X-ray structures of Sap1 and Sap5: structural comparison of the secreted aspartic proteinases from Candida albicans.

Proteolytic activity is an important virulence factor for Candida albicans (C. albicans). It is attributed to the family of the secreted aspartic proteinases (Saps) from C. albicans with a minimum of 10 members. Saps show controlled expression and regulation for the individual stages of the infection process. Distinct isoenzymes can be responsible for adherence and tissue damage of local infections, while others cause systemic diseases. Earlier, only the structures of Sap2 and Sap3 were known. In our research, we have now succeeded in solving the X-ray crystal structures of the apoenzyme of Sap1 and Sap5 in complex with pepstatin A at 2.05 and 2.5 A resolution, respectively. With the structure of Sap1, we have completed the set of structures of isoenzyme subgroup Sap1-3. Of subgroup Sap4-6, the structure of the enzyme Sap5 is the first structure that has been described up to now. This facilitates comparison of structural details as well as inhibitor binding modes among the different subgroup members. Structural analysis reveals a highly conserved overall secondary structure of Sap1-3 and Sap5. However, Sap5 clearly differs from Sap1-3 by its electrostatic overall charge as well as through structural conformation of its entrance to the active site cleft. Design of inhibitors specific for Sap5 should concentrate on the S4 and S3 pockets, which significantly differ from Sap1-3 in size and electrostatic charge. Both Sap1 and Sap5 seem to play a major part in superficial Candida infections. Determination of the isoenzymes' structures can contribute to the development of new Sap-specific inhibitors for the treatment of superficial infections with a structure-based drug design program.

Oligonucleotide-polyethylenimine complexes targeting retinal cells: structural analysis and application to anti-TGFbeta-2 therapy.

PURPOSE: The aim of this study was to characterize oligonucleotide-polyethylenimine (ODN/PEI) complex preparation for potential transfection of retinal cells in vitro and in vivo. METHODS: The effect of medium preparation [HEPES-buffered saline (HBS), water] on particle size and morphology was evaluated. Cultured Lewis rat retinal Müller glial (RMG) cells were transfected using fluorescein isothiocyanate (FITC)-ODN/PEI complexes specifically directed at transforming growth factor beta (TGFbeta)-2. Efficacy of transfection was evaluated using confocal microscopy, and regulation of gene expression was assayed using quantitative real-time RT-PCR and ELISA assay. One, 24, and 72 h after injection of FITC-ODN/PEI complexes into the vitreous of rat eyes, their distribution was analyzed on eye sections. RESULTS: Complexes prepared in HBS were smaller than complexes prepared in pure water and presented a core-shell structure. These particles showed a high cellular internalization efficacy, along with a significant and specific down-regulation of TGFbeta-2 expression and production in RMG cells, correlating with specific inhibition of cell growth at 72 h. In vivo, complexes efficiently transfect retinal cells and follow a transretinal migration at 24 h. After 72 h, ODN seems to preferentially target RMG cells without inducing any detectable toxicity. CONCLUSIONS: Specific down-regulation of TGFbeta-2 expression using ODN/PEI complexes may have potential interest for the treatment of retinal diseases associated with glial proliferation.

The 16p11.2 locus modulates brain structures common to autism, schizophrenia and obesity

Anatomical structures and mechanisms linking genes to neuropsychiatric disorders are not deciphered. Reciprocal copy number variants at the 16p11.2 BP4-BP5 locus offer a unique opportunity to study the intermediate phenotypes in carriers at high risk for autism spectrum disorder (ASD) or schizophrenia (SZ). We investigated the variation in brain anatomy in 16p11.2 deletion and duplication carriers. Beyond gene dosage effects on global brain metrics, we show that the number of genomic copies negatively correlated to the gray matter volume and white matter tissue properties in cortico-subcortical regions implicated in reward, language and social cognition. Despite the near absence of ASD or SZ diagnoses in our 16p11.2 cohort, the pattern of brain anatomy changes in carriers spatially overlaps with the well-established structural abnormalities in ASD and SZ. Using measures of peripheral mRNA levels, we confirm our genomic copy number findings. This combined molecular, neuroimaging and clinical approach, applied to larger datasets, will help interpret the relative contributions of genes to neuropsychiatric conditions by measuring their effect on local brain anatomy.Molecular Psychiatry advance online publication, 25 November 2014; doi:10.1038/mp.2014.145.

Tectonics of the Simplon massif and Lepontine gneiss dome: deformation structures due to collision between the underthrusting European plate and the Adriatic indenter

this study presents a review of published geological data, combined with original observations on the tectonics of the simplon massif and the Lepontine gneiss dome in the Western Alps. New observations concern the geometry of the Oligocene Vanzone back fold, formed under amphibolite facies conditions, and of its root between Domodossola and Locarno, which is cut at an acute angle by the Miocene, epi- to anchizonal, dextral centovalli strike-slip fault. the structures of the simplon massif result from collision over 50 Ma between two plate boundaries with a different geometry: the underthrusted European plate and the Adriatic indenter. Detailed mapping and analysis of a complex structural interference pattern, combined with observations on the metamorphic grade of the superimposed structures and radiometric data, allow a kinematic model to be developed for this zone of oblique continental collision. the following main Alpine tectonic phases and structures may be distinguished: 1. NW-directed nappe emplacement, starting in the Early Eocene (similar to 50 Ma); 2. W, SW and S- verging transverse folds; 3. transpressional movements on the dextral simplon ductile shear zone since similar to 32 Ma; 4. formation of the Bergell - Vanzone backfolds and of the southern steep belt during the Oligocene, emplacement of the mantle derived 31 - 29 Ma Bergell and Biella granodiorites and porphyritic andesites as well as intrusions of 29-25 Ma crustal aplites and pegmatites; 5. formation of the dextral discrete Rhone-Simplon line and the centovalli line during the Miocene, accompanied by the pull-apart development of the Lepontine gneiss dome - Dent blanche (Valpelline) depression. It is suggested that movements of shortening in fan shaped NW, W and sW directions accompanied the more regular NW- to WNW-directed displacement of the Adriatic indenter during continental collision.

TCRep 3D: an automated in silico approach to study the structural properties of TCR repertoires.

TCRep 3D is an automated systematic approach for TCR-peptide-MHC class I structure prediction, based on homology and ab initio modeling. It has been considerably generalized from former studies to be applicable to large repertoires of TCR. First, the location of the complementary determining regions of the target sequences are automatically identified by a sequence alignment strategy against a database of TCR Vα and Vβ chains. A structure-based alignment ensures automated identification of CDR3 loops. The CDR are then modeled in the environment of the complex, in an ab initio approach based on a simulated annealing protocol. During this step, dihedral restraints are applied to drive the CDR1 and CDR2 loops towards their canonical conformations, described by Al-Lazikani et. al. We developed a new automated algorithm that determines additional restraints to iteratively converge towards TCR conformations making frequent hydrogen bonds with the pMHC. We demonstrated that our approach outperforms popular scoring methods (Anolea, Dope and Modeller) in predicting relevant CDR conformations. Finally, this modeling approach has been successfully applied to experimentally determined sequences of TCR that recognize the NY-ESO-1 cancer testis antigen. This analysis revealed a mechanism of selection of TCR through the presence of a single conserved amino acid in all CDR3β sequences. The important structural modifications predicted in silico and the associated dramatic loss of experimental binding affinity upon mutation of this amino acid show the good correspondence between the predicted structures and their biological activities. To our knowledge, this is the first systematic approach that was developed for large TCR repertoire structural modeling.

Analysis of susceptibility to human immunodeficiency virus 1 (HIV-1) by comparative genetics

Summary [résumé français voir ci-dessous] From the beginning of the 20th century the world population has been confronted with the human immune deficiency virus 1 (HIV-1). This virus has the particularity to mutate fast, and could thus evade and adapt to the human host. Our closest evolutionary related organisms, the non-human primates, are less susceptible to HIV-1. In a broader sense, primates are differentially susceptible to various retrovirus. Species specificity may be due to genetic differences among primates. In the present study we applied evolutionary and comparative genetic techniques to characterize the evolutionary pattern of host cellular determinants of HIV-1 pathogenesis. The study of the evolution of genes coding for proteins participating to the restriction or pathogenesis of HIV-1 may help understanding the genetic basis of modern human susceptibility to infection. To perform comparative genetics analysis, we constituted a collection of primate DNA and RNA to allow generation of de novo sequence of gene orthologs. More recently, release to the public domain of two new primate complete genomes (bornean orang-utan and common marmoset) in addition of the three previously available genomes (human, chimpanzee and Rhesus monkey) help scaling up the evolutionary and comparative genome analysis. Sequence analysis used phylogenetic and statistical methods for detecting molecular adaptation. We identified different selective pressures acting on host proteins involved in HIV-1 pathogenesis. Proteins with HIV-1 restriction properties in non-human primates were under strong positive selection, in particular in regions of interaction with viral proteins. These regions carried key residues for the antiviral activity. Proteins of the innate immunity presented an evolutionary pattern of conservation (purifying selection) but with signals of relaxed constrain if we compared them to the average profile of purifying selection of the primate genomes. Large scale analysis resulted in patterns of evolutionary pressures according to molecular function, biological process and cellular distribution. The data generated by various analyses served to guide the ancestral reconstruction of TRIM5a a potent antiviral host factor. The resurrected TRIM5a from the common ancestor of Old world monkeys was effective against HIV-1 and the recent resurrected hominoid variants were more effective against other retrovirus. Thus, as the result of trade-offs in the ability to restrict different retrovirus, human might have been exposed to HIV-1 at a time when TRIM5a lacked the appropriate specific restriction activity. The application of evolutionary and comparative genetic tools should be considered for the systematical assessment of host proteins relevant in viral pathogenesis, and to guide biological and functional studies. Résumé La population mondiale est confrontée depuis le début du vingtième siècle au virus de l'immunodéficience humaine 1 (VIH-1). Ce virus a un taux de mutation particulièrement élevé, il peut donc s'évader et s'adapter très efficacement à son hôte. Les organismes évolutivement le plus proches de l'homme les primates nonhumains sont moins susceptibles au VIH-1. De façon générale, les primates répondent différemment aux rétrovirus. Cette spécificité entre espèces doit résider dans les différences génétiques entre primates. Dans cette étude nous avons appliqué des techniques d'évolution et de génétique comparative pour caractériser le modèle évolutif des déterminants cellulaires impliqués dans la pathogenèse du VIH- 1. L'étude de l'évolution des gènes, codant pour des protéines impliquées dans la restriction ou la pathogenèse du VIH-1, aidera à la compréhension des bases génétiques ayant récemment rendu l'homme susceptible. Pour les analyses de génétique comparative, nous avons constitué une collection d'ADN et d'ARN de primates dans le but d'obtenir des nouvelles séquences de gènes orthologues. Récemment deux nouveaux génomes complets ont été publiés (l'orang-outan du Bornéo et Marmoset commun) en plus des trois génomes déjà disponibles (humain, chimpanzé, macaque rhésus). Ceci a permis d'améliorer considérablement l'étendue de l'analyse. Pour détecter l'adaptation moléculaire nous avons analysé les séquences à l'aide de méthodes phylogénétiques et statistiques. Nous avons identifié différentes pressions de sélection agissant sur les protéines impliquées dans la pathogenèse du VIH-1. Des protéines avec des propriétés de restriction du VIH-1 dans les primates non-humains présentent un taux particulièrement haut de remplacement d'acides aminés (sélection positive). En particulier dans les régions d'interaction avec les protéines virales. Ces régions incluent des acides aminés clé pour l'activité de restriction. Les protéines appartenant à l'immunité inné présentent un modèle d'évolution de conservation (sélection purifiante) mais avec des traces de "relaxation" comparé au profil général de sélection purifiante du génome des primates. Une analyse à grande échelle a permis de classifier les modèles de pression évolutive selon leur fonction moléculaire, processus biologique et distribution cellulaire. Les données générées par les différentes analyses ont permis la reconstruction ancestrale de TRIM5a, un puissant facteur antiretroviral. Le TRIM5a ressuscité, correspondant à l'ancêtre commun entre les grands singes et les groupe des catarrhiniens, est efficace contre le VIH-1 moderne. Les TRIM5a ressuscités plus récents, correspondant aux ancêtres des grands singes, sont plus efficaces contre d'autres rétrovirus. Ainsi, trouver un compromis dans la capacité de restreindre différents rétrovirus, l'homme aurait été exposé au VIH-1 à une période où TRIM5a manquait d'activité de restriction spécifique contre celui-ci. L'application de techniques d'évolution et de génétique comparative devraient être considérées pour l'évaluation systématique de protéines impliquées dans la pathogenèse virale, ainsi que pour guider des études biologiques et fonctionnelles

Using graphical probability analysis (Bayes Nets) to evaluate a conditional DNA inclusion

This paper discusses the analysis of cases in which the inclusion or exclusion of a particular suspect, as a possible contributor to a DNA mixture, depends on the value of a variable (the number of contributors) that cannot be determined with certainty. It offers alternative ways to deal with such cases, including sensitivity analysis and object-oriented Bayesian networks, that separate uncertainty about the inclusion of the suspect from uncertainty about other variables. The paper presents a case study in which the value of DNA evidence varies radically depending on the number of contributors to a DNA mixture: if there are two contributors, the suspect is excluded; if there are three or more, the suspect is included; but the number of contributors cannot be determined with certainty. It shows how an object-oriented Bayesian network can accommodate and integrate varying perspectives on the unknown variable and how it can reduce the potential for bias by directing attention to relevant considerations and distinguishing different sources of uncertainty. It also discusses the challenge of presenting such evidence to lay audiences.

How can we teach EBM in clinical practice? : an analysis of barriers to implementation of on-the-job EBM teaching and learning.

Introduction: Evidence-based medicine (EBM) improves the quality of health care. Courses on how to teach EBM in practice are available, but knowledge does not automatically imply its application in teaching. We aimed to identify and compare barriers and facilitators for teaching EBM in clinical practice in various European countries. Methods: A questionnaire was constructed listing potential barriers and facilitators for EBM teaching in clinical practice. Answers were reported on a 7-point Likert scale ranging from not at all being a barrier to being an insurmountable barrier. Results: The questionnaire was completed by 120 clinical EBM teachers from 11 countries. Lack of time was the strongest barrier for teaching EBM in practice (median 5). Moderate barriers were the lack of requirements for EBM skills and a pyramid hierarchy in health care management structure (median 4). In Germany, Hungary and Poland, reading and understanding articles in English was a higher barrier than in the other countries. Conclusion: Incorporation of teaching EBM in practice faces several barriers to implementation. Teaching EBM in clinical settings is most successful where EBM principles are culturally embedded and form part and parcel of everyday clinical decisions and medical practice.

Decision curve analysis revisited: overall net benefit, relationships to ROC curve analysis, and application to case-control studies.

ABSTRACT: BACKGROUND: Decision curve analysis has been introduced as a method to evaluate prediction models in terms of their clinical consequences if used for a binary classification of subjects into a group who should and into a group who should not be treated. The key concept for this type of evaluation is the "net benefit", a concept borrowed from utility theory. METHODS: We recall the foundations of decision curve analysis and discuss some new aspects. First, we stress the formal distinction between the net benefit for the treated and for the untreated and define the concept of the "overall net benefit". Next, we revisit the important distinction between the concept of accuracy, as typically assessed using the Youden index and a receiver operating characteristic (ROC) analysis, and the concept of utility of a prediction model, as assessed using decision curve analysis. Finally, we provide an explicit implementation of decision curve analysis to be applied in the context of case-control studies. RESULTS: We show that the overall net benefit, which combines the net benefit for the treated and the untreated, is a natural alternative to the benefit achieved by a model, being invariant with respect to the coding of the outcome, and conveying a more comprehensive picture of the situation. Further, within the framework of decision curve analysis, we illustrate the important difference between the accuracy and the utility of a model, demonstrating how poor an accurate model may be in terms of its net benefit. Eventually, we expose that the application of decision curve analysis to case-control studies, where an accurate estimate of the true prevalence of a disease cannot be obtained from the data, is achieved with a few modifications to the original calculation procedure. CONCLUSIONS: We present several interrelated extensions to decision curve analysis that will both facilitate its interpretation and broaden its potential area of application.

Construction of Pseudomonas strains with improved biocontrol activity

Selected strains of fluorescent pseudomonads suppress various plant diseases caused by soil-borne pathogenic fungi, by a blend of several mechanisms including aggressive root colonization, antibiosis, competition for nutrients, induction of resistance in the plant, and enzymatic attack of the pathogen. These traits are amenable to genetic analysis and, therefore, to modification by genetic engineering. Biocontrol activities of Pseudomonas spp. have been enhanced in two ways: (i) by overexpression of traits known to involved in diseaese suppression, and (ii) by introduction of additional beneficial traits into strains having basal biocontrol activities. Under experimental conditions in microcosms, a number of genetically modified Pseudomonas strains have given promising results. It remains to be seen whether such strains will be superior to the best naturally occurring strains, applied singly or in combination, under greenhouse and field conditions.

Side effects of genome structural changes.

The first extensive catalog of structural human variation was recently released. It showed that large stretches of genomic DNA that vary considerably in copy number were extremely abundant. Thus it is conceivable that they play a major role in functional variation. Consistently, genomic insertions and deletions were shown to contribute to phenotypic differences by modifying not only the expression levels of genes within the aneuploid segments but also of normal copy-number neighboring genes. In this report, we review the possible mechanisms behind this latter effect.

Structures and failure mechanisms analysis of Turtle Mountain

The 30 M m3 rockslide that occurred on the east face of Turtle Mountain in the Crowsnest Pass area (Alberta) in 1903 is one of the most famous landslides in the world. In this paper, the structural features of the South part of Turtle Mountain are investigated in order to understand the present-day scar morphology and to identify the most important failure mechanisms. The structural features were mapped using a high resolution digital elevation model (DEM) in order to have a large overview of the relevant structural features. At the same time, a field survey was carried out and small scale fractures were analyzed in different parts of southern Turtle Mountain in order to confirm the DEM analysis. Results allow to identify six main discontinuity sets that influence the Turtle Mountain morphology. These discontinuity sets were then used to identify the potential failure mechanisms affecting Third Peak and South Peak area.

A 2D/3D image analysis system to track fluorescently labeled structures in rod-shaped cells: application to measure spindle pole asymmetry during mitosis.

BACKGROUND: The yeast Schizosaccharomyces pombe is frequently used as a model for studying the cell cycle. The cells are rod-shaped and divide by medial fission. The process of cell division, or cytokinesis, is controlled by a network of signaling proteins called the Septation Initiation Network (SIN); SIN proteins associate with the SPBs during nuclear division (mitosis). Some SIN proteins associate with both SPBs early in mitosis, and then display strongly asymmetric signal intensity at the SPBs in late mitosis, just before cytokinesis. This asymmetry is thought to be important for correct regulation of SIN signaling, and coordination of cytokinesis and mitosis. In order to study the dynamics of organelles or large protein complexes such as the spindle pole body (SPB), which have been labeled with a fluorescent protein tag in living cells, a number of the image analysis problems must be solved; the cell outline must be detected automatically, and the position and signal intensity associated with the structures of interest within the cell must be determined. RESULTS: We present a new 2D and 3D image analysis system that permits versatile and robust analysis of motile, fluorescently labeled structures in rod-shaped cells. We have designed an image analysis system that we have implemented as a user-friendly software package allowing the fast and robust image-analysis of large numbers of rod-shaped cells. We have developed new robust algorithms, which we combined with existing methodologies to facilitate fast and accurate analysis. Our software permits the detection and segmentation of rod-shaped cells in either static or dynamic (i.e. time lapse) multi-channel images. It enables tracking of two structures (for example SPBs) in two different image channels. For 2D or 3D static images, the locations of the structures are identified, and then intensity values are extracted together with several quantitative parameters, such as length, width, cell orientation, background fluorescence and the distance between the structures of interest. Furthermore, two kinds of kymographs of the tracked structures can be established, one representing the migration with respect to their relative position, the other representing their individual trajectories inside the cell. This software package, called "RodCellJ", allowed us to analyze a large number of S. pombe cells to understand the rules that govern SIN protein asymmetry. CONCLUSIONS: "RodCell" is freely available to the community as a package of several ImageJ plugins to simultaneously analyze the behavior of a large number of rod-shaped cells in an extensive manner. The integration of different image-processing techniques in a single package, as well as the development of novel algorithms does not only allow to speed up the analysis with respect to the usage of existing tools, but also accounts for higher accuracy. Its utility was demonstrated on both 2D and 3D static and dynamic images to study the septation initiation network of the yeast Schizosaccharomyces pombe. More generally, it can be used in any kind of biological context where fluorescent-protein labeled structures need to be analyzed in rod-shaped cells. AVAILABILITY: RodCellJ is freely available under http://bigwww.epfl.ch/algorithms.html, (after acceptance of the publication).

The effects of catechin rich teas and caffeine on energy expenditure and fat oxidation: a meta-analysis.

Different outcomes of the effect of catechin-caffeine mixtures and caffeine-only supplementation on energy expenditure and fat oxidation have been reported in short-term studies. Therefore, a meta-analysis was conducted to elucidate whether catechin-caffeine mixtures and caffeine-only supplementation indeed increase thermogenesis and fat oxidation. First, English-language studies measuring daily energy expenditure and fat oxidation by means of respiration chambers after catechin-caffeine mixtures and caffeine-only supplementation were identified through PubMed. Six articles encompassing a total of 18 different conditions fitted the inclusion criteria. Second, results were aggregated using random/mixed-effects models and expressed in terms of the mean difference in 24 h energy expenditure and fat oxidation between the treatment and placebo conditions. Finally, the influence of moderators such as BMI and dosage on the results was examined as well. The catechin-caffeine mixtures and caffeine-only supplementation increased energy expenditure significantly over 24 h (428.0 kJ (4.7%); P < 0.001 and 429.1 kJ (4.8%); P < 0.001, respectively). However, 24 h fat oxidation was only increased by catechin-caffeine mixtures (12.2 g (16.0%); P < 0.02 and 9.5 g (12.4%); P = 0.11, respectively). A dose-response effect on 24 h energy expenditure and fat oxidation occurred with a mean increase of 0.53 kJ mg(-1) (P < 0.01) and 0.02 g mg(-1) (P < 0.05) for catechin-caffeine mixtures and 0.44 kJ mg(-1) (P < 0.001) and 0.01 g mg(-1) (P < 0.05) for caffeine-only. In conclusion, catechin-caffeine mixtures or a caffeine-only supplementation stimulates daily energy expenditure dose-dependently by 0.4-0.5 kJ mg(-1) administered. Compared with placebo, daily fat-oxidation was only significantly increased after catechin-caffeine mixtures ingestion.