A novel study of copy number variations in Hirschsprung disease using the multiple ligation-dependent probe amplification (MLPA) technique.

BACKGROUND Hirschsprung disease (HSCR) is a congenital malformation of the hindgut produced by a disruption in neural crest cell migration during embryonic development. HSCR has a complex genetic etiology and mutations in several genes, mainly the RET proto-oncogene, have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. METHODS In this study we have aimed to analyze the presence of CNVs in some HSCR genes (RET, EDN3, GDNF and ZFHX1B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. RESULTS Two alterations in the MLPA profiles of RET and EDN3 were detected, but a detailed inspection showed that the decrease in the corresponding dosages were due to point mutations affecting the hybridization probes regions. CONCLUSION Our results indicate that CNVs of the gene coding regions analyzed here are not a common molecular cause of Hirschsprung disease. However, further studies are required to determine the presence of CNVs affecting non-coding regulatory regions, as well as other candidate genes.

Using 3D surface datasets to understand landslide evolution: From analogue models to real case study

Early detection of landslide surface deformation with 3D remote sensing techniques, as TLS, has become a great challenge during last decade. To improve our understanding of landslide deformation, a series of analogue simulation have been carried out on non-rigid bodies coupled with 3D digitizer. All these experiments have been carried out under controlled conditions, as water level and slope angle inclination. We were able to follow 3D surface deformation suffered by complex landslide bodies from precursory deformation still larger failures. These experiments were the basis for the development of a new algorithm for the quantification of surface deformation using automatic tracking method on discrete points of the slope surface. To validate the algorithm, comparisons were made between manually obtained results and algorithm surface displacement results. Outputs will help in understanding 3D deformation during pre-failure stages and failure mechanisms, which are fundamental aspects for future implementation of 3D remote sensing techniques in early warning systems.

Sources of pre-analytical variations in yield of DNA extracted from blood samples: analysis of 50,000 DNA samples in EPIC.

The European Prospective Investigation into Cancer and nutrition (EPIC) is a long-term, multi-centric prospective study in Europe investigating the relationships between cancer and nutrition. This study has served as a basis for a number of Genome-Wide Association Studies (GWAS) and other types of genetic analyses. Over a period of 5 years, 52,256 EPIC DNA samples have been extracted using an automated DNA extraction platform. Here we have evaluated the pre-analytical factors affecting DNA yield, including anthropometric, epidemiological and technical factors such as center of subject recruitment, age, gender, body-mass index, disease case or control status, tobacco consumption, number of aliquots of buffy coat used for DNA extraction, extraction machine or procedure, DNA quantification method, degree of haemolysis and variations in the timing of sample processing. We show that the largest significant variations in DNA yield were observed with degree of haemolysis and with center of subject recruitment. Age, gender, body-mass index, cancer case or control status and tobacco consumption also significantly impacted DNA yield. Feedback from laboratories which have analyzed DNA with different SNP genotyping technologies demonstrate that the vast majority of samples (approximately 88%) performed adequately in different types of assays. To our knowledge this study is the largest to date to evaluate the sources of pre-analytical variations in DNA extracted from peripheral leucocytes. The results provide a strong evidence-based rationale for standardized recommendations on blood collection and processing protocols for large-scale genetic studies.

Development and validation of a risk score predicting substantial weight gain over 5 years in middle-aged European men and women.

BACKGROUND Identifying individuals at high risk of excess weight gain may help targeting prevention efforts at those at risk of various metabolic diseases associated with weight gain. Our aim was to develop a risk score to identify these individuals and validate it in an external population. METHODS We used lifestyle and nutritional data from 53°758 individuals followed for a median of 5.4 years from six centers of the European Prospective Investigation into Cancer and Nutrition (EPIC) to develop a risk score to predict substantial weight gain (SWG) for the next 5 years (derivation sample). Assuming linear weight gain, SWG was defined as gaining ≥ 10% of baseline weight during follow-up. Proportional hazards models were used to identify significant predictors of SWG separately by EPIC center. Regression coefficients of predictors were pooled using random-effects meta-analysis. Pooled coefficients were used to assign weights to each predictor. The risk score was calculated as a linear combination of the predictors. External validity of the score was evaluated in nine other centers of the EPIC study (validation sample). RESULTS Our final model included age, sex, baseline weight, level of education, baseline smoking, sports activity, alcohol use, and intake of six food groups. The model's discriminatory ability measured by the area under a receiver operating characteristic curve was 0.64 (95% CI = 0.63-0.65) in the derivation sample and 0.57 (95% CI = 0.56-0.58) in the validation sample, with variation between centers. Positive and negative predictive values for the optimal cut-off value of ≥ 200 points were 9% and 96%, respectively. CONCLUSION The present risk score confidently excluded a large proportion of individuals from being at any appreciable risk to develop SWG within the next 5 years. Future studies, however, may attempt to further refine the positive prediction of the score.

National Health System Virtual Library Viability Project (Spain)

Introduction: The ICT progress and development are making the set up of virtual libraries easier, with great advantages for users in terms of immediate access to vast amounts of library information, online resources, and services. Most of the Autonomous Spanish Regions are presently working on the establishment of virtual libraries with the aim of optimising economic resources destined to subscribe bibliographic information sources and offering qualified documentary services to Public Health System Professionals. The Ministry of Health and Social Policy, an institution unifying health matters, issued this Project to study the National Health System Virtual Library creation viability, in order to guarantee relevant scientific information access equity. Objectives: - To study the National Health System (NHS) Virtual Library set up viability. - To elaborate the Spanish territory health information resource map. - To identify the appropriate technological model. - To identify the documentary services to be offered. - To identify the more optimum functional, structural model. - To identify the economic model. Method: - To create a chart organization for the project´s management. - To organize Work groups. - To elaborate a standardised survey model for the libraries of the autonomous regions. Results: - Identification of the different Autonomous Region libraries of health models. - Recommendations for the NHS Virtual Library: - Functional, structural model - Technological model - Financial-economic model Conclusions: The fact that the Virtual Library would be an invaluable space for access to quality scientific information, as well as that the minimum services could be offered to every NHS user regardless of geographical situation, is confirmed. Accordingly, we thought about three different models to develop the Virtual Library, depending on this initial analysis, which will allow us to establish the most suitable model for the Spanish National Health System, considering its economic, functional and technological recommendations. It would be necessary to do a study for the NHS Virtual Library Set Up following the recommendations arising from this Project.

"I don't know how you are" or indirect questioning in oncology interviews: an ongoing exploratory study

Background: The exploratory study is part of an evaluation of the pre-graduate teaching of communication skills (Lausanne Medical School). It is based on the data of a project highlighting the impact of individualized vs. group training for medicine students in breaking bad news to simulated patients who are diagnosed with cancer. The analysis of the video-taped interviews of the students (N=63) with the RIAS has shown a current usage of utterances such as I don't know if -you have any plans for the future / you have already heard about chemotherapy / ... or I don't know how -you are feeling today after this surgery / you like that all this stuff takes place / ...Aim: The present study questions the specificity of these assertive utterances used as questions (indirect), the specificity of their content, and their intentionality - specific vs. exploratory.Methods: The mentioned utterances are qualitatively analyzed (content analysis, intentionality analysis, etc).Results: 26 students (41%) used 1 to 6 times I don't know utterances during the interviews that contain 53 of such utterances in total. In contrast, they are atypical in an oncologist sample who conducted similar interviews (N=31; 4 oncologist used them 1 to 2 times). In more than half of the cases (29/53), simulated patients interpret I don't know questions as giving them a space to speak (open responses). Conclusions: The atypicality of the I don't know utterances in the oncologist sample may have linguistic explanations in terms of generational marker, but the specificity of the content suggests psychological explanations in terms of defense mechanism as well (marker of "toning down" or insecurity as regards the discussed topic).Keywords: Breaking bad news, communication skills, oncology, pre-graduate medical education, indirect questioning

Long-term outcome of patients after a single interruption of antiretroviral therapy: a cohort study.

BACKGROUND To describe the long term outcome of patients who interrupted highly active antiretroviral therapy (HAART) once, identify the variables associated with earlier need to re-start HAART, and the response when therapy was resumed. A retrospective observational cohort of 66 adult patients with HIV-1 infection who interrupted HAART with a CD4+cell count ≥ 350 cells/μL and undetectable viral load (VL) was performed. The pre-established CD4+ cell count for restarting therapy was 300cells/μL. Cox regression was used to analyse the variables associated with earlier HAART reinitiation. RESULTS The median follow-up was 209 weeks (range, 64-395). Rates of HIV-related or possible HIV-related events were 0.37 (one case of acute retroviral syndrome) and 1.49 per 100 patient-years, respectively. Two patients died after re-starting therapy and having reached undetectable VL. Three patients suffered a sexually transmitted disease while off therapy. Fifty patients (76%) resumed therapy after a median of 97 weeks (range, 17-267). Age, a nadir of CD4+ <250 cells/μL, and a mean VL during interruption of >10,000 copies/ml were independent predictors for earlier re-start. The intention-to-treat success rate of the first HAART resumed regimen was 85.4%. There were no differences by regimen used, nor between regimens that were the same as or different from the one that had been interrupted. CONCLUSIONS Our data suggest highly active antiretroviral therapy may be interrupted in selected patients because in these patients, when the HAART is restarted, the viral and clinical response may be achieved.

Characterization of SKI-1/S1P-mediated processing of Arenavirus glycoprotein precursors

Arenaviruses are rodent-born world-wide distributed negative strand RNA viruses that comprise a number of important human pathogens including Lassa virus (LASV) which causes more than 3 00'000 infections annually in Western Africa. Lymphocytic choriomeningitis virus (LCMV) is the prototypic member of the arenavirus family, which is divided in two major subgroups according to serological properties and geographical distribution, the Old World and New World arenaviruses. The envelope glycoprotein precursors (GPCs) of arenaviruses have to undergo proteolytic processing to acquire biological function and to be incorporated into progeny virions. A cellular enzyme is responsible for this processing: the Subtilisin Kexin Isozyme-1 or Site-1 protease (SKI- 1/S1P). In this thesis we have studied the relationship between SKI-1/S1P and the envelope GPs of arenaviruses. In a first project, we investigated the molecular interactions between SKI-1/SIP and arenavirus GPCs. Using SKI-1/SIP mutants, we confirmed previously published observations locating LCMV GPC and LASV GPC processing in the Late Golgi/TGN and ER/cis-Golgi, respectively. A single mutation in the cleavage site of LCMV was sufficient to re-locate SKI- 1/SIP-mediated processing from the late Golgi/TGN to the ER/cis-Golgi. We then demonstrated that the transmembrane domain, the C-terminal tail and the phosphorylation sites of SKI-1/S1P are dispensable for GPC processing. Additionally we identified a SKI- 1/S1P mutant defective for autoprocessing at site Β, B' that was selectively impaired in processing of viral GPCs but not cellular substrates. We also showed that a soluble variant of SKI-1/SIΡ was unable to cleave envelope GPs at the cell surface when added in the culture medium. This study highlighted a new target for small molecule inhibitors that would specifically impair GPC but not cellular substrate processing. In a second project, we identified and characterized two residues: LASV GPC Y253 and SKI-1/S1P Y285 that are important for the SKI-1/SIP-mediated LASV GPC cleavage. An alignment of GPC sequences revealed a conserved aromatic residue in P7 position in the GPCs of Old World and Clade C of New World arenaviruses. Mutations in GPC at position P7 impaired processing efficiency. In SKI-1/S1P, mutating Y285 into A negatively affected processing of substrates containing aromatic residues in P7, without affecting others. This property could be used to develop specific drugs targeting SKI-1/SIP-mediated cleavage of LASV GPC without affecting cellular substrates. As a third project we studied the role of the SKI-1/SIP-mediated processing and the unusual stable signal peptide (SSP) for the folding and secretion of soluble forms of the ectodomain of LASV and LCMV glycoproteins. We provide evidence that the transmembrane domain and the cytosolic tail are crucial for the stability of the prefusion conformation of arenavirus GP and that the SSP is required for transport and processing of full-length GP, but not the soluble ectodomain per se. Taken together, these results will lead to a better understanding of the complex interactions between arenavirus GPCs and SKI-1/S IP, paving the avenue for the development of novel anti-arenaviral therapeutics. - Les Arenavirus sont des virus à ARN négatif distribués mondialement et portés par les rongeurs. Cette famille de virus comprend des virus hautement pathogènes pour l'homme comme le virus de Lassa (LASV) qui cause plus de 300Ό00 infections par année en Afrique de l'Ouest. Le virus de la chorioméningite lymphocytaire (LCMV) est le représentant de cette famille qui est divisée en deux sous-groupes selon des critères sérologiques et de distributions géographiques: arenavirus du Nouveau et de l'Ancien monde. Les glycoprotéines d'enveloppe de ces virus (GPCs) doivent être clivées pour être incorporées dans le virus et ainsi lui permettre d'être infectieux. Une enzyme cellulaire est responsable de ce clivage : la Subtilisin Kexin Isozyme-1 ou protéase Site-1 (SKI-l/SlP). Dans cette thèse, nous avons étudié la relation entre cette enzyme cellulaire et les GPs des arenavirus. Dans un premier temps, nous avons étudié les interactions moléculaires entre SKI- 1/S1P et GPC. A l'aide de mutants de SKI-l/SlP, nous avons confirmé des résultats précédemment publiés montrant que les glycoprotéines d'enveloppe de LASV sont clivés dans le réticulum endoplasmique/cis-Golgi alors que celles de LCMV sont clivées dans le Golgi tardif/TGN. Une seule mutation dans le site de clivage de la glycoprotéine de LCMV est suffisante pour changer le compartiment cellulaire dans lequel est clivée cette glycoprotéine. Ensuite, nous avons démontré que le domaine transmembranaire, la partie cytosolique C-terminale ainsi que les sites de phosphorylations de cette enzyme ne sont pas indispensables pour permettre le clivage de GPC. De plus, nous avons identifié un mutant de SKI-l/SlP dans lequel Γ autoprocessing au site B,B' est impossible, incapable de cliver GPC mais toujours pleinement fonctionnelle envers ses substrats cellulaires. Nous avons également démontré qu'une forme soluble de SKI-l/SlP ajoutée dans le milieu de culture n'est pas capable de couper GPC à la surface de la cellule. Cette étude a défini une nouvelle cible potentielle pour un médicament qui inhiberait le clivage des glycoprotéines des arenavirus sans affecter les processus normaux de la cellule. Dans un second project, nous avons identifié deux acides aminés, LASV GPC Y253 et SKI-l/SlP Y285, qui sont important pour le clivage de LASV GPC. Un alignement des séquences de clivage des GPCs a montré qu'un résidu aromatique est conservé en position P7 du site de clivage chez tous les arenavirus de l'Ancien monde et dans le clade C des arenavirus du Nouveau monde. Une mutation de cet acide aminée dans GPC réduit l'efficacité de clivage par SKI-l/SlP. Mutation de la tyrosine 285 de SKI-l/SlP en alanine affecte négativement le clivage des substrats contenant un résidu aromatique en position P7 sans affecter les autres. Cette propriété pourrait être utilisée pour le développement de médicaments spécifiques ciblant le clivage de GPC. Finalement, nous avons étudié le rôle du processing accomplit par SKI-l/SlP et du signal peptide pour le pliage et la sécrétion de formes solubles des glycoprotéines de LASV et LCMV. Nous avons montré que le domaine transmembranaire et la partie cytosolique de GP sont crucials pour la stabilité de la conformation pre-fusionnelle des GPs et que SSP est nécessaire pour le transport et le processing de GP, mais pas de son ecto-domaine soluble. En conclusion, les résultats obtenus durant cette thèse permettrons de mieux comprendre les interactions complexes entre SKI-l/SlP et les glycoprotéines des arenavirus, ouvrant le chemin pour le développement de nouveaux médicaments anti-arénaviraux.

A Sustainable Development Strategy for the Pyrenees-Mediterranean Euroregion: Basic Guidelines

L'estudi presenta té per objecte formular els requisits necessaris per posar en marxa una estratègia per al desenvolupament sostenible de l'Euroregió Pirineus-Mediterrània. Aquest és un projecte de cooperació transfronterera regional promogut per l'expresident de la Generalitat, Honorable Sr Pasqual Maragall, que, a més de Catalunya, inclou Aragó, Illes Balears, Llenguadoc-Rosselló i Migdia-Pirineus. A la Unió Europea hi ha més de 70 euroregions, que han estat en funcionant per diferents periodes de temps, després d'haver estat creat per regions o entitats locals, o ambdós

Fosfomycin versus meropenem in bacteraemic urinary tract infections caused by extended-spectrum β-lactamase-producing Escherichia coli (FOREST): study protocol for an investigator-driven randomised controlled trial.

INTRODUCTION Finding therapeutic alternatives to carbapenems in infections caused by extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) is imperative. Although fosfomycin was discovered more than 40 years ago, it was not investigated in accordance with current standards and so is not used in clinical practice except in desperate situations. It is one of the so-called neglected antibiotics of high potential interest for the future. METHODS AND ANALYSIS The main objective of this project is to demonstrate the clinical non-inferiority of intravenous fosfomycin with regard to meropenem for treating bacteraemic urinary tract infections (UTI) caused by ESBL-EC. This is a 'real practice' multicentre, open-label, phase III randomised controlled trial, designed to compare the clinical and microbiological efficacy, and safety of intravenous fosfomycin (4 g/6 h) and meropenem (1 g/8 h) as targeted therapy for this infection; a change to oral therapy is permitted after 5 days in both arms, in accordance with predetermined options. The study design follows the latest recommendations for designing trials investigating new options for multidrug-resistant bacteria. Secondary objectives include the study of fosfomycin concentrations in plasma and the impact of both drugs on intestinal colonisation by multidrug-resistant Gram-negative bacilli. ETHICS AND DISSEMINATION Ethical approval was obtained from the Andalusian Coordinating Institutional Review Board (IRB) for Biomedical Research (Referral Ethics Committee), which obtained approval from the local ethics committees at all participating sites in Spain (22 sites). Data will be presented at international conferences and published in peer-reviewed journals. DISCUSSION This project is proposed as an initial step in the investigation of an orphan antimicrobial of low cost with high potential as a therapeutic alternative in common infections such as UTI in selected patients. These results may have a major impact on the use of antibiotics and the development of new projects with this drug, whether as monotherapy or combination therapy. TRIAL REGISTRATION NUMBER NCT02142751. EudraCT no: 2013-002922-21. Protocol V.1.1 dated 14 March 2014.

The joint R&D project: The case of the first Brazilian microcontroller chip

ABSTRACT The interorganizational cooperation, through joint efforts with various actors, allows the high-tech companies to complement resources, especially in R&D projects. Collaborative projects have been identified in many studies as an important strategy to produce complex products and services in uncertain and competitive environments. Thus, this research aims at deepening the understanding of how the development dynamics of a collaborative R&D project in an industry of high technology occur. In order to achieve the proposed objective, the R&D project of the first microcontroller in the Brazilian semiconductor industry was defined as the object of analysis. The empirical choice is justified by the uniqueness of the case, besides bringing a diversity of actors and a level of complementarity of resources that were significant to the success of the project. Given the motivation to know who the actors were and what the main forms of interorganizational coordination were used in this project, interviews were carried out and a questionnaire was also made, besides other documents related to the project. The results presented show a network of nine actors and their roles in the interorganizational collaboration process, as well as the forms of social and temporal overlapping, used in the coordination of collective efforts. Focusing on the mechanisms of temporal and social integration highlighted throughout the study, the inclusion of R&D projects in the typology for interorganizational projects is proposed in this paper, which was also proposed by Jones and Lichtenstein (2008).

Development of a Manual Crack Quantification and Automated Crack Measurment System, Project TR-457 Final Report, 2005

Final report produced by DOT on development of manual crack quantification and automatic crack measurment system.