Amiodarone biokinetics, the formation of its major oxidative metabolite and neurotoxicity after acute and repeated exposure of brain cell cultures.

The difficulty in mimicking nervous system complexity and cell-cell interactions as well as the lack of kinetics information has limited the use of in vitro neurotoxicity data. Here, we assessed the biokinetic profile as well as the neurotoxicity of Amiodarone after acute and repeated exposure in two advanced rodent brain cell culture models, consisting of both neurons and glial cells organized in 2 or 3 dimensions to mimic the brain histiotypic structure and function. A strategy was applied to evidence the abiotic processes possibly affecting Amiodarone in vitro bioavailability, showing its ability to adsorb to the plastic devices. At clinically relevant Amiodarone concentrations, known to induce neurotoxicity in some patients during therapeutic treatment, a complete uptake was observed in both models in 24h, after single exposure. After repeated treatments, bioaccumulation was observed, especially in the 3D cell model, together with a greater alteration of neurotoxicity markers. After 14days, Amiodarone major oxidative metabolite (mono-N-desethylamiodarone) was detected at limited levels, indicating the presence of active drug metabolism enzymes (i.e. cytochrome P450) in both models. The assessment of biokinetics provides useful information on the relevance of in vitro toxicity data and should be considered in the design of an Integrated Testing Strategy aimed to identify specific neurotoxic alerts, and to improve the neurotoxicity assay predictivity for human acute and repeated exposure.

Between-year variation in population sex ratio increases with complexity of the breeding system in Hymenoptera.

While adaptive adjustment of sex ratio in the function of colony kin structure and food availability commonly occurs in social Hymenoptera, long-term studies have revealed substantial unexplained between-year variation in sex ratio at the population level. In order to identify factors that contribute to increased between-year variation in population sex ratio, we conducted a comparative analysis across 47 Hymenoptera species differing in their breeding system. We found that between-year variation in population sex ratio steadily increased as one moved from solitary species, to primitively eusocial species, to single-queen eusocial species, to multiple-queen eusocial species. Specifically, between-year variation in population sex ratio was low (6.6% of total possible variation) in solitary species, which is consistent with the view that in solitary species, sex ratio can vary only in response to fluctuations in ecological factors such as food availability. In contrast, we found significantly higher (19.5%) between-year variation in population sex ratio in multiple-queen eusocial species, which supports the view that in these species, sex ratio can also fluctuate in response to temporal changes in social factors such as queen number and queen-worker control over sex ratio, as well as factors influencing caste determination. The simultaneous adjustment of sex ratio in response to temporal fluctuations in ecological and social factors seems to preclude the existence of a single sex ratio optimum. The absence of such an optimum may reflect an additional cost associated with the evolution of complex breeding systems in Hymenoptera societies.

Kinematics and dynamic complexity of postural transitions in frail elderly subjects.

The aim of this study was to propose a methodology allowing a detailed characterization of body sit-to-stand/stand-to-sit postural transition. Parameters characterizing the kinematics of the trunk movement during sit-to-stand (Si-St) postural transition were calculated using one initial sensor system fixed on the trunk and a data logger. Dynamic complexity of these postural transitions was estimated by fractal dimension of acceleration-angular velocity plot. We concluded that this method provides a simple and accurate tool for monitoring frail elderly and to objectively evaluate the efficacy of a rehabilitation program.

Ubiquitination and the Ubiquitin-Proteasome System as regulators of transcription and transcription factorsin epithelial mesenchymal transition of cancer.

Epithelial to Mesenchymal Transition (EMT) in cancer is a process that allows cancer cells to detach from neighboring cells, become mobile and metastasize and shares many signaling pathways with development. Several molecular mechanisms which regulate oncogenic properties in neoplastic cells such as proliferation, resistance to apoptosis and angiogenesis through transcription factors or other mediators are also regulators of EMT. These pathways and downstream transcription factors are, in their turn, regulated by ubiquitination and the Ubiquitin-Proteasome System (UPS). Ubiquitination, the covalent link of the small 76-amino acid protein ubiquitin to target proteins, serves as a signal for protein degradation by the proteasome or for other outcomes such as endocytosis, degradation by the lysosome or directing these proteins to specific cellular compartments. This review discusses aspects of the regulation of EMT by ubiquitination and the UPS and underlines its complexity focusing on transcription and transcription factors regulating EMT and are being regulated by ubiquitination.

How useful is satellite positioning system (GPS) to track gait parameters? A review.

Over the last century, numerous techniques have been developed to analyze the movement of humans while walking and running. The combined use of kinematics and kinetics methods, mainly based on high speed video analysis and forceplate, have permitted a comprehensive description of locomotion process in terms of energetics and biomechanics. While the different phases of a single gait cycle are well understood, there is an increasing interest to know how the neuro-motor system controls gait form stride to stride. Indeed, it was observed that neurodegenerative diseases and aging could impact gait stability and gait parameters steadiness. From both clinical and fundamental research perspectives, there is therefore a need to develop techniques to accurately track gait parameters stride-by-stride over a long period with minimal constraints to patients. In this context, high accuracy satellite positioning can provide an alternative tool to monitor outdoor walking. Indeed, the high-end GPS receivers provide centimeter accuracy positioning with 5-20 Hz sampling rate: this allows the stride-by-stride assessment of a number of basic gait parameters--such as walking speed, step length and step frequency--that can be tracked over several thousand consecutive strides in free-living conditions. Furthermore, long-range correlations and fractal-like pattern was observed in those time series. As compared to other classical methods, GPS seems a promising technology in the field of gait variability analysis. However, relative high complexity and expensiveness--combined with a usability which requires further improvement--remain obstacles to the full development of the GPS technology in human applications.

Automation in clinical bacteriology: what system to choose?

With increased activity and reduced financial and human resources, there is a need for automation in clinical bacteriology. Initial processing of clinical samples includes repetitive and fastidious steps. These tasks are suitable for automation, and several instruments are now available on the market, including the WASP (Copan), Previ-Isola (BioMerieux), Innova (Becton-Dickinson) and Inoqula (KIESTRA) systems. These new instruments allow efficient and accurate inoculation of samples, including four main steps: (i) selecting the appropriate Petri dish; (ii) inoculating the sample; (iii) spreading the inoculum on agar plates to obtain, upon incubation, well-separated bacterial colonies; and (iv) accurate labelling and sorting of each inoculated media. The challenge for clinical bacteriologists is to determine what is the ideal automated system for their own laboratory. Indeed, different solutions will be preferred, according to the number and variety of samples, and to the types of sample that will be processed with the automated system. The final choice is troublesome, because audits proposed by industrials risk being biased towards the solution proposed by their company, and because these automated systems may not be easily tested on site prior to the final decision, owing to the complexity of computer connections between the laboratory information system and the instrument. This article thus summarizes the main parameters that need to be taken into account for choosing the optimal system, and provides some clues to help clinical bacteriologists to make their choice.

Prediction of 18-month survival in patients with primary myelodysplastic syndrome. A regression model and scoring system based on the combination of chromosome findings and the Bournemouth score.

The predictive potential of six selected factors was assessed in 72 patients with primary myelodysplastic syndrome using univariate and multivariate logistic regression analysis of survival at 18 months. Factors were age (above median of 69 years), dysplastic features in the three myeloid bone marrow cell lineages, presence of chromosome defects, all metaphases abnormal, double or complex chromosome defects (C23), and a Bournemouth score of 2, 3, or 4 (B234). In the multivariate approach, B234 and C23 proved to be significantly associated with a reduction in the survival probability. The similarity of the regression coefficients associated with these two factors means that they have about the same weight. Consequently, the model was simplified by counting the number of factors (0, 1, or 2) present in each patient, thus generating a scoring system called the Lausanne-Bournemouth score (LB score). The LB score combines the well-recognized and easy-to-use Bournemouth score (B score) with the chromosome defect complexity, C23 constituting an additional indicator of patient outcome. The predicted risk of death within 18 months calculated from the model is as follows: 7.1% (confidence interval: 1.7-24.8) for patients with an LB score of 0, 60.1% (44.7-73.8) for an LB score of 1, and 96.8% (84.5-99.4) for an LB score of 2. The scoring system presented here has several interesting features. The LB score may improve the predictive value of the B score, as it is able to recognize two prognostic groups in the intermediate risk category of patients with B scores of 2 or 3. It has also the ability to identify two distinct prognostic subclasses among RAEB and possibly CMML patients. In addition to its above-described usefulness in the prognostic evaluation, the LB score may bring new insights into the understanding of evolution patterns in MDS. We used the combination of the B score and chromosome complexity to define four classes which may be considered four possible states of myelodysplasia and which describe two distinct evolutional pathways.

Validated prediction of clinical outcome in sarcomas and multiple types of cancer on the basis of a gene expression signature related to genome complexity.

Sarcomas are heterogeneous and aggressive mesenchymal tumors. Histological grading has so far been the best predictor for metastasis-free survival, but it has several limitations, such as moderate reproducibility and poor prognostic value for some histological types. To improve patient grading, we performed genomic and expression profiling in a training set of 183 sarcomas and established a prognostic gene expression signature, complexity index in sarcomas (CINSARC), composed of 67 genes related to mitosis and chromosome management. In a multivariate analysis, CINSARC predicts metastasis outcome in the training set and in an independent 127 sarcomas validation set. It is superior to the Fédération Francaise des Centres de Lutte Contre le Cancer grading system in determining metastatic outcome for sarcoma patients. Furthermore, it also predicts outcome for gastrointestinal stromal tumors (GISTs), breast carcinomas and lymphomas. Application of the signature will permit more selective use of adjuvant therapies for people with sarcomas, leading to decreased iatrogenic morbidity and improved outcomes for such individuals.

Large scale assessment of regulatory evolution and transcriptome complexity in mammals

AbstractIn addition to genetic changes affecting the function of gene products, changes in gene expression have been suggested to underlie many or even most of the phenotypic differences among mammals. However, detailed gene expression comparisons were, until recently, restricted to closely related species, owing to technological limitations. Thus, we took advantage of the latest technologies (RNA-Seq) to generate extensive qualitative and quantitative transcriptome data for a unique collection of somatic and germline tissues from representatives of all major mammalian lineages (placental mammals, marsupials and monotremes) and birds, the evolutionary outgroup.In the first major project of my thesis, we performed global comparative analyses of gene expression levels based on these data. Our analyses provided fundamental insights into the dynamics of transcriptome change during mammalian evolution (e.g., the rate of expression change across species, tissues and chromosomes) and allowed the exploration of the functional relevance and phenotypic implications of transcription changes at a genome-wide scale (e.g., we identified numerous potentially selectively driven expression switches).In a second project of my thesis, which was also based on the unique transcriptome data generated in the context of the first project we focused on the evolution of alternative splicing in mammals. Alternative splicing contributes to transcriptome complexity by generating several transcript isoforms from a single gene, which can, thus, perform various functions. To complete the global comparative analysis of gene expression changes, we explored patterns of alternative splicing evolution. This work uncovered several general and unexpected patterns of alternative splicing evolution (e.g., we found that alternative splicing evolves extremely rapidly) as well as a large number of conserved alternative isoforms that may be crucial for the functioning of mammalian organs.Finally, the third and final project of my PhD consisted in analyzing in detail the unique functional and evolutionary properties of the testis by exploring the extent of its transcriptome complexity. This organ was previously shown to evolve rapidly both at the phenotypic and molecular level, apparently because of the specific pressures that act on this organ and are associated with its reproductive function. Moreover, my analyses of the amniote tissue transcriptome data described above, revealed strikingly widespread transcriptional activity of both functional and nonfunctional genomic elements in the testis compared to the other organs. To elucidate the cellular source and mechanisms underlying this promiscuous transcription in the testis, we generated deep coverage RNA-Seq data for all major testis cell types as well as epigenetic data (DNA and histone methylation) using the mouse as model system. The integration of these complete dataset revealed that meiotic and especially post-meiotic germ cells are the major contributors to the widespread functional and nonfunctional transcriptome complexity of the testis, and that this "promiscuous" spermatogenic transcription is resulting, at least partially, from an overall transcriptionally permissive chromatin state. We hypothesize that this particular open state of the chromatin results from the extensive chromatin remodeling that occurs during spermatogenesis which ultimately leads to the replacement of histones by protamines in the mature spermatozoa. Our results have important functional and evolutionary implications (e.g., regarding new gene birth and testicular gene expression evolution).Generally, these three large-scale projects of my thesis provide complete and massive datasets that constitute valuables resources for further functional and evolutionary analyses of mammalian genomes.

Case complexity and clinical outcome in diabetes mellitus. A prospective study using the INTERMED.

The aim of this study was to assess a population of patients with diabetes mellitus by means of the INTERMED, a classification system for case complexity integrating biological, psychosocial and health care related aspects of disease. The main hypothesis was that the INTERMED would identify distinct clusters of patients with different degrees of case complexity and different clinical outcomes. Patients (n=61) referred to a tertiary reference care centre were evaluated with the INTERMED and followed 9 months for HbA1c values and 6 months for health care utilisation. Cluster analysis revealed two clusters: cluster 1 (62%) consisting of complex patients with high INTERMED scores and cluster 2 (38%) consisting of less complex patients with lower INTERMED. Cluster 1 patients showed significantly higher HbA1c values and a tendency for increased health care utilisation. Total INTERMED scores were significantly related to HbA1c and explained 21% of its variance. In conclusion, different clusters of patients with different degrees of case complexity were identified by the INTERMED, allowing the detection of highly complex patients at risk for poor diabetes control. The INTERMED therefore provides an objective basis for clinical and scientific progress in diabetes mellitus. Ongoing intervention studies will have to confirm these preliminary data and to evaluate if management strategies based on the INTERMED profiles will improve outcomes.

Das GRADE-System: ein internationaler Ansatz zur Vereinheitlichung der Graduierung von Evidenz und Empfehlungen in Leitlinien. [The GRADE System: an international approach to standardize the graduation of evidence and recommendations in guidelines]

Clinical practice guidelines have become an important source of information to support clinicians in the management of individual patients. However, current guideline methods have limitations that include the lack of separating the quality of evidence from the strength of recommendations. The Grading of Recommendations, Assessment, Development and Evaluation (GRADE) working group, an international collaboration of guideline developers, methodologists, and clinicians have developed a system that addresses these shortcomings. Core elements include transparent methodology for grading the quality of evidence, the distinction between quality of the evidence and strength of a recommendation, an explicit balancing of benefits and harms of health care interventions, an explicit recognition of the values and preferences that underlie recommendations. The GRADE system has been piloted in various practice settings to ensure that it captures the complexity involved in evidence assessment and grading recommendations while maintaining simplicity and practicality. Many guideline organizations and medical societies have endorsed the system and adopted it for their guideline processes.

From pathogen defence to food allergy; The multiple facets of the mucosal immune system

Résumé destiné à un large public Le système immunitaire associé aux muqueuses gastro-intestinales doit être capable de protéger notre organisme contre l'invasion de pathogènes. Parallèlement, il doit identifier en Cant que tels, des composés inoffensifs comme la nourriture ou les milliards de bactéries qui résident dans notre intestin. Le travail présenté ici aborde ces deux aspects essentiels au bon fonctionnement de notre muqueuse intestinale. Dans une première partie, la protéine nommée pièce sécrétoire a été étudiée pour ses propriétés protectrices contre le pathogène viral rotavirus. Le rôle de la pièce sécrétoire est de transporter les anticorps que nous produisons vers la surface des muqueuses. En dehors de cette fonction bien connue, il se peut que cette protéine soit également capable de protéger notre organisme contre certains virus. L'hypothèse de travail était donc que la pièce sécrétoire se lie directement au virus, l'empêchant ainsi d'infecter des cellules épithéliales de l'intestin. En utilisant différentes techniques biochimiques, cette hypothèse s'est révélée fausse car aucune interaction entre la pièce sécrétoire et le virus n'a pu être observée, et logiquement, aucune protection n'a pu prendre place. En revanche, la pièce sécrétoire se lie à d'autres structures pathogéniques et permet ainsi de neutraliser leurs effets néfastes. La pièce sécrétoire participe donc activement à la protection de nos muqueuses, en plus de son rôle de transporteur. La deuxième partie de ce travail avait pour sujet les réactions inappropriées que le système immunitaire induit parfois contre un aliment, ou, autrement dit, les allergies alimentaires. Un modèle d'allergie alimentaire à donc été développé chez la souris et a permis de mesurer plusieurs symptômes et facteurs liés à l'allergie. Puis, ce modèle a été utilisé afin de tester les effets bénéfiques d'une bactérie lactique, dite probiotique, sur le développement de l'allergie. Il a été observé que, sous certaines circonstances, l'administration de la bactérie lactique protégeait entièrement les souris contre les réactions allergiques. L'effet bénéfique dépend donc du probiotique mais également d'autres facteurs encore inconnus â ce jour. Cette étude ouvre la voie sur la compréhension des mécanismes liés aux allergies alimentaires et sur l'impact que peuvent avoir les bactéries probiotiques sur cette maladie. Résumé Le système immunitaire associé aux muqueuses intestinales doit être capable de différencier les antigènes inoffensifs tels que 1a nourriture ou les bactéries commensales des microorganismes potentiellement dangereux. Cet aspect est essentiel pour le maintien de l'homéostase intestinale et fait l'objet du travail présenté ici. Dans un premier projet, les propriétés protectrices de la protéine appelée pièce sécrétoire (SC) ont été étudiées. SC est une protéine connue pour le transport des immunoglobulines à la surface des muqueuses. Cette protéine est fortement glycosylée paz des sucres complexes, ce qui nous a mené à postuler que SC puisse interagir avec le pathogène rotavirus. Cette hypothèse était soutenue par le fait que ce virus adhère aux cellules épithéliales par des résidus glycosylés. Des analyses biochimiques et biologiques ont démontré qu'aucune interaction entre SC et le virus ne prenait place, et que par conséquent SC n'offrait aucune protection contre ce pathogène. En revanche, SC interagit avec d'autres structures pathogéniques, comme la toxine A de Clostridium difficile, et la molécule d'adhésion intimine de la bactérie entéropathogène Escherichia coli. La liaison se fait par l'intermédiaire des sucres et confère ainsi une protection contre ces pathogènes. Ainsi, SC a été identifié comme agent neutralisant au niveau de l'intestin. La deuxième partie de ce travail abordait le sujet des allergies alimentaires, et avait pour but de tester les effets bénéfiques potentiels d'une bactérie probiotique, Lactobacillus paracasei NCC2461, contre les réactions allergiques. Un modèle marin d'allergie alimentaire a été mis au point, permettant de mesurer des immunoglobulines E, des symptômes allergiques, et la dégranulation de mastocytes. Lorsque le probiotique a été administré aux souris, celles-ci ont été complètement protégées des réactions allergiques dans une première expérience. Cependant, cette protection n'a pas été reproduite et suggère que des facteurs environnementaux encore inconnus sont critiques pour que le probiotique agisse positivement. Ce travail a permis de mettre en évidence la complexité de l'approche des traitements liés aux probiotiques et ouvre la voie sur la compréhension des mécanismes liés à l'allergie. Abstract The mucosal immune system associated to the gastrointestinal mucosa must efficiently distinguish between innocuous antigens, such as food proteins and commensal bacteria and potentially infectious agents. The work presented here deals with these two essential aspects guaranteeing intestinal homeostasis. In the first part of this work, the protective properties of secretory component (SC) toward the pathogen rotavirus were investigated. SC, which allows the transport of polymeric immunoglobulins (Ig) to mucosal surfaces, is highly glycosylated with complex glycan structures. The abundance and the nature of these carbohydrates led us to speculate that SC might interact with rotavirus, which is known to bind target cells with glycan receptors. Using various biological and biochemical techniques, we demonstrated that SC did not interact with rotaviruses, nor protected epithelial cells from infection. However, SC was shown to bind to Clostridium difficile toxin A and to the enteropathogenic Echerischia coli adhesion molecule intimin in a glycan-dependent fashion. These interactions allow in vitro protection of epithelial cells using physiological concentrations of SC. These data identify SC as a microbial scavenger at mucosal surfaces, and in the context of secretory IgA, further enhance the neutralising properties of the complex. The second project was inscribed in the domain of food allergy and aimed to test the modulatory functions of a probiotic strain of Lactobacillus paracasei toward allergic reactions. A model of food-mediated allergy was developed in the mouse using mucosal sensitisation. Several parameters associated to allergy were quantified after allergen challenge, and included allergen-specific IgE, allergic signs like diarrhea and temperature drop, and degranulation of mast cells. Administration of the probiotic strain was shown to completely protect mice from allergic reactions. However, these data were not reproduced, suggesting that unknown environmental factors are required so that protection mediated by the probiotic strain occurs. This study paves the way to the understanding of the mechanisms associated to allergy, and highlights the tremendous complexity that probiotic treatments will have to face.