Structural, Ligand Binding and Redox Studies of hPDI fragments using NMR spectroscopy

Human Protein Disulphide Isomerase (hPDI) is an important protein folding catalyst involved in the reduction, oxidation and isomerisation of disulphide bonds, a major rate-limiting step in the formation of many proteins. hPDI also exhibits molecular chaperone activity, selectively binding small, unfolded peptides and a large range of mis-folded protein intermediates. hPDI is the founding member of a family of 21 structurally related proteins, and consists of domain subunits a and a' (which are catalytically active) and b and b' (which are not). b domain is thought to confer structural stability to the protein, and b' contains the primary ligand binding site. There is also an x-linker region, and a C-terminal acidic tail c, in the complete domain structure abb'xa'c. Despite a recently solved crystal structure of hPDI abb'xa' (4EKZ.pdb) and over 50 years of research, we still have limited knowledge of how hPDI works, and more specifically how it interacts with ligands. Obtaining more detailed information is of significant importance; not only is hPDI associated with several human disease states, but it shows great potential as a therapeutic treatment, and is also used industrially to improve recombinant protein yields in mammalian and bacterial cell expression systems. Nonetheless, physiological ligand specificity of hPDI is still poorly understood. This study focuses on characterising the ligand binding ability and specificity of hPDI using a model binding peptide, ?-somatostatin (?-som), and two recombinantly expressed fragments of hPDI: b'x, the smallest monomeric fragment of hPDI capable of ligand binding, and abb'x, a larger fragment previously uncharacterised by NMR. Backbone and side-chain resonance assignments were carried out, and allowed hPDI binding affinity to be quantified using both protein-observed and ligand-observed NMR methods. Peptide affinity was in agreement with the previously estimated 0.1-1 mM affinity, and was found to be independent of neighbouring domains and protein redox state. Fluorination of the peptide at key aromatic residues and subsequent Ala substitutions showed that the three Phe residues of the peptide all contributed in some part to hPDI binding, with substitution of the C-terminal Phe having the largest negative effect. Substitution of the three Phe residues to Ala abolished binding to hPDI, suggesting that unlike the family member PDIp (pancreas-specific Protein Disulphide Isomerase), hPDI ligand specificity may be mediated by Phe residues. Investigation of abb'x redox potential by NMR supported recently published values of full- length protein as determined by mass spectrometry, but also highlighted how the redox state of a domain becomes progressively more reducing upon addition of subsequent domains to the protein, further confirming cross-talk among the four domains of hPDI. Recombinant fluoroindole incorporation into abb'x showed the incorporation rate was not as extensive as previously reported for b'x fragment. Preliminary work showed the fluorine probe did not significantly alter ligand binding affinity, but in contrast reported a markedly more reducing redox potential, suggesting care must be taken to consider the potential impact protein fluorine modification can infer

Intra and intersession reliability of the Run RocketTM in recreationally trained participants

Sprint performance plays an important role in the success of many sports including track and field and team-based sports. Resisted sprint equipment has shown to be an effective method to increase sprint velocity and acceleration. The aim of the study was to determine the intrasession and intersession (7 days) reliability of a commercially available resisted sprint machine in recreationally trained individuals for two resistance settings. Fourteen recreationally active participants partook in the study (male = 10, female = 4) over a 7-day period. Three maximal 15m sprints, at two resistance levels (R0 and R5), were undertaken in a randomised order (6 sprints in total at each trial). Intrasession (comparison of the first 3 sprints for each trial) and intersession (mean of the 3 sprints for both trials) correlation coefficient (ICC), coefficients of variation (%CV), average variability, SEM and minimal detectable difference were calculated for 5 and 15m for both resistance levels. Intrasession reliability was very large to nearly perfect across both distances and resistance levels (ICC range 0.79-0.98), %CV ranged between 2.4-5.8% with larger values seen during the first trial for three of the four indices. Intersession reliability was very large to nearly perfect across all variables (ICC range 0.87-0.97), %CV was small and ranged between 2.0-4.1%. Average variability was small for all measurements. The Run RocketTM showed high intra and intersession reliability. The results show that this equipment could be reliably used within a sprint programme for recreationally trained individuals

Exploitation of the Escherichia coli lac operon promoter for controlled recombinant protein production

The Escherichia coli lac operon promoter is widely used as a tool to control recombinant protein production in bacteria. Here we give a brief review of how it functions, how it is regulated, and how, based on this knowledge, a suite of lac promoter derivatives has been developed to give controlled expression that is suitable for diverse biotechnology applications

19F NMR spectroscopy monitors ligand binding to recombinantly fluorine-labelled b'x from human protein disulphide isomerase (hPDI)

We report a protein-observe (19)F NMR-based ligand titration binding study of human PDI b'x with ?-somatostatin that also emphasises the need to optimise recombinant protein fluorination when using 5- or 6-fluoroindole. This study highlights a recombinant preference for 5-fluoroindole over 6-fluoroindole; most likely due to the influence of fluorine atomic packing within the folded protein structure. Fluorination affords a single (19)F resonance probe to follow displacement of the protein x-linker as ligand is titrated and provides a dissociation constant of 23 ± 4 ?M

Little evidence for an effect of smoking on multiple sclerosis risk:A Mendelian Randomization study

The causes of multiple sclerosis (MS) remain unknown. Smoking has been associated with MS in observational studies and is often thought of as an environmental risk factor. We used two-sample Mendelian randomization (MR) to examine whether this association is causal using genetic variants identified in genome-wide association studies (GWASs) as associated with smoking. We assessed both smoking initiation and lifetime smoking behaviour (which captures smoking duration, heaviness, and cessation). There was very limited evidence for a meaningful effect of smoking on MS susceptibility as measured using summary statistics from the International Multiple Sclerosis Genetics Consortium (IMSGC) meta-analysis, including 14,802 cases and 26,703 controls. There was no clear evidence for an effect of smoking on the risk of developing MS (smoking initiation: odds ratio [OR] 1.03, 95% confidence interval [CI] 0.92-1.61; lifetime smoking: OR 1.10, 95% CI 0.87-1.40). These findings suggest that smoking does not have a detrimental consequence on MS susceptibility. Further work is needed to determine the causal effect of smoking on MS progression

Rheumatoid leptomeningitis presenting with an acute neuropsychiatric disorder

Leptomeningitis is a rare central nervous system manifestation of rheumatoid arthritis, generally in patients with established chronic rheumatoid disease. We report a 41-year-old man without previous rheumatoid arthritis or psychiatric disorder who presented with an acute neuropsychiatric disturbance and polyarthralgia. His MR scan of brain showed asymmetric bifrontal leptomeningitis, confirmed on (18F)-fluoro-D-glucose-positron emission tomography. Other investigations showed highly positive serum and cerebrospinal fluid anti-cyclic citrullinated peptide. A leptomeningeal biopsy showed necrotising leptomeningeal inflammation with ill-defined granulomas and lymphoplasmacytic infiltrate without organisms. Prolonged high-dose corticosteroids and then rituximab resulted in recovery. Chronic leptomeningitis can present with an acute neuropsychiatric disorder. We highlight that early rheumatoid disease can, rarely, cause a chronic leptomeningitis, reversible with immunotherapy

Pandemic (H1N1) 2009 Risk for Frontline Health Care Workers

To determine whether frontline health care workers (HCWs) are at greater risk for contracting pandemic (H1N1) 2009 than nonclinical staff, we conducted a study of 231 HCWs and 215 controls. Overall, 79 (17.7%) of 446 had a positive antibody titer by hemagglutination inhibition, with 46 (19.9%) of 231 HCWs and 33 (15.3%) of 215 controls positive (OR 1.37, 95% confidence interval 0.84–2.22). Of 87 participants who provided a second serum sample, 1 showed a 4-fold rise in antibody titer; of 45 patients who had a nose swab sample taken during a respiratory illness, 7 had positive results. Higher numbers of children in a participant’s family and working in an intensive care unit were risk factors for infection; increasing age, working at hospital 2, and wearing gloves were protective factors. This highly exposed group of frontline HCWs was no more likely to contract pandemic (H1N1) 2009 influenza infection than nonclinical staff, which suggests that personal protective measures were adequate in preventing transmission

Modelling the spread and control of African swine fever in domestic and feral pigs

African swine fever (ASF) represents a significant threat to the Australian pork sector and the economy in general. Estimates of the economic damages from a large multi-state outbreak of ASF in Australia exceed $A2 billion. ASF outbreaks are widespread and increasing in number in Asia and Europe. Although ASF is not present in Australia, detections of ASF viral fragments in undeclared pork products intercepted at the Australian border and the recent spread of the disease to neighbouring Papua New Guinea demonstrate the significance of the threat. The AADIS model (Bradhurst et al., 2015), simulates the spread and control of contagious emergency animal diseases such as foot-and-mouth disease. The ability to evaluate different outbreak scenarios in time and space, and trial various control measures, assists the development of animal health policy. This project expanded the AADIS modelling framework to simulate the potential spread and control of ASF in Queensland domestic and feral pig populations. Of particular interest was the epidemiological interface between domestic and feral pigs and the potential role of ASF-infectious feral pig carcasses in transmission. The upgraded model will provide a useful decision support tool to assist with preparedness and planning for ASF outbreaks. The report provides a literature review on ASF, feral pigs in Australia, and ASF decision support tools. Case studies on the spread and control of ASF in domestic and feral pigs demonstrate the functionality of the new model. Queensland was selected as the test case study area due to the wide distribution and high numbers of feral pigs and the availability of local expertise and data from Biosecurity Queensland, Department of Agriculture and Fisheries, Australian Pork Limited and SunPork Group Pty Ltd. The model was parameterised from the literature review and expert opinion that incorporated local knowledge of Australian production systems and environmental conditions. Note that the model is only parameterised for Queensland and will be scaled up to a national model through Biosecurity Innovation Program project 182021