Computational Analysis and Experimental Validation of Gene Predictions in Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular protozoan that infects 20 to 90% of the population. It can cause both acute and chronic infections, many of which are asymptomatic, and, in immunocompromised hosts, can cause fatal infection due to reactivation from an asymptomatic chronic infection. An essential step towards understanding molecular mechanisms controlling transitions between the various life stages and identifying candidate drug targets is to accurately characterize the T. gondii proteome.We have explored the proteome of T. gondii tachyzoites with high throughput proteomics experiments and by comparison to publicly available cDNA sequence data. Mass spectrometry analysis validated 2,477 gene coding regions with 6,438 possible alternative gene predictions; approximately one third of the T. gondii proteome. The proteomics survey identified 609 proteins that are unique to Toxoplasma as compared to any known species including other Apicomplexan. Computational analysis identified 787 cases of possible gene duplication events and located at least 6,089 gene coding regions. Commonly used gene prediction algorithms produce very disparate sets of protein sequences, with pairwise overlaps ranging from 1.4% to 12%. Through this experimental and computational exercise we benchmarked gene prediction methods and observed false negative rates of 31 to 43%.This study not only provides the largest proteomics exploration of the T. gondii proteome, but illustrates how high throughput proteomics experiments can elucidate correct gene structures in genomes

Novel Protein-Lipid Conformations of High Density Lipoproteins through Molecular Dynamics

A detailed understanding of the lipid-associated structure of apolipoprotein (apo) A-I, the major protein of the anti-atherogenic high density lipoprotein (HDL), is crucial because the lipid environment of HDL modulates apoA-I structure and function. Recently, this lab proposed an atomic resolution double belt model for apoA-I associated with discoidal HDL, in which two apoA-I molecules were wrapped beltwise around a discoidal lipid bilayer containing 160 palmitoyloleoylphosphatidylcholine (POPC) molecules. Recently, our lab has shown that apoA-I complexed with POPC forms three different sized discoidal HDL particles containing 2 apoA-I. To understand the structure of these discrete particles, we performed molecular dynamics (MD) simulations of model discoidal HDL particles formed by systematically removing POPC molecules from an initial particle with 160 POPC and a double belt of two antiparallel lipid-associating domains of apoA-I (residues 41-243). After a few nanoseconds of MD simulation, independent of either of two starting particles and either of two methods of size reduction, the flat disc is transformed to a saddle-shaped bilayer structure approximating an Enneper’s minimal surface with the apoA-I double belt twisted to conform to the surface edge. Since POPC molecules seek to minimize their surface curvature forming flat bilayers (trivial minimal surfaces) the conversion to an Enneper’s minimal surface should involve minimal energy. Further, each of the simulated double belts forms a close approximation of the non-planar, amphipathic alpha helical structure of the X-ray structure of lipid-free apoA-I. Most importantly, the MD simulated apoA-I conformation was independent of the starting particle and the method of particle size reduction. We conclude that apoA-I represents a unique lipid-scavenging nanomachine capable of assembling variable numbers of phospholipid molecules into stable minimal surface particles

Novel changes in discoidal high density lipoprotein morphology: A molecular dynamics study

ApoA-l is a uniquely flexible lipid-scavenging protein capable of incorporating phospholipids into stable particles. Here we report molecular dynamics simulations on a series of progressively smaller discoidal high density lipoprotein particles produced by incremental removal of palmitoyloleoylphosphatidylcholine via four different pathways. The starting model contained 160 palmitoyloleoylphosphatidylcholines and a belt of two antiparallel amphipathic helical lipid-associating domains of apolipoprotein (apo) A-l. The results are particularly compelling. After a few nanoseconds of molecular dynamics simulation, independent of the starting particle and method of size reduction, all simulated double belts of the four lipidated apoA-l particles have helical domains that impressively approximate the x-ray crystal structure of lipid-free apoA-l, particularly between residues 88 and 186. These results provide atomic resolution models for two of the particles produced by in vitro reconstitution of nascent high density lipoprotein particles. These particles, measuring 95 angstrom and 78 angstrom by nondenaturing gradient gel electrophoresis, correspond in composition and in size/shape (by negative stain electron microscopy) to the simulated particles with molar ratios of 100:2 and 50:2, respectively. The lipids of the 100:2 particle family form minimal surfaces at their monolayer-monolayer interface, whereas the 50:2 particle family displays a lipid pocket capable of binding a dynamic range of phospholipid molecules

Mortality after surgery in Europe: a 7 day cohort study

SummaryBackgroundClinical outcomes after major surgery are poorly described at the national level. Evidence of heterogeneity between hospitals and health-care systems suggests potential to improve care for patients but this potential remains unconfirmed. The European Surgical Outcomes Study was an international study designed to assess outcomes after non-cardiac surgery in Europe.MethodsWe did this 7 day cohort study between April 4 and April 11, 2011. We collected data describing consecutive patients aged 16 years and older undergoing inpatient non-cardiac surgery in 498 hospitals across 28 European nations. Patients were followed up for a maximum of 60 days. The primary endpoint was in-hospital mortality. Secondary outcome measures were duration of hospital stay and admission to critical care. We used χ2 and Fisher's exact tests to compare categorical variables and the t test or the Mann-Whitney U test to compare continuous variables. Significance was set at p<0·05. We constructed multilevel logistic regression models to adjust for the differences in mortality rates between countries.FindingsWe included 46 539 patients, of whom 1855 (4%) died before hospital discharge. 3599 (8%) patients were admitted to critical care after surgery with a median length of stay of 1·2 days (IQR 0·9–3·6). 1358 (73%) patients who died were not admitted to critical care at any stage after surgery. Crude mortality rates varied widely between countries (from 1·2% [95% CI 0·0–3·0] for Iceland to 21·5% [16·9–26·2] for Latvia). After adjustment for confounding variables, important differences remained between countries when compared with the UK, the country with the largest dataset (OR range from 0·44 [95% CI 0·19–1·05; p=0·06] for Finland to 6·92 [2·37–20·27; p=0·0004] for Poland).InterpretationThe mortality rate for patients undergoing inpatient non-cardiac surgery was higher than anticipated. Variations in mortality between countries suggest the need for national and international strategies to improve care for this group of patients.FundingEuropean Society of Intensive Care Medicine, European Society of Anaesthesiology