First-generation linkage map for the European tree frog (Hyla arborea) with utility in congeneric species.

BACKGROUND: Western Palearctic tree frogs (Hyla arborea group) represent a strong potential for evolutionary and conservation genetic research, so far underexploited due to limited molecular resources. New microsatellite markers have recently been developed for Hyla arborea, with high cross-species utility across the entire circum-Mediterranean radiation. Here we conduct sibship analyses to map available markers for use in future population genetic applications. FINDINGS: We characterized eight linkage groups, including one sex-linked, all showing drastically reduced recombination in males compared to females, as previously documented in this species. Mapping of the new 15 markers to the ~200 My diverged Xenopus tropicalis genome suggests a generally conserved synteny with only one confirmed major chromosome rearrangement. CONCLUSIONS: The new microsatellites are representative of several chromosomes of H. arborea that are likely to be conserved across closely-related species. Our linkage map provides an important resource for genetic research in European Hylids, notably for studies of speciation, genome evolution and conservation.

Generation and validation of a normative, age-specific reference curve for lumbar spine trabecular bone score (TBS) in French women.

Age-related changes in lumbar vertebral microarchitecture are evaluated, as assessed by trabecular bone score (TBS), in a cohort of 5,942 French women. The magnitude of TBS decline between 45 and 85 years of age is piecewise linear in the spine and averaged 14.5 %. TBS decline rate increases after 65 years by 50 %. INTRODUCTION: This study aimed to evaluate age-related changes in lumbar vertebral microarchitecture, as assessed by TBS, in a cohort of French women aged 45-85 years. METHODS: An all-comers cohort of French Caucasian women was selected from two clinical centers. Data obtained from these centers were cross-calibrated for TBS and bone mineral density (BMD). BMD and TBS were evaluated at L1-L4 and for all lumbar vertebrae combined using GE-Lunar Prodigy densitometer images. Weight, height, and body mass index (BMI) also were determined. To validate our all-comers cohort, the BMD normative data of our cohort and French Prodigy data were compared. RESULTS: A cohort of 5,942 French women aged 45 to 85 years was created. Dual-energy X-ray absorptiometry normative data obtained for BMD from this cohort were not significantly different from French prodigy normative data (p = 0.15). TBS values at L1-L4 were poorly correlated with BMI (r = -0.17) and weight (r = -0.14) and not correlated with height. TBS values obtained for all lumbar vertebra combined (L1, L2, L3, L4) decreased with age. The magnitude of TBS decline at L1-L4 between 45 and 85 years of age was piecewise linear in the spine and averaged 14.5 %, but this rate increased after 65 years by 50 %. Similar results were obtained for other region of interest in the lumbar spine. As opposed to BMD, TBS was not affected by spinal osteoarthrosis. CONCLUSION: The age-specific reference curve for TBS generated here could therefore be used to help clinicians to improve osteoporosis patient management and to monitor microarchitectural changes related to treatment or other diseases in routine clinical practice.

Modulation of proteasomal activity required for the generation of a cytotoxic T lymphocyte-defined peptide derived from the tumor antigen MAGE-3.

We have analyzed the presentation of human histocompatability leukocyte antigen-A*0201-associated tumor peptide antigen MAGE-3271-279 by melanoma cells. We show that specific cytotoxic T lymphocyte (CTL)-recognizing cells transfected with a minigene encoding the preprocessed fragment MAGE-3271-279 failed to recognize cells expressing the full length MAGE-3 protein. Digestion of synthetic peptides extended at the NH2 or COOH terminus of MAGE-3271-279 with purified human proteasome revealed that the generation of the COOH terminus of the antigenic peptide was impaired. Surprisingly, addition of lactacystin to purified proteasome, though partially inhibitory, resulted in the generation of the antigenic peptide. Furthermore, treatment of melanoma cells expressing the MAGE-3 protein with lactacystin resulted in efficient lysis by MAGE-3271-279-specific CTL. We therefore postulate that the generation of antigenic peptides by the proteasome in cells can be modulated by the selective inhibition of certain of its enzymaticactivities.

Generation and characterization of a Notch1 signaling-specific reporter mouse line.

Signaling through the Notch1 receptor is essential for the control of numerous developmental processes during embryonic life as well as in adult tissue homeostasis and disease. Since the outcome of Notch1 signaling is highly context-dependent, and its precise physiological and pathological role in many organs is unclear, it is of great interest to localize and identify the cells that receive active Notch1 signals in vivo. Here, we report the generation and characterization of a BAC-transgenic mouse line, N1-Gal4VP16, that when crossed to a Gal4-responsive reporter mouse line allowed the identification of cells undergoing active Notch1 signaling in vivo. Analysis of embryonic and adult N1-Gal4VP16 mice demonstrated that the activation pattern of the transgene coincides with previously observed activation patterns of the endogenous Notch1 receptor. Thus, this novel reporter mouse line provides a unique tool to specifically investigate the spatial and temporal aspects of Notch1 signaling in vivo. genesis 50:700-710, 2012. © 2012 Wiley Periodicals, Inc.

Generation of a tightly regulated all-cis beta cell-specific tetracycline-inducible vector.

Ability to induce protein expression at will in a cell is a powerful strategy used by scientists to better understand the function of a protein of interest. Various inducible systems have been designed in eukaryotic cells to achieve this goal. Most of them rely on two distinct vectors, one encoding a protein that can regulate transcription by binding a compound X, and one hosting the cDNA encoding the protein of interest placed downstream of promoter sequences that can bind the protein regulated by compound X (e.g., tetracycline, ecdysone). The commercially available systems are not designed to allow cell- or tissue-specific regulated expression. Additionally, although these systems can be used to generate stable clones that can be induced to express a given protein, extensive screening is often required to eliminate the clones that display poor induction or high basal levels. In the present report, we aimed to design a pancreatic beta cell-specific tetracycline-inducible system. Since the classical two-vector based tetracycline-inducible system proved to be unsatisfactory in our hands, a single vector was eventually designed that allowed tight beta cell-specific tetracycline induction in unselected cell populations.

Rapid cohort generation and analysis of disease spectrum of large animal model of cone dystrophy.

Large animal models are an important resource for the understanding of human disease and for evaluating the applicability of new therapies to human patients. For many diseases, such as cone dystrophy, research effort is hampered by the lack of such models. Lentiviral transgenesis is a methodology broadly applicable to animals from many different species. When conjugated to the expression of a dominant mutant protein, this technology offers an attractive approach to generate new large animal models in a heterogeneous background. We adopted this strategy to mimic the phenotype diversity encounter in humans and generate a cohort of pigs for cone dystrophy by expressing a dominant mutant allele of the guanylate cyclase 2D (GUCY2D) gene. Sixty percent of the piglets were transgenic, with mutant GUCY2D mRNA detected in the retina of all animals tested. Functional impairment of vision was observed among the transgenic pigs at 3 months of age, with a follow-up at 1 year indicating a subsequent slower progression of phenotype. Abnormal retina morphology, notably among the cone photoreceptor cell population, was observed exclusively amongst the transgenic animals. Of particular note, these transgenic animals were characterized by a range in the severity of the phenotype, reflecting the human clinical situation. We demonstrate that a transgenic approach using lentiviral vectors offers a powerful tool for large animal model development. Not only is the efficiency of transgenesis higher than conventional transgenic methodology but this technique also produces a heterogeneous cohort of transgenic animals that mimics the genetic variation encountered in human patients.

Generation of TALEN-mediated GRdim knock-in rats by homologous recombination.

Transcription Activator-Like Effector Nucleases (TALEN) are potential tools for precise genome engineering of laboratory animals. We report the first targeted genomic integration in the rat using TALENs (Transcription Activator-Like Effector Nucleases) by homology-derived recombination (HDR). We assembled TALENs and designed a linear donor insert targeting a pA476T mutation in the rat Glucocorticoid Receptor (Nr3c1) namely GR(dim), that prevents receptor homodimerization in the mouse. TALEN mRNA and linear double-stranded donor were microinjected into rat one-cell embryos. Overall, we observed targeted genomic modifications in 17% of the offspring, indicating high TALEN cutting efficiency in rat zygotes.

Role of β-catenin and γ-catenin in haematopoiesis : Generation of floxed Delta like 4 gene targeted mice

Résumé La voie de signalisation de Wnt est extrêmement conservée au cours de l'évolution. Les protéines Wnt sont des molécules sécrétées qui se lient à la famille de récepteurs Frizzled. Cette interaction mène à la stabilisation de la protéine β-caténine, qui va s'accumuler dans le cytoplasme puis migrer dans le noyau où elle peut s'hétérodimériser avec les facteurs de transcription de la famille TCF/LEF. Il a été démontré que cette voie de signalisation joue un rôle important durant la lymphopoïèse et de récents résultats suggèrent un rôle clé de cette voie dans le renouvellement des Cellules Souches Hématopoïétique (CSH). Des études se basant sur un système de surexpression de protéines montrent clairement que la voie Wnt peut influencer l'hématopoïèse. Cependant, le rôle de la protéine β-caténine dans le système hématopoïétique n'a jamais été testé directement. Ce projet de thèse se propose d'étudier la fonction de la protéine β-caténine par sa délétion inductible via le système Cre-loxP. De façon surprenante, nous avons pu démontrer que les progéniteurs de la moelle osseuse, déficients en β-caténine, ne montrent aucune altération dans leur capacité à s'auto-renouveler et/ou à reconstituer toutes les lignées hématopoïétiques (myéloïde, érythroïde et lymphoïde) dans les souris-chimères. De plus, le développement, la survie des thymocytes ainsi que la prolifération des cellules T périphériques induite par un antigène, sont indépendants de β-caténine. Ces résultats suggèrent soit que la protéine β-caténine ne joue pas un rôle primordial dans le système hématopoiétique, soit que son absence pourrait être compensée par une autre protéine. Un candidat privilégié susceptible de se substituer à β-caténine, serait plakoglobine, aussi connu sous le nom de γ-caténine. En effet, ces deux protéines partagent de multiples caractéristiques structurelles. Afin de démontrer que la protéine γ-caténine peut compenser l'absence de β-caténine, nous avons généré des souris dans lesquelles, le système hématopoïétique est déficient pour ces deux protéines. Cette déficience combinée de β- caténine et γ-caténine ne perturbe pas la capacité des Cellules Souche Hématopoïétique-Long Terme (CSH-LT) de se renouveler, par contre elle agit sur un progéniteur précoce déjà différencié de la moelle osseuse. Ces résultats mettent en évidence que la protéine γ-caténine est capable de compenser l'absence de protéine β-caténine dans le système hématopoïétique. Par conséquent, ce travail contribue à une meilleure connaissance de la cascade Wnt dans l'hématopoïèse. Summary The canonical Wnt signal transduction pathway is a developmentally highly conserved. Wnts are secreted molecules which bind to the family of Frizzled receptors in a complex with the low density lipoprotein receptor related protein (LRP-5/6). This initial activation step leads to the stabilization and accumulation of β-catenin, first in the cytoplasm and subsequently in the nucleus where it forms heterodimers with TCF/LEF transcription factor family members. Wnt signalling has been shown to be important during early lymphopoiesis and has more recently, been suggested to be a key player in self-renewal of haematopoietic stem cells (HSCs). Although mostly gain of function studies indicate that components of the Wnt signalling pathway can influence the haematopoietic system, the role of β-catenin has never been directly investigated. The aim of this thesis project is to investigate the putatively critical role of β-catenin in vivo using the Cre-loxP mediated conditional loss of function approach. Surprisingly, β-catenin deficient bone marrow (BM) progenitors arc not impaired in their ability to self-renew and/or to reconstitute all haematopoietic lineages (myeloid, erythroid and lymphoid) in both mixed and straight bone marrow chimeras. In addition, both thymocyte development and survival, and antigen-induced proliferation of peripheral T cells are β- catenin independent. Our results do not necessarily exclude the possibility of an important function for β-catenin mediated Wnt signalling in the haematopoietic system, it rather raises the question that β-catenin is compensated for by another protein. A prime candidate that may take over the function of β-catenin in its absence, is the close relative plakoglobin, also know as γ-catenin. This protein shares multiple structural features with β-catenin. In order to investigate whether γ-catenin can compensate for the loss of β-catenin we have generated mice in which the haematopoietic compartment is deficient for both proteins. Combined deficiency of β-catenin and γ-catenin does not perturb Long Term-Haematopoietic Stem Cells (LT-HSC) self renewal, but affects an already lineage committed progenitor population within the BM. Our results demonstrate that y-catenin can indeed compensate for the loss of β-catenin within the haematopoietie system.

Nonspecific PCR amplification by high-fidelity polymerases: implications for next-generation sequencing of AFLP markers.

High-fidelity 'proofreading' polymerases are often used in library construction for next-generation sequencing projects, in an effort to minimize errors in the resulting sequence data. The increased template fidelity of these polymerases can come at the cost of reduced template specificity, and library preparation methods based on the AFLP technique may be particularly susceptible. Here, we compare AFLP profiles generated with standard Taq and two versions of a high-fidelity polymerase. We find that Taq produces fewer and brighter peaks than high-fidelity polymerase, suggesting that Taq performs better at selectively amplifying templates that exactly match the primer sequences. Because the higher accuracy of proofreading polymerases remains important for sequencing applications, we suggest that it may be more effective to use alternative library preparation methods.

Evaluation of the user-friendliness of seven new generation intensive care ventilators.

OBJECTIVE: To explore the user-friendliness and ergonomics of seven new generation intensive care ventilators. DESIGN: Prospective task-performing study. SETTING: Intensive care research laboratory, university hospital. METHODS: Ten physicians experienced in mechanical ventilation, but without prior knowledge of the ventilators, were asked to perform eight specific tasks [turning the ventilator on; recognizing mode and parameters; recognizing and setting alarms; mode change; finding and activating the pre-oxygenation function; pressure support setting; stand-by; finding and activating non-invasive ventilation (NIV) mode]. The time needed for each task was compared to a reference time (by trained physiotherapist familiar with the devices). A time >180 s was considered a task failure. RESULTS: For each of the tests on the ventilators, all physicians' times were significantly higher than the reference time (P < 0.001). A mean of 13 +/- 8 task failures (16%) was observed by the ventilator. The most frequently failed tasks were mode and parameter recognition, starting pressure support and finding the NIV mode. Least often failed tasks were turning on the pre-oxygenation function and alarm recognition and management. Overall, there was substantial heterogeneity between machines, some exhibiting better user-friendliness than others for certain tasks, but no ventilator was clearly better that the others on all points tested. CONCLUSIONS: The present study adds to the available literature outlining the ergonomic shortcomings of mechanical ventilators. These results suggest that closer ties between end-users and manufacturers should be promoted, at an early development phase of these machines, based on the scientific evaluation of the cognitive processes involved by users in the clinical setting.

Magnetoelectricity in the ferrimagnetic Cu2OSeO3: symmetry analysis and Raman scattering study

sublattices ferrimagnet Cu2OSeO3 with a cubic symmetry and a linear magnetoelectric effect. There is no spectroscopic evidence for structural lattice distortions below T-C=60 K, which are expected due to magnetoelectric coupling. Using symmetry arguments we explain this observation by considering a special type of ferrimagnetic ground state which does not generate a spontaneous electric polarization. Interestingly, Raman scattering shows a strong increase of electric polarization of media through a dynamic magnetoelectric effect as a remarkable enhancement of the scattering intensity below T-C. New lines of purely magnetic origin have been detected in the magnetically ordered state. A part of them are attributed as scattering on exchange magnons. Using this observation and further symmetry considerations we argue for strong Dzyaloshinskii-Moriya interaction existing in the Cu2OSeO3. (c) 2010 American Institute of Physics. [doi:10.1063/1.3455808]

Herbivory in the previous generation primes plants for enhanced insect resistance.

Inducible defenses, which provide enhanced resistance after initial attack, are nearly universal in plants. This defense signaling cascade is mediated by the synthesis, movement, and perception of jasmonic acid and related plant metabolites. To characterize the long-term persistence of plant immunity, we challenged Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) with caterpillar herbivory, application of methyl jasmonate, or mechanical damage during vegetative growth and assessed plant resistance in subsequent generations. Here, we show that induced resistance was associated with transgenerational priming of jasmonic acid-dependent defense responses in both species, caused caterpillars to grow up to 50% smaller than on control plants, and persisted for two generations in Arabidopsis. Arabidopsis mutants that are deficient in jasmonate perception (coronatine insensitive1) or in the biogenesis of small interfering RNA (dicer-like2 dicer-like3 dicer-like4 and nuclear RNA polymerase d2a nuclear RNA polymerase d2b) do not exhibit inherited resistance. The observation of inherited resistance in both the Brassicaceae and Solanaceae suggests that this trait may be more widely distributed in plants. Epigenetic resistance to herbivory thus represents a phenotypically plastic mechanism for enhanced defense across generations.

La Generation Sandwich en Suisse Romande: mieux comprendre les facteurs associés avec la santé perçue afin de mieux agir en promotion de la santé [Sandwich Generation in Roman Switzerland : a better understanding of factors linked with perceived health for better acting in health promotion].

The so-called < Sandwich Generation > (SG) is characterized by concurrent and competing professional, familial, and informal caregiving workloads. These stressors pose potential health risks. However, the current knowledge about SG characteristics and perceived state of health are insufficient to allow occupational health nurses to develop evidence-based interventions designed for health promotion. We aimed to describe this population and examine the relationships between these coexisting workloads and their perceived health. This study is based on a descriptive, correlational design. Employees of a Swiss public administration completed an electronic questionnaire. Of 844 respondents, 23 % are SG members. Ages of frailed parents or parents-in-law, co-residence with the latters, children still living at home predict that employees could be members of the SG. Perceived physical health status of SG members is rated better than mental health status. The heterogeneity of SG is reflected in three clusters. Finally, physical health score is the only that differs from the other health scores adjusting for clusters and sex. This study provides a foundation for developing preventive interventions targeting the SG.