Validity of Gō models: comparison with a solvent-shielded empirical energy decomposition

Do Gō-type model potentials provide a valid approach for studying protein folding? They have been widely used for this purpose because of their simplicity and the speed of simulations based on their use. The essential assumption in such models is that only contact interactions existing in the native state determine the energy surface of a polypeptide chain, even for non-native configurations sampled along folding trajectories. Here we use an all-atom molecular mechanics energy function to investigate the adequacy of Gō-type potentials. We show that, although the contact approximation is accurate, non-native contributions to the energy can be significant. The assumed relation between residue-residue interaction energies and the number of contacts between them is found to be only approximate. By contrast, individual residue energies correlate very well with the number of contacts. The results demonstrate that models based on the latter should give meaningful results (e.g., as used to interpret phi values), whereas those that depend on the former are only qualitative, at best.     

Beta-alanine protection against hypoxic liver injury in the rat

Liver hypoxia still represents an important cause of liver injury during shock and liver transplantation. We have investigated the protective effects of beta-alanine against hypoxic injury using isolated perfused rat livers and isolated rat hepatocyte suspensions. Perfusion with hypoxic Krebs-Henseleit buffer increased liver weight and caused a progressive release of lactate dehydrogenase (LDH) in the effluent perfusate. The addition of 5 mmol/l beta-alanine to the perfusion buffer completely prevented both weight increase and LDH leakage. These findings were confirmed by histological examinations showing that beta-alanine blocked the staining by trypan blue of either liver parenchymal and sinusoidal cells. Studies performed in isolated hepatocytes revealed that beta-alanine exerted its protective effects by interfering with Na+ accumulation induced by hypoxia. The addition of gamma-amino-butyric acid, which interfered with beta-alanine uptake by the hepatocytes or of Na+/H+ ionophore monensin, reverted beta-alanine protection in either hepatocyte suspensions or isolated perfused livers. We also observed that liver receiving beta-alanine were also protected against LDH leakage and weight increase caused by the perfusion with an hyposmotic (205 mosm) hypoxic buffer obtained by decreasing NaCl content from 118 to 60 mmol/l. This latter effect was not reverted by blocking K+ efflux from hepatocyte with BaCl(2) (1mmol/l). Altogether these results indicated that beta-alanine protected against hypoxic liver injury by preventing Na+ overload and by increasing liver resistance to osmotic stress consequent to the impairment of ion homeostasis during hypoxia.     

Enhanced activity of the plasma membrane oxidoreductase in circulating lymphocytes from insulin-dependent diabetes mellitus patients

Circulating human lymphocytes contain a transmembrane oxidoreductase (PMOR) capable of reducing dichlorophenol indophenol (DCIP) by endogenous reductants, presumably NADH. Membranes from lymphocytes obtained from buffy coats contain a NADH DCIP reductase having a K(m) of about 1 microM and almost insensible to dicoumarol. The PMOR of lymphocytes from insulin-dependent diabetic patients is higher than that from age-matched controls and, in addition, has a dicoumarol-sensitive component, lacking in most controls, presumably due to membrane association of DT-diaphorase. The increase of PMOR in diabetes is likely due to overexpression of the enzyme, in view of the very low K(m) for NADH indicating that, in intact cells, the enzyme is practically saturated with the reductant substrate.     

Pulling geometry defines the mechanical resistance of a beta-sheet protein

Proteins show diverse responses when placed under mechanical stress. The molecular origins of their differing mechanical resistance are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to have an important function. Here, by using a method of protein immobilization that allows force to be applied to the same all-beta protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is markedly anisotropic. These results, in combination with molecular dynamics (MD) simulations, reveal that the unfolding pathway depends on the pulling geometry and is associated with unfolding forces that differ by an order of magnitude. Thus, the mechanical resistance of a protein is not dictated solely by amino acid sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.     

Learning Environment,Preparedness and Satisfaction in Osteopathy in Europe: The PreSS Study

Objective

1) to assess the preparedness to practice and satisfaction in learning environment amongst new graduates from European osteopathic institutions; 2) to compare the results of preparedness to practice and satisfaction in learning environment between and within countries where osteopathy is regulated and where regulation is still to be achieved; 3) to identify possible correlations between learning environment and preparedness to practice.

Method

Osteopathic education providers of full-time education located in Europe were enrolled, and their final year students were contacted to complete a survey. Measures used were: Dundee Ready Educational Environment Measure (DREEM), the Association of American Medical Colleges (AAMC) and a demographic questionnaire. Scores were compared across institutions using one-way ANOVA and generalised linear model.

Results

Nine European osteopathic education institutions participated in the study (4 located in Italy, 2 in the UK, 1 in France, 1 in Belgium and 1 in the Netherlands) and 243 (77%) of their final-year students completed the survey. The DREEM total score mean was 121.4 (SEM: 1.66) whilst the AAMC was 17.58 (SEM:0.35). A generalised linear model found a significant association between not-regulated countries and total score as well as subscales DREEM scores (p<0.001). Learning environment and preparedness to practice were significantly positively correlated (r=0.76; p<0.01).

Discussion

A perceived higher level of preparedness and satisfaction was found amongst students from osteopathic institutions located in countries without regulation compared to those located in countries where osteopathy is regulated; however, all institutions obtained a ‘more positive than negative’ result. Moreover, in general, cohorts with fewer than 20 students scored significantly higher compared to larger student cohorts. Finally, an overall positive correlation between students’ preparedness and satisfaction were found across all institutions recruited.     

The Role of High-Dimensional Diffusive Search,Stabilization, and Frustration in Protein Folding

Proteins are polymeric molecules with many degrees of conformational freedom whose internal energetic interactions are typically screened to small distances. Therefore, in the high-dimensional conformation space of a protein, the energy landscape is locally relatively flat, in contrast to low-dimensional representations, where, because of the induced entropic contribution to the full free energy, it appears funnel-like. Proteins explore the conformation space by searching these flat subspaces to find a narrow energetic alley that we call a hypergutter and then explore the next, lower-dimensional, subspace. Such a framework provides an effective representation of the energy landscape and folding kinetics that does justice to the essential characteristic of high-dimensionality of the search-space. It also illuminates the important role of nonnative interactions in defining folding pathways. This principle is here illustrated using a coarse-grained model of a family of three-helix bundle proteins whose conformations, once secondary structure has formed, can be defined by six rotational degrees of freedom. Two folding mechanisms are possible, one of which involves an intermediate. The stabilization of intermediate subspaces (or states in low-dimensional projection) in protein folding can either speed up or slow down the folding rate depending on the amount of native and nonnative contacts made in those subspaces. The folding rate increases due to reduced-dimension pathways arising from the mere presence of intermediate states, but decreases if the contacts in the intermediate are very stable and introduce sizeable topological or energetic frustration that needs to be overcome. Remarkably, the hypergutter framework, although depending on just a few physically meaningful parameters, can reproduce all the types of experimentally observed curvature in chevron plots for realizations of this fold.     

Nutritional parameters associated with prolonged hospital stay among ambulatory adult patients

Background

Comprehensive evaluations of the nutritional parameters associated with length of hospital stay are lacking. We investigated the association between malnutrition and length of hospital stay in a cohort of ambulatory adult patients.

Methods

From September 2006 to June 2009, we systematically evaluated 1274 ambulatory adult patients admitted to hospital for medical or surgical treatment. We evaluated the associations between malnutrition and prolonged hospital stay (> 17 days [> 75th percentile of distribution]) using multivariable log-linear models adjusted for several potential nutritional and clinical confounders recorded at admission and collected during and at the end of the hospital stay.

Results

Nutritional factors associated with a prolonged hospital stay were a Nutritional Risk Index score of less than 97.5 (relative risk [RR] 1.64, 95% confidence interval [CI] 1.31–2.06) and an in-hospital weight loss of 5% or greater (RR 1.60, 95% CI 1.30–1.97). Sensitivity analysis of data for patients discharged alive and who had a length of stay of at least three days (n = 1073) produced similar findings (adjusted RR 1.51, 95% CI 1.20–1.89, for Nutritional Risk Index score < 97.5). A significant association was also found with in-hospital starvation of three or more days (RR 1.14, 95% CI 1.01–1.28).

Interpretation

Nutritional risk at admission was strongly associated with a prolonged hospital stay among ambulatory adult patients. Another factor associated with length of stay was worsening nutritional status during the hospital stay, whose cause–effect relationship with length of stay should be clarified in intervention trials. Clinicians need to be aware of the impact of malnutrition and of the potential role of worsening nutritional status in prolonging hospital stay.Choosing the most appropriate approach to clinical management for patients admitted to hospital may not only improve clinical outcomes but also result in early discharge.^1–4 Several factors associated with prolonged hospital stay include the clinical setting, the type and the severity of disease, the presence of comorbidities, the quality and number of interventions, and the patient’s age.^5,6 There is a growing body of evidence that nutritional factors, both related and unrelated to the leading diseases, also affect length of hospital stay and overall health care costs.^7–11 A poor nutritional status at the time of admission can contribute to a prolonged hospital stay, and inadequate nutritional support may negatively affect both nutritional status and prognosis.^7,8 However, these factors have been frequently analyzed independently, and comprehensive and multivariable evaluations of the nutritional parameters associated with a prolonged hospital stay are lacking. Moreover, the potential effect of other confounders occurring during the hospital stay, such as worsening nutritional status, is unknown.We identified the nutritional parameters associated with prolonged hospital stay in a representative sample of ambulatory adult patients. We investigated the association between nutritional risk at the time of admission and length of stay after controlling for several confounders recorded at admission and during the hospital stay.     

Pinus sylvestris forest regeneration under different post-fire restoration practices in the northwestern Italian Alps

It is frequently believed that a post-fire environment requires immediate actions in order to be restored. Salvage logging followed by plantation is a common post-fire restoration practice in many forests of the northwestern Italian Alps.The objectives of this study were to assess the impact of active and passive management techniques on the restoration of a burned area of the Aosta Valley and to determine which approach is the most suitable for enhancing Pinus sylvestris regeneration after stand replacing wildfires.The influence of five management options (no intervention; salvage logging; broadleaves plantation; Larix decidua plantation; P. sylvestris or Pseudotsuga menziesii plantation) and environmental variables on natural regeneration structure and composition was evaluated through direct gradient analysis.Pinus sylvestris and Populus tremula were the dominant tree species (40 and 29%, respectively) in the regeneration layer. Density, size, and structural diversity of natural regeneration were higher in the no intervention area. The proximity to forest edge was found to be the most important environmental variable.This study provided evidence that taking advantage of natural restoration processes may be a suitable alternative strategy to the active restoration practices adopted according to the Aosta Valley policy of post-fire management.     

Aberrant 5' splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization

Despite a growing number of splicing mutations found in hereditary diseases, utilization of aberrant splice sites and their effects on gene expression remain challenging to predict. We compiled sequences of 346 aberrant 5′splice sites (5′ss) that were activated by mutations in 166 human disease genes. Mutations within the 5′ss consensus accounted for 254 cryptic 5′ss and mutations elsewhere activated 92 de novo 5′ss. Point mutations leading to cryptic 5′ss activation were most common in the first intron nucleotide, followed by the fifth nucleotide. Substitutions at position +5 were exclusively G>A transitions, which was largely attributable to high mutability rates of C/G>T/A. However, the frequency of point mutations at position +5 was significantly higher than that observed in the Human Gene Mutation Database, suggesting that alterations of this position are particularly prone to aberrant splicing, possibly due to a requirement for sequential interactions with U1 and U6 snRNAs. Cryptic 5′ss were best predicted by computational algorithms that accommodate nucleotide dependencies and not by weight-matrix models. Discrimination of intronic 5′ss from their authentic counterparts was less effective than for exonic sites, as the former were intrinsically stronger than the latter. Computational prediction of exonic de novo 5′ss was poor, suggesting that their activation critically depends on exonic splicing enhancers or silencers. The authentic counterparts of aberrant 5′ss were significantly weaker than the average human 5′ss. The development of an online database of aberrant 5′ss will be useful for studying basic mechanisms of splice-site selection, identifying splicing mutations and optimizing splice-site prediction algorithms.