respiratory, liver, renal and cognitive function, diabetes mellitus and quality of life. Recruitment was from 30 November 2017 to 31 May 2020; last follow-up measurements is due on 31 May 2023. The primary outcome measure is the incidence of new-onset type 3c diabetes mellitus during follow-up. Secondary outcome measures include: quality of life analyses (SF-36, Gastrointestinal Quality of Life Index); montreal cognitive assessment; organ system physiological performance; multiomics predictors of AP severity, detection of premature cellular senescence. In a nested cohort within the main cohort, individuals may also consent to multiparameter MRI scan, echocardiography, pulmonary function testing, cardiopulmonary exercise testing and pulse-wave analysis.Ethics and dissemination This study has received the following approvals: UK IRAS Number 178615; South-east Scotland Research Ethics Committee number 16/SS/0065. Results will be made available to AP survivors, caregivers, funders and other researchers. Publications will be open-access.Trial registration numbers ClinicalTrials.gov Registry (NCT03342716) and ISRCTN50581876; Pre-results

The emotional impact of verbal irony: eye-tracking evidence for a two-stage process

In this paper we investigate the socio-emotional functions of verbal irony. Specifically, we use eye-tracking while reading to assess moment-to-moment processing of a character’s emotional response to ironic versus literal criticism. In Experiment 1, participants read stories describing a character being upset following criticism from another character. Results showed that participants initially more easily integrated a hurt response following ironic criticism; but later found it easier to integrate a hurt response following literal criticism. In Experiment 2, characters were instead described as having an amused response, which participants ultimately integrated more easily following ironic criticism. From this we propose a two-stage process of emotional responding to irony: Whilst readers may initially expect a character to be more hurt by ironic than literal criticism, they ultimately rationalise ironic criticism as being less hurtful, and more amusing

Impact of primary kidney disease on the effects of empagliflozin in patients with chronic kidney disease: secondary analyses of the EMPA-KIDNEY trial

Background: The EMPA KIDNEY trial showed that empagliflozin reduced the risk of the primary composite outcome of kidney disease progression or cardiovascular death in patients with chronic kidney disease mainly through slowing progression. We aimed to assess how effects of empagliflozin might differ by primary kidney disease across its broad population. Methods: EMPA-KIDNEY, a randomised, controlled, phase 3 trial, was conducted at 241 centres in eight countries (Canada, China, Germany, Italy, Japan, Malaysia, the UK, and the USA). Patients were eligible if their estimated glomerular filtration rate (eGFR) was 20 to less than 45 mL/min per 1·73 m2, or 45 to less than 90 mL/min per 1·73 m2 with a urinary albumin-to-creatinine ratio (uACR) of 200 mg/g or higher at screening. They were randomly assigned (1:1) to 10 mg oral empagliflozin once daily or matching placebo. Effects on kidney disease progression (defined as a sustained ≥40% eGFR decline from randomisation, end-stage kidney disease, a sustained eGFR below 10 mL/min per 1·73 m2, or death from kidney failure) were assessed using prespecified Cox models, and eGFR slope analyses used shared parameter models. Subgroup comparisons were performed by including relevant interaction terms in models. EMPA-KIDNEY is registered with ClinicalTrials.gov, NCT03594110. Findings: Between May 15, 2019, and April 16, 2021, 6609 participants were randomly assigned and followed up for a median of 2·0 years (IQR 1·5–2·4). Prespecified subgroupings by primary kidney disease included 2057 (31·1%) participants with diabetic kidney disease, 1669 (25·3%) with glomerular disease, 1445 (21·9%) with hypertensive or renovascular disease, and 1438 (21·8%) with other or unknown causes. Kidney disease progression occurred in 384 (11·6%) of 3304 patients in the empagliflozin group and 504 (15·2%) of 3305 patients in the placebo group (hazard ratio 0·71 [95% CI 0·62–0·81]), with no evidence that the relative effect size varied significantly by primary kidney disease (pheterogeneity=0·62). The between-group difference in chronic eGFR slopes (ie, from 2 months to final follow-up) was 1·37 mL/min per 1·73 m2 per year (95% CI 1·16–1·59), representing a 50% (42–58) reduction in the rate of chronic eGFR decline. This relative effect of empagliflozin on chronic eGFR slope was similar in analyses by different primary kidney diseases, including in explorations by type of glomerular disease and diabetes (p values for heterogeneity all >0·1). Interpretation: In a broad range of patients with chronic kidney disease at risk of progression, including a wide range of non-diabetic causes of chronic kidney disease, empagliflozin reduced risk of kidney disease progression. Relative effect sizes were broadly similar irrespective of the cause of primary kidney disease, suggesting that SGLT2 inhibitors should be part of a standard of care to minimise risk of kidney failure in chronic kidney disease. Funding: Boehringer Ingelheim, Eli Lilly, and UK Medical Research Council

Insights into SusCD-mediated glycan import by a prominent gut symbiont

In Bacteroidetes, one of the dominant phyla of the mammalian gut, active uptake of large nutrients across the outer membrane is mediated by SusCD protein complexes via a “pedal bin” transport mechanism. However, many features of SusCD function in glycan uptake remain unclear, including ligand binding, the role of the SusD lid and the size limit for substrate transport. Here we characterise the β2,6 fructo-oligosaccharide (FOS) importing SusCD from Bacteroides thetaiotaomicron (Bt1762-Bt1763) to shed light on SusCD function. Co-crystal structures reveal residues involved in glycan recognition and suggest that the large binding cavity can accommodate several substrate molecules, each up to ~2.5 kDa in size, a finding supported by native mass spectrometry and isothermal titration calorimetry. Mutational studies in vivo provide functional insights into the key structural features of the SusCD apparatus and cryo-EM of the intact dimeric SusCD complex reveals several distinct states of the transporter, directly visualising the dynamics of the pedal bin transport mechanism

Structural and functional insights into oligopeptide acquisition by the RagAB transporter from Porphyromonas gingivalis

Porphyromonas gingivalis, an asaccharolytic member of the Bacteroidetes, is a keystone pathogen in human periodontitis that may also contribute to the development of other chronic inflammatory diseases. P. gingivalis utilizes protease-generated peptides derived from extracellular proteins for growth, but how these peptides enter the cell is not clear. Here, we identify RagAB as the outer-membrane importer for these peptides. X-ray crystal structures show that the transporter forms a dimeric RagA2B2 complex, with the RagB substrate-binding surface-anchored lipoprotein forming a closed lid on the RagA TonB-dependent transporter. Cryo-electron microscopy structures reveal the opening of the RagB lid and thus provide direct evidence for a ‘pedal bin’ mechanism of nutrient uptake. Together with mutagenesis, peptide-binding studies and RagAB peptidomics, our work identifies RagAB as a dynamic, selective outer-membrane oligopeptide-acquisition machine that is essential for the efficient utilization of proteinaceous nutrients by P. gingivalis