Structural and functional characterisation of novel bacterial Toll/Interleukin-1 receptor (TIR)-like proteins.

The extracellular domain of Toll-like receptors (TLRs) recognises highly specific pathogen-associated molecular patterns in innate immunity. This causes molecular rearrangement of the intracellular Toll/Interleukin-1 (TIR) domains of the TLRs allowing recruitment of downstream adaptor proteins via heterotypic TIR-TIR protein interactions. This in turn initiates a signalling cascade leading to proinflammatory immune responses. Recent work has indicated that TIR-like proteins (TLPs)from pathogenic bacteria contain TIR domains and interfere with host TLR signalling. Bacterial TLPs are suggested to bind to the TIR domains of host TLRs and/or adaptor proteins, thereby inhibiting intracellular signalling. This project focuses on characterisation of the TLPs from the highly pathogenic bacteria Yersinia pestis (YpTLP) and Burkholderia pseudomallei (BpTLP). The aim of the project was to produce soluble, pure and stable TLPs of yields suitable for functional and structural studies. Bacterial TLPs were expressed in E. coli. Expression of the full-length YpTLP and BpTLP using pWaldo-GFPe yielded 2.78 mg/l and 2.52 mg/l respectively. Protein purification steps were complicated by protein precipitation, possible degradation and misfolding. Subsequent efforts focused on the expression of the TIR only domain/homologue regions: YpTIR and BpTIR. Soluble YpTIR and BpTIR were expressed using pET26b. GST pull down assays indicated positive interactions between His-tagged YpTIR/BpTIR and GST-tagged human MyD88-TIR, a major adaptor protein, revealing MyD88 as a potential target of the bacterial TLPs. However expression yields of pure protein were too low to allow further studies. YpTIR and BpTIR were then expressed as fusions with an N-terminal GB1 tag using GEV2. Subsequent purification produced highly pure GB1-YpTIR (5 mg/l). NMR analysis indicated that the protein was folded and likely to be in a dimeric form, a finding confirmed by gel filtration. Native and derivative crystals of YpTIR were obtained, and native diffraction datasets collected to 2.95 A. Further work includes obtaining the phase information. It is anticipated that the crystal structure of YpTIR will provide insight into the molecular basis of TIR signalling and evidence of evolutionary conservation among TIR domains

Association of respiratory symptoms and lung function with occupation in the multinational Burden of Obstructive Lung Disease (BOLD) study

Background Chronic obstructive pulmonary disease has been associated with exposures in the workplace. We aimed to assess the association of respiratory symptoms and lung function with occupation in the Burden of Obstructive Lung Disease study. Methods We analysed cross-sectional data from 28 823 adults (≥40 years) in 34 countries. We considered 11 occupations and grouped them by likelihood of exposure to organic dusts, inorganic dusts and fumes. The association of chronic cough, chronic phlegm, wheeze, dyspnoea, forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1)/FVC with occupation was assessed, per study site, using multivariable regression. These estimates were then meta-analysed. Sensitivity analyses explored differences between sexes and gross national income. Results Overall, working in settings with potentially high exposure to dusts or fumes was associated with respiratory symptoms but not lung function differences. The most common occupation was farming. Compared to people not working in any of the 11 considered occupations, those who were farmers for ≥20 years were more likely to have chronic cough (OR 1.52, 95% CI 1.19–1.94), wheeze (OR 1.37, 95% CI 1.16–1.63) and dyspnoea (OR 1.83, 95% CI 1.53–2.20), but not lower FVC (β=0.02 L, 95% CI −0.02–0.06 L) or lower FEV1/FVC (β=0.04%, 95% CI −0.49–0.58%). Some findings differed by sex and gross national income. Conclusion At a population level, the occupational exposures considered in this study do not appear to be major determinants of differences in lung function, although they are associated with more respiratory symptoms. Because not all work settings were included in this study, respiratory surveillance should still be encouraged among high-risk dusty and fume job workers, especially in low- and middle-income countries.publishedVersio

Cohort Profile: Burden of Obstructive Lung Disease (BOLD) study

The Burden of Obstructive Lung Disease (BOLD) study was established to assess the prevalence of chronic airflow obstruction, a key characteristic of chronic obstructive pulmonary disease, and its risk factors in adults (≥40 years) from general populations across the world. The baseline study was conducted between 2003 and 2016, in 41 sites across Africa, Asia, Europe, North America, the Caribbean and Oceania, and collected high-quality pre- and post-bronchodilator spirometry from 28 828 participants. The follow-up study was conducted between 2019 and 2021, in 18 sites across Africa, Asia, Europe and the Caribbean. At baseline, there were in these sites 12 502 participants with high-quality spirometry. A total of 6452 were followed up, with 5936 completing the study core questionnaire. Of these, 4044 also provided high-quality pre- and post-bronchodilator spirometry. On both occasions, the core questionnaire covered information on respiratory symptoms, doctor diagnoses, health care use, medication use and ealth status, as well as potential risk factors. Information on occupation, environmental exposures and diet was also collected

Tandem Facial Amphiphiles for Membrane Protein Stabilization

We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles

Novel Tripod Amphiphiles for Membrane Protein Analysis

Integral membrane proteins play central roles in controlling the flow of information and molecules across membranes. Our understanding of membrane protein structure and function, however, is seriously limited, mainly due to difficulties in handling and analyzing these proteins in aqueous solution. The use of a detergent or other amphipathic agents is required to overcome the intrinsic incompatibility between the large lipophilic surfaces displayed by membrane proteins in their native forms and polar solvent molecules. Here we introduce new tripod amphiphiles displaying favourable behaviours toward several membrane protein systems, leading to enhanced protein solubilization and stabililization compared to both conventional detergents and previously-described tripod amphiphiles

Glucose-neopentyl glycol (GNG) amphiphiles for membrane protein study

The development of a new class of surfactants for membrane protein manipulation, “GNG amphiphiles”, is reported. These amphiphiles display promising behavior for membrane proteins, as demonstrated recently by the high resolution structure of a sodium-pumping pyrophosphatase reported by Kellosalo et al

Predicting Liver Allograft Discard: The Discard Risk Index

Background An index that predicts liver allograft discard can effectively grade allografts and can be used to preferentially allocate marginal allografts to aggressive centers. The aim of this study is to devise an index to predict liver allograft discard using only risk factors available at the time of initial DonorNet offer. Methods Using univariate and multivariate analyses on a training set of 72 297 deceased donors, we identified independent risk factors for liver allograft discard. Multiple imputation was used to account for missing variables. Results We identified 15 factors as significant predictors of liver allograft discard; the most significant risk factors were: total bilirubin > 10 mg/dL (odds ratio [OR], 25.23; confidence interval [CI], 17.32-36.77), donation after circulatory death (OR, 14.13; CI, 13.30-15.01), and total bilirubin 5 to 10 mg/dL (OR, 7.57; 95% CI, 6.32-9.05). The resulting Discard Risk Index (DSRI) accurately predicted the risk of liver discard with a C statistic of 0.80. We internally validated the model with a validation set of 37 243 deceased donors and also achieved a 0.80 C statistic. At a DSRI at the 90th percentile, the discard rate was 50% (OR, 32.34; CI, 28.63-36.53), whereas at a DSRI at 10th percentile, only 3% of livers were discarded. Conclusions The use of the DSRI can help predict liver allograft discard. The DSRI can be used to effectively grade allografts and preferentially allocate marginal allografts to aggressive centers to maximize the donor yield and expedite allocation