Quantification of nicotine, cotinine, trans-3'-hydroxycotinine and varenicline in human plasma by a sensitive and specific UPLC-tandem mass-spectrometry procedure for a clinical study on smoking cessation.

A sensitive and specific ultra performance liquid chromatography-tandem mass spectrometry method for the simultaneous quantification of nicotine, its metabolites cotinine and trans-3'-hydroxycotinine and varenicline in human plasma was developed and validated. Sample preparation was realized by solid phase extraction of the target compounds and of the internal standards (nicotine-d4, cotinine-d3, trans-3'-hydroxycotinine-d3 and CP-533,633, a structural analog of varenicline) from 0.5mL of plasma, using a mixed-mode cation exchange support. Chromatographic separations were performed on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1×100mm, 1.7μm). A gradient program was used, with a 10mM ammonium formate buffer pH 3/acetonitrile mobile phase at a flow of 0.4mL/min. The compounds were detected on a triple quadrupole mass spectrometer, operated with an electrospray interface in positive ionization mode and quantification was performed using multiple reaction monitoring. Matrix effects were quantitatively evaluated with success, with coefficients of variation inferior to 8%. The procedure was fully validated according to Food and Drug Administration guidelines and to Société Française des Sciences et Techniques Pharmaceutiques. The concentration range was 2-500ng/mL for nicotine, 1-1000ng/mL for cotinine, 2-1000ng/mL for trans-3'-hydroxycotinine and 1-500ng/mL for varenicline, according to levels usually measured in plasma. Trueness (86.2-113.6%), repeatability (1.9-12.3%) and intermediate precision (4.4-15.9%) were found to be satisfactory, as well as stability in plasma. The procedure was successfully used to quantify nicotine, its metabolites and varenicline in more than 400 plasma samples from participants in a clinical study on smoking cessation.

Reconstituted human polyclonal plasma-derived secretory-like IgM and IgA maintain the barrier function of epithelial cells infected with an enteropathogen.

Intravenous administration of polyclonal and monoclonal antibodies has proven to be a clinically valid approach in the treatment, or at least relief, of many acute and chronic pathologies, such as infection, immunodeficiency, and a broad range of autoimmune conditions. Plasma-derived IgG or recombinant IgG are most frequently used for intravenous or subcutaneous administration, whereas a few IgM-based products are available as well. We have established recently that secretory-like IgA and IgM can be produced upon association of plasma-derived polymeric IgA and IgM with a recombinant secretory component. As a next step toward potential future mucosal administration, we sought to unravel the mechanisms by which these secretory Igs protect epithelial cells located at the interface between the environment and the inside of the body. By using polarized epithelial Caco-2 cell monolayers and Shigella flexneri as a model enteropathogen, we found that polyspecific plasma-derived SIgA and SIgM fulfill many protective functions, including dose-dependent recognition of the antigen via formation of aggregated immune complexes, reduction of bacterial infectivity, maintenance of epithelial cell integrity, and inhibition of proinflammatory cytokine/chemokine production by epithelial cells. In this in vitro model devoid of other cellular or molecular interfering partners, IgM and secretory IgM showed stronger bacterial neutralization than secretory IgA. Together, these data suggest that mucosally delivered antibody preparations may be most effective when combining both secretory-like IgA and IgM, which, together, play a crucial role in preserving several levels of epithelial cell integrity.

Assessing protein stability of the dimeric DNA-binding domain of E2 human papillomavirus 18 with molecular dynamics

The objective of this study is to understand the structural flexibility and curvature of the E2 protein of human papillomavirus type 18 using molecular dynamics (6 ns). E2 is required for viral DNA replication and its disruption could be an anti-viral strategy. E2 is a dimer, with each monomer folding into a stable open-faced β-sandwich. We calculated the mobility of the E2 dimer and found that it was asymmetric. These different mobilities of E2 monomers suggest that drugs or vaccines could be targeted to the interface between the two monomers.

Magnetic pill tracking : a novel non-invasive tool for investigation of human digestive motility

RESUME Les améliorations méthodologiques des dernières décennies ont permis une meilleure compréhension de la motilité gastro-intestinale. Il manque toutefois une méthode qui permette de suivre la progression du chyme le long du tube gastro-intestinal. Pour permettre l'étude de la motilité de tout le tractus digestif humain, une nouvelle technique, peu invasive, a été élaborée au Département de Physiologie, en collaboration avec l'EPFL. Appelée "Magnet Tracking", la technique est basée sur la détection du champ magnétique généré par des matériaux ferromagnétiques avalés. A cet usage, une pilule magnétique, une matrice de capteurs et un logiciel ont été développés. L'objet de ce travail est de démontrer la faisabilité d'un examen de la motilité gastro-intestinale chez l'Homme par cette méthode. L'aimant est un cylindre (ø 6x7 mm, 0.2 cm3) protégé par une gaine de silicone. Le système de mesure est constitué d'une matrice de 4x4 capteurs et d'un ordinateur portable. Les capteurs fonctionnent sur l'effet Hall. Grâce à l'interface informatique, l'évolution de la position de l'aimant est suivie en temps réel à travers tout le tractus digestif. Sa position est exprimée en fonction du temps ou reproduite en 3-D sous forme d'une trajectoire. Différents programmes ont été crées pour analyser la dynamique des mouvements de l'aimant et caractériser la motilité digestive. Dix jeunes volontaires en bonne santé ont participé à l'étude. L'aimant a été avalé après une nuit de jeûne et son séjour intra digestif suivi pendant 2 jours consécutifs. Le temps moyen de mesure était de 34 heures. Chaque sujet a été examiné une fois sauf un qui a répété sept fois l'expérience. Les sujets restaient en décubitus dorsal, tranquilles et pouvaient interrompre la mesure s'ils le désiraient. Ils sont restés à jeûne le premier jour. L'évacuation de l'aimant a été contrôlée chez tous les sujets. Tous les sujets ont bien supporté l'examen. Le marqueur a pu être détecté de l'oesophage au rectum. La trajectoire ainsi constituée représente une conformation de l'anatomie digestive : une bonne superposition de celle-ci à l'anatomie est obtenue à partir des images de radiologie conventionnelle (CT-scan, lavement à la gastrografine). Les mouvements de l'aimant ont été caractérisés selon leur périodicité, leur amplitude ou leur vitesse pour chaque segment du tractus digestif. Ces informations physiologiques sont bien corrélées à celles obtenues par des méthodes établies d'étude de la motilité gastro-intestinale. Ce travail démontre la faisabilité d'un examen de la motilité gastro-intestinal chez l'Homme par la méthode de Magnet Tracking. La technique fournit les données anatomiques et permet d'analyser en temps réel la dynamique des mouvements du tube digestif. Cette méthode peu invasive ouvre d'intéressantes perspectives pour l'étude de motilité dans des conditions physiologiques et pathologiques. Des expériences visant à valider cette approche en tant que méthode clinique sont en voie de réalisation dans plusieurs centres en Suisse et à l'étranger. SUMMARY Methodological improvements realised over the last decades have permitted a better understanding of gastrointestinal motility. Nevertheless, a method allowing a continuous following of lumina' contents is still lacking. In order to study the human digestive tract motility, a new minimally invasive technique was developed at the Department of Physiology in collaboration with Swiss Federal Institute of Technology. The method is based on the detection of magnetic field generated by swallowed ferromagnetic materials. The aim of our work was to demonstrate the feasibility of this new approach to study the human gastrointestinal motility. The magnet used was a cylinder (ø6x7mm, 0.2 cm3) coated with silicon. The magnet tracking system consisted of a 4x4 matrix of sensors based on the Hall effect Signals from the sensors were digitised and sent to a laptop computer for processing and storage. Specific software was conceived to analyse in real time the progression of the magnet through the gastrointestinal tube. Ten young and healthy volunteers were enrolled in the study. After a fasting period of 12 hours, they swallowed the magnet. The pill was then tracked for two consecutive days for 34 hours on average. Each subject was studied once except one who was studied seven times. Every subject laid on his back for the entire experiment but could interrupt it at anytime. Evacuation of the magnet was controlled in all subjects. The examination was well tolerated. The pill could be followed from the esophagus to the rectum. The trajectory of the magnet represented a "mould" of the anatomy of the digestive tube: a good superimposition with radiological anatomy (gastrografin contrast and CT) was obtained. Movements of the magnet were characterized by periodicity, velocity, and amplitude of displacements for every segment of the digestive tract. The physiological information corresponded well to data from current methods of studying gastrointestinal motility. This work demonstrates the feasibility of the new approach in studies of human gastrointestinal motility. The technique allows to correlate in real time the dynamics of digestive movements with the anatomical data. This minimally invasive method is ready for studies of human gastrointestinal motility under physiological as well as pathological conditions. Studies aiming at validation of this new approach as a clinically relevant tool are being realised in several centres in Switzerland and abroad. Abstract: A new minimally invasive technique allowing for anatomical mapping and motility studies along the entire human digestive system is presented. The technique is based on continuous tracking of a small magnet progressing through the digestive tract. The coordinates of the magnet are calculated from signals recorded by 16 magnetic field sensors located over the abdomen. The magnet position, orientation and trajectory are displayed in real time. Ten young healthy volunteers were followed during 34 h. The technique was well tolerated and no complication was encountered, The information obtained was 3-D con-figuration of the digestive tract and dynamics of the magnet displacement (velocity, transit time, length estimation, rhythms). In the same individual, repea-ted examination gave very reproducible results. The anatomical and physiological information obtained corresponded well to data from current methods and imaging. This simple, minimally invasive technique permits examination of the entire digestive tract and is suitable for both research and clinical studies. In combination with other methods, it may represent a useful tool for studies of Cl motility with respect to normal and pathological conditions.

Ultra high throughput sequencing in human DNA variation detection: a comparative study on the NDUFA3-PRPF31 region.

BACKGROUND: Ultra high throughput sequencing (UHTS) technologies find an important application in targeted resequencing of candidate genes or of genomic intervals from genetic association studies. Despite the extraordinary power of these new methods, they are still rarely used in routine analysis of human genomic variants, in part because of the absence of specific standard procedures. The aim of this work is to provide human molecular geneticists with a tool to evaluate the best UHTS methodology for efficiently detecting DNA changes, from common SNPs to rare mutations. METHODOLOGY/PRINCIPAL FINDINGS: We tested the three most widespread UHTS platforms (Roche/454 GS FLX Titanium, Illumina/Solexa Genome Analyzer II and Applied Biosystems/SOLiD System 3) on a well-studied region of the human genome containing many polymorphisms and a very rare heterozygous mutation located within an intronic repetitive DNA element. We identify the qualities and the limitations of each platform and describe some peculiarities of UHTS in resequencing projects. CONCLUSIONS/SIGNIFICANCE: When appropriate filtering and mapping procedures are applied UHTS technology can be safely and efficiently used as a tool for targeted human DNA variations detection. Unless particular and platform-dependent characteristics are needed for specific projects, the most relevant parameter to consider in mainstream human genome resequencing procedures is the cost per sequenced base-pair associated to each machine.

Enjeux éthiques des nouvelles technologies de la reproduction : une conception de la justice à l'interface de l'éthique du "care" et de l'approche des "capabilités"

L'émergence des nouvelles technologies de la reproduction (NTR) est allée de pair avec un certain nombre de discours. Un discours promettant d'une part une extension de la palette de choix reproductifs des individus, une extension de leur liberté et de leur autonomie reproductives, dont la forme la plus extrême peut se traduire par la formule : un enfant quand je veux et comme je veux. D'autre part, un discours annonçant une série de « catastrophes » à venir, telles que l'effondrement de l'institution de la famille et la modification de l'espèce humaine. En d'autres termes, une tension entre promesses et catastrophes qui place les sociétés contemporaines face à de nombreux défis sociaux, politiques et éthiques, notamment quant à la question de la régulation de la PMA (procréation médicalement assistée) : qui peut y avoir accès ? Quelles techniques doit-on autoriser ? Ou au contraire limiter ? Tant de questions auxquelles aucune réponse simple et évidente n'existe. La diversité des réponses législatives quant à ces questions illustre cette complexité. L'éthique peut, ici, jouer un rôle fondamental. Sans toutefois prétendre donner des réponses toutes faites et facilement applicables, elle offre un espace de réflexion, le privilège de prendre une certaine distance face à des enjeux contemporains. C'est dans cette perspective que nous avons ancré ce travail de recherche en questionnant les enjeux éthiques de la PMA à partir d'une perspective de justice. Toutefois, au sein des études en bioéthique, majoritairement issues de la tradition libérale, la tension énoncée précédemment mène la bioéthique à justifier un certain nombre d'inégalités plutôt que de veiller à les dépasser. Ainsi, une évaluation de la pratique de la PMA à partir d'une perspective de la justice, exige, au préalable, une réévaluation du concept même de justice. Ce faisant, par une articulation entre l'éthique du care de Joan Tronto et l'approche des capabilités de Martha Nussbaum qui placent la vulnérabilité au coeur de la personne, nous avons proposé une conception de la justice fondée sur une anthropologie de la vulnérabilité. Cette conception nous permet d'identifier, dans le cadre de la pratique de la PMA en Suisse et en partant de la loi sur la procréation assistée (LPMA), les constructions normatives qui mènent à la non-reconnaissance et, ce faisant, à la mise à l'écart, de certaines formes de vulnérabilité : une vulnérabilité générique et une vulnérabilité socio-économique. Traitant la question de la vulnérabilité générique principalement, nos analyses ont une incidence sur les conceptions de la famille, du bien de l'enfant, de la femme et de la nature, telles qu'elles sont actuellement véhiculées par une conception naturalisée de la PMA. Répondre aux vulnérabilités identifiées, en veillant à leur donner une place, signifie alors déplacer ces conceptions naturalisées, afin que les vulnérabilités soient intégrées aux pratiques sociales et que les exigences de justice soient ainsi remplies. - The emergence of assisted reproductive technologies (ART) came along with several discourses. On the one hand a discourse promising an extension of the individuals' reproductive choices, their procreative liberty and autonomy. On the other hand a discourse announced a series of disasters to come such as the collapse of the family institution and the modification of human kind. In other words, a growing tension appears between promises and disasters and contemporary societies are facing inevitable social, political and ethical challenges, in particular with regard to the issue of ART regulation: who has access? What procedures should be authorized? Which ones should be limited? These complex questions have no simple or obvious answers. The variety of legislative responses to these questions highlights complexity. Ethics can play a fundamental role, and without claiming to give simple answers, also offer a space for reflection as well as the privilege to distance itself with regard to contemporary issues. It is in this perspective that this study questions the ethical considerations of ART in a perspective of justice. However, in previous studies in bioethics mainly following a liberal tradition, previously mentioned tension has lead bioethics to justify some inequalities instead of trying to overcome them. As a consequence, evaluating practices of ART from a perspective of justice requires to first reevaluate the concept of justice itself. In doing so we offer a conception of justice founded on the anthropology of vulnerability. This conception draws on an articulation of the ethic of care of Joan Tronto and the capability approach of Martha Nussbaum, which places vulnerability at the center of the person. This conception allows us to identify, within the framework of ARTS in Switzerland and starting with the laws of medically assisted procreation (LPMA), some normative constructions. These constructions lead to the non-recognition and the disregard of some forms of vulnerability: a generic vulnerability as well as socio-economic counterpart. Focusing mainly on the issue of generic vulnerability, our analysis has implications for the conceptions of family, the best interests of the child, woman, and nature in the way they are defined in a naturalized conception of ART. Responding to such failures by taking into account these vulnerabilities thus means to move these conceptions in order for vulnerabilities to be integrated in social practices and requirements for justice to be fulfilled.

Quantification of typical antipsychotics in human plasma by ultra-high performance liquid chromatography tandem mass spectrometry for therapeutic drug monitoring.

A selective and sensitive method was developed for the simultaneous quantification of seven typical antipsychotic drugs (cis-chlorprothixene, flupentixol, haloperidol, levomepromazine, pipamperone, promazine and zuclopenthixol) in human plasma. Ultra-high performance liquid chromatography (UHPLC) was used for complete separation of the compounds in less than 4.5min on an Acquity UPLC BEH C18 column (2.1mm×50mm; 1.7μm), with a gradient elution of ammonium formate buffer pH 4.0 and acetonitrile at a flow rate of 400μl/min. Detection was performed on a tandem quadrupole mass spectrometer (MS/MS) equipped with an electrospray ionization interface. A simple protein precipitation procedure with acetonitrile was used for sample preparation. Thanks to the use of stable isotope-labeled internal standards for all analytes, internal standard-normalized matrix effects were in the range of 92-108%. The method was fully validated to cover large concentration ranges of 0.2-90ng/ml for haloperidol, 0.5-90ng/ml for flupentixol, 1-450ng/ml for levomepromazine, promazine and zuclopenthixol and 2-900ng/ml for cis-chlorprothixene and pipamperone. Trueness (89.1-114.8%), repeatability (1.8-9.9%), intermediate precision (1.9-16.3%) and accuracy profiles (<30%) were in accordance with the latest international recommendations. The method was successfully used in our laboratory for routine quantification of more than 500 patient plasma samples for therapeutic drug monitoring. To the best of our knowledge, this is the first UHPLC-MS/MS method for the quantification of the studied drugs with a sample preparation based on protein precipitation.

Personalized modeling for drug concentration prediction using Support Vector Machine

Building a personalized model to describe the drug concentration inside the human body for each patient is highly important to the clinical practice and demanding to the modeling tools. Instead of using traditional explicit methods, in this paper we propose a machine learning approach to describe the relation between the drug concentration and patients' features. Machine learning has been largely applied to analyze data in various domains, but it is still new to personalized medicine, especially dose individualization. We focus mainly on the prediction of the drug concentrations as well as the analysis of different features' influence. Models are built based on Support Vector Machine and the prediction results are compared with the traditional analytical models.

PANACEA (Platform for Automatic, Normalised Annotation and Cost-Effective Acquisition of Language Resources for Human Language Technologies)

The objective of PANACEA is to build a factory of LRs that automates the stages involved in the acquisition, production, updating and maintenance of LRs required by MT systems and by other applications based on language technologies, and simplifies eventual issues regarding intellectual property rights. This automation will cut down the cost, time and human effort significantly. These reductions of costs and time are the only way to guarantee the continuous supply of LRs that MT and other language technologies will be demanding in the multilingual Europe.

Towards a user-friendly webservice architecture for statistical machine translation in the PANACEA project

This paper presents a webservice architecture for Statistical Machine Translation aimed at non-technical users. A workfloweditor allows a user to combine different webservices using a graphical user interface. In the current state of this project,the webservices have been implemented for a range of sentential and sub-sententialaligners. The advantage of a common interface and a common data format allows the user to build workflows exchanging different aligners.

Monoubiquitination and Activity of the Paracaspase MALT1 Requires Glutamate 549 in the Dimerization Interface.

The mucosa-associated lymphoid tissue protein-1 (MALT1, also known as paracaspase) is a protease whose activity is essential for the activation of lymphocytes and the growth of cells derived from human diffuse large B-cell lymphomas of the activated B-cell subtype (ABC DLBCL). Crystallographic approaches have shown that MALT1 can form dimers via its protease domain, but why dimerization is relevant for the biological activity of MALT1 remains largely unknown. Using a molecular modeling approach, we predicted Glu 549 (E549) to be localized within the MALT1 dimer interface and thus potentially relevant. Experimental mutation of this residue into alanine (E549A) led to a complete impairment of MALT1 proteolytic activity. This correlated with an impaired capacity of the mutant to form dimers of the protease domain in vitro, and a reduced capacity to promote NF-κB activation and transcription of the growth-promoting cytokine interleukin-2 in antigen receptor-stimulated lymphocytes. Moreover, this mutant could not rescue the growth of ABC DLBCL cell lines upon MALT1 silencing. Interestingly, the MALT1 mutant E549A was unable to undergo monoubiquitination, which we identified previously as a critical step in MALT1 activation. Collectively, these findings suggest a model in which E549 at the dimerization interface is required for the formation of the enzymatically active, monoubiquitinated form of MALT1.

The State of the Immigrant Body and the Body of the State: Negotiations at the Interface

Using an ethnographic analysis of the social interfaces between state agents and Cape Verdean students in Portugal, observed through participant observation in medical appointments, social work, immigration services and legal support to immigrants, this article aims to examine disciplinary state practices and the negotiations and power struggles that take place. The ethnographic cases discussed demonstrate how the idea of a fair and neutral state is simultaneously reproduced and denied in practice, thus elucidating the state as a symbol of union of an effective disunity. The ethnographic examples also indicate other dimensions of state practice, besides micro-disciplinary powers, which create room for flexibility and adaptation. And it is in this sense that ethnographies of interfaces between state and citizen offer a more relative perspective of excessively systematic interpretations of governmentality, illustrating how the effects of contradictory state practices are as unpredictable as human action itself.

Reactivation of protein aggregates by mortalin and Tid1-the human mitochondrial Hsp70 chaperone system.

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.

OpenFluDB, a database for human and animal influenza virus.

Although research on influenza lasted for more than 100 years, it is still one of the most prominent diseases causing half a million human deaths every year. With the recent observation of new highly pathogenic H5N1 and H7N7 strains, and the appearance of the influenza pandemic caused by the H1N1 swine-like lineage, a collaborative effort to share observations on the evolution of this virus in both animals and humans has been established. The OpenFlu database (OpenFluDB) is a part of this collaborative effort. It contains genomic and protein sequences, as well as epidemiological data from more than 27,000 isolates. The isolate annotations include virus type, host, geographical location and experimentally tested antiviral resistance. Putative enhanced pathogenicity as well as human adaptation propensity are computed from protein sequences. Each virus isolate can be associated with the laboratories that collected, sequenced and submitted it. Several analysis tools including multiple sequence alignment, phylogenetic analysis and sequence similarity maps enable rapid and efficient mining. The contents of OpenFluDB are supplied by direct user submission, as well as by a daily automatic procedure importing data from public repositories. Additionally, a simple mechanism facilitates the export of OpenFluDB records to GenBank. This resource has been successfully used to rapidly and widely distribute the sequences collected during the recent human swine flu outbreak and also as an exchange platform during the vaccine selection procedure. Database URL: http://openflu.vital-it.ch.

Type I IFNs at the interface between cutaneous immunity and epidermal remodeling.

Type I IFNs are key cytokines in antiviral host defense. Preferentially expressed by plasmacytoid dendritic cells, type I IFNs are induced by viral infection and in common skin wounds. In this issue, Tohyama et al. identify a new link between type I IFNs and epidermal remodeling, by showing that type I IFNs specifically upregulate IL-22R expression on keratinocytes and, thereby, IL-22-mediated Stat3 phosphorylation in keratinocytes. The findings suggest that type I IFNs play dual roles in human skin: first, they induce immune activation with the induction of IL-22-producing T cells; second, they provide the interface between immune activation and epidermal remodeling by increasing keratinocyte responsiveness to IL-22.