Copper binding of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase in Staphylococcus aureus

PhD ThesisStaphylococcus aureus is a Gram positive bacterium which is predominantly commensal, but which can also be a human and animal pathogen, able to cause serious infections. It is becoming an increasing problem due to it becoming resistant to modern antibiotics. In common with most bacteria, S. aureus requires small quantities of the essential metal copper, but they also experience toxicity when exposed to high concentrations of copper, a metal that has been used for its antimicrobial properties for centuries. However, the mechanism of such toxicity remains elusive. Here, the effect of copper toxicity on S. aureus has been investigated in order to understand how excess copper ions affect its physiology. The growth of S. aureus was found to be inhibited in media containing elevated copper, and the extent of this inhibition was shown to be dependent on the type of growth medium used. Analysis of soluble extracts from S. aureus cells exposed to elevated copper led to the identification of a cytoplasmic enzyme, GapA, which binds copper. GapA is a member of the wellcharacterised family of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) enzymes, and is not a metalloprotein. Copper inhibition of the activity of the S. aureus GapA enzyme was demonstrated, both in S. aureus cell extracts and with recombinant enzyme in vitro, using a specific enzyme activity assay. Analysis of the purified recombinant GapA enzyme in vitro demonstrated a copper binding stoichiometry of one Cu(I) ion to each GapA monomer using analytical sizeexclusion chromatography and spectrophotometry, and evidence is presented that suggests high affinity Cu(I) binding of biochemical relevance. Some preliminary evidence is provided that the Cu(I) binding site on the GapA protein includes the thiol group of cysteine 151 located within the active site, consistent with predictions based on published crystal structures, and explaining the observed copper-dependent inhibition of enzyme activity. This is the first evidence of copper binding to a non-metalloprotein within the cytoplasm of S. aureus and adds to mounting evidence that aberrant binding of copper to cytosolic proteins contributes to bacterial copper toxicity

New Formation Models for the Kepler-36 System

Formation of the planets in the Kepler-36 system is modeled by detailed numerical simulations according to the core-nucleated accretion scenario. The standard model is updated to include the dissolution of accreting rocky planetesimals in the gaseous envelope of the planet, leading to substantial enrichment of the envelope mass in heavy elements and a non-uniform composition with depth. For Kepler-36 c, models involving in situ formation and models involving orbital migration are considered. The results are compared with standard formation models. The calculations include the formation (accretion) phase, as well as the subsequent cooling phase, up to the age of Kepler-36 (7 Gyr). During the latter phase, mass loss induced by stellar XUV radiation is included. In all cases, the results fit the measured mass, 7.84 M$_\oplus$, and radius, 3.68 R$_\oplus$, of Kepler-36 c. Two parameters are varied to obtain these fits: the disk solid surface density at the formation location, and the "efficiency" factor in the XUV mass loss rate. The updated models are hotter and therefore less dense in the silicate portion of the planet and in the overlying layers of H/He, as compared with standard models. The lower densities mean that only about half as much H/He is needed to be accreted to fit the present-day mass and radius constraints. For Kepler-36 b, an updated in situ calculation shows that the entire H/He envelope is lost, early in the cooling phase, in agreement with observation.Comment: 21 pages, 18 figures, 1 table. Accepted for publication in The Astrophysical Journa

Scaling a hippocampus model with GPU parallelisation and test-driven refactoring

The hippocampus is the brain area used for localisation, mapping and episodic memory. Humans and animals can outperform robotic systems in these tasks, so functional models of hippocampus may be useful to improve robotic navigation, such as for self-driving cars. Previous work developed a biologically plausible model of hippocampus based on Unitary Coherent Particle Filter (UCPF) and Temporal Restricted Boltzmann Machine, which was able to learn to navigate around small test environments. However it was implemented in serial software, which becomes very slow as the environments and numbers of neurons scale up. Modern GPUs can parallelize execution of neural networks. The present Neural Software Engineering study develops a GPU accelerated version of the UCPF hippocampus software, using the formal Software Engineering techniques of profiling, optimisation and test-driven refactoring. Results show that the model can greatly benefit from parallel execution, which may enable it to scale from toy environments and applications to real-world ones such as self-driving car navigation. The refactored parallel code is released to the community as open source software as part of this publication

Hippocampal CA1 gamma power predicts the precision of spatial memory judgments.

The hippocampus plays a critical role in spatial memory. However, the exact neural mechanisms underlying high-fidelity spatial memory representations are unknown. We report findings from presurgical epilepsy patients with bilateral hippocampal depth electrodes performing an object-location memory task that provided a broad range of spatial memory precision. During encoding, patients were shown a series of objects along the circumference of an invisible circle. At test, the same objects were shown at the top of the circle (0°), and patients used a dial to move the object to its location shown during encoding. Angular error between the correct location and the indicated location was recorded as a continuous measure of performance. By registering pre- and postimplantation MRI scans, we were able to localize the electrodes to specific hippocampal subfields. We found a correlation between increased gamma power, thought to reflect local excitatory activity, and the precision of spatial memory retrieval in hippocampal CA1 electrodes. Additionally, we found a similar relationship between gamma power and memory precision in the dorsolateral prefrontal cortex and a directional relationship between activity in this region and in the CA1, suggesting that the dorsolateral prefrontal cortex is involved in postretrieval processing. These results indicate that local processing in hippocampal CA1 and dorsolateral prefrontal cortex supports high-fidelity spatial memory representations

Evaluation of known and novel inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a Fluorescence Imaging Plate Reader assay

The Orai1 Ca2+ permeable ion channel is an important component of store operated Ca2+ entry (SOCE) in cells. It's over-expression in basal molecular subtype breast cancers has been linked with poor prognosis, making it a potential target for drug development. We pharmacologically characterised a number of reported inhibitors of SOCE in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader (FLIPR) assay, and show that the rank order of their potencies in this assay is the same as those reported in a wide range of published assays. The assay was also used in a screening project seeking novel inhibitors. Following a broad literature survey of classes of calcium channel inhibitors we used simplified ligand structures to query the ZINC on-line database, and following two iterations of refinement selected a novel Orai1-selective dichlorophenyltriazole hit compound. Analogues of this were synthesized and evaluated in the FLIPR assay to develop structure-activity relationships (SAR) for the three domains of the hit; triazole (head), dichlorophenyl (body) and substituted phenyl (tail). For this series, the results suggested the need for a lipophilic tail domain and an out-of-plane twist between the body and tail domains. (C) 2016 Elsevier Ltd. All rights reserved

Whey protein consumption after resistance exercise reduces energy intake at a post-exercise meal

Purpose - Protein consumption after resistance exercise potentiates muscle protein synthesis, but its effects on subsequent appetite in this context are unknown. This study examined appetite and energy intake following consumption of protein- and carbohydrate-containing drinks after resistance exercise. Methods - After familiarisation, 15 resistance training males (age 21 ± 1 years, body mass 78.0 ± 11.9 kg, stature 1.78 ± 0.07 m) completed two randomised, double-blind trials, consisting of lower-body resistance exercise, followed by consumption of a whey protein (PRO 23.9 ± 3.6 g protein) or dextrose (CHO 26.5 ± 3.8 g carbohydrate) drink in the 5 min post-exercise. An ad libitum meal was served 60 min later, with subjective appetite measured throughout. Drinks were flavoured and matched for energy content and volume. The PRO drink provided 0.3 g/kg body mass protein. Results - Ad libitum energy intake (PRO 3742 ± 994 kJ; CHO 4172 ± 1132 kJ; P = 0.007) and mean eating rate (PRO 339 ± 102 kJ/min; CHO 405 ± 154 kJ/min; P = 0.009) were lower during PRO. The change in eating rate was associated with the change in energy intake (R = 0.661, P = 0.007). No interaction effects were observed for subjective measures of appetite. The PRO drink was perceived as creamier and thicker, and less pleasant, sweet and refreshing (P < 0.05). Conclusion - These results suggest whey protein consumption after resistance exercise reduces subsequent energy intake, and this might be partially mediated by a reduced eating rate. Whilst this reduced energy intake is unlikely to impair hypertrophy, it may be of value in supporting an energy deficit for weight loss

Scale-independent mixing angles

A radiatively-corrected mixing angle has to be independent of the choice of renormalization scale to be a physical observable. At one-loop in MS-bar, this only occurs for a particular value, p*, of the external momentum in the two-point functions used to define the mixing angle: p*^2=(M1^2+M2^2)/2, where M1, M2 are the physical masses of the two mixed particles. We examine two important applications of this to the Minimal Supersymmetric Standard Model: the mixing angle for a) neutral Higgs bosons and b) stops. We find that this choice of external momentum improves the scale independence (and therefore provides a more reliable determination) of these mixing angles.Comment: 14 pages, 11 ps figures Version to appear in PR

Escherichia coli TatA and TatB Proteins Have N-out, C-in Topology in Intact Cells

The twin arginine protein transport (Tat) system translocates folded proteins across the cytoplasmic membrane of prokaryotes and the thylakoid membrane of chloroplasts. In Escherichia coli, TatA, TatB, and TatC are essential components of the machinery. A complex of TatB and TatC acts as the substrate receptor, whereas TatA is proposed to form the Tat transport channel. TatA and TatB are related proteins that comprise an N-terminal transmembrane helix and an adjacent amphipathic helix. Previous studies addressing the topological organization of TatA have given conflicting results. In this study, we have addressed the topological arrangement of TatA and TatB in intact cells by labeling of engineered cysteine residues with the membrane-impermeable thiol reagent methoxypolyethylene glycol maleimide. Our results show that TatA and TatB share an N-out, C-in topology, with no evidence that the amphipathic helices of either protein are exposed at the periplasmic side of the membrane. We further show that the N-out, C-in topology of TatA is fixed and is not affected by the absence of other Tat components or by the overproduction of a Tat substrate. These data indicate that topological reorganization of TatA is unlikely to accompany Tat-dependent protein transport