The genome sequence of taurine cattle: a window to ruminant biology and evolution.

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.

Molecular evidence for ancient asexuality in timema stick insects.

Asexuality is rare in animals in spite of its apparent advantage relative to sexual reproduction, indicating that it must be associated with profound costs [1-9]. One expectation is that reproductive advantages gained by new asexual lineages will be quickly eroded over time [3, 5-7]. Ancient asexual taxa that have evolved and adapted without sex would be "scandalous" exceptions to this rule, but it is often difficult to exclude the possibility that putative asexuals deploy some form of "cryptic" sex, or have abandoned sex more recently than estimated from divergence times to sexual relatives [10]. Here we provide evidence, from high intraspecific divergence of mitochondrial sequence and nuclear allele divergence patterns, that several independently derived Timema stick-insect lineages have persisted without recombination for more than a million generations. Nuclear alleles in the asexual lineages displayed significantly higher intraindividual divergences than in related sexual species. In addition, within two asexuals, nuclear allele phylogenies suggested the presence of two clades, with sequences from the same individual appearing in both clades. These data strongly support ancient asexuality in Timema and validate the genus as an exceptional opportunity to attack the question of how asexual reproduction can be maintained over long periods of evolutionary time.

Hepatitis C virus infection: in vivo and in vitro models.

Major advances in the understanding of the molecular biology of hepatitis C virus (HCV) have been made recently. While the chimpanzee is the only established animal model of HCV infection, several in vivo and in vitro models have been established that allow us to study various aspects of the viral life cycle. In particular, the replicon system and the production of recombinant infectious virions revolutionized the investigation of HCV-RNA replication and rendered all steps of the viral life cycle, including entry and release of viral particles, amenable to systematic analysis. In the following we will review the different in vivo and in vitro models of HCV infection.

Strategies and tools for preventing neurotoxicity: to test, to predict and how to do it

A change in paradigm is needed in the prevention of toxic effects on the nervous system, moving from its present reliance solely on data from animal testing to a prediction model mostly based on in vitro toxicity testing and in silico modeling. According to the report published by the National Research Council (NRC) of the US National Academies of Science, high-throughput in vitro tests will provide evidence for alterations in"toxicity pathways" as the best possible method of large scale toxicity prediction. The challenges to implement this proposal are enormous, and provide much room for debate. While many efforts address the technical aspects of implementing the vision, many questions around it need also to be addressed. Is the overall strategy the only one to be pursued? How can we move from current to future paradigms? Will we ever be able to reliably model for chronic and developmental neurotoxicity in vitro? This paper summarizes four presentations from a symposium held at the International Neurotoxicology Conference held in Xi"an, China, in June 2011. A. Li reviewed the current guidelines for neurotoxicity and developmental neurotoxicity testing, and discussed the major challenges existing to realize the NCR vision for toxicity testing. J. Llorens reviewed the biology of mammalian toxic avoidance in view of present knowledge on the physiology and molecular biology of the chemical senses, taste and smell. This background information supports the hypothesis that relating in vivo toxicity to chemical epitope descriptors that mimic the chemical encoding performed by the olfactory system may provide a way to the long term future of complete in silico toxicity prediction. S. Ceccatelli reviewed the implementation of rodent and human neural stem cells (NSCs) as models for in vitro toxicity testing that measures parameters such as cell proliferation, differentiation and migration. These appear to be sensitive endpoints that can identify substances with developmental neurotoxic potential. C. Sun ol reviewed the use of primary neuronal cultures in testing for neurotoxicity of environmental pollutants, including the study of the effects of persistent exposures and/or in differentiating cells, which allow recording of effects that can be extrapolated to human developmental neurotoxicity.

ENFIN--A European network for integrative systems biology.

Integration of biological data of various types and the development of adapted bioinformatics tools represent critical objectives to enable research at the systems level. The European Network of Excellence ENFIN is engaged in developing an adapted infrastructure to connect databases, and platforms to enable both the generation of new bioinformatics tools and the experimental validation of computational predictions. With the aim of bridging the gap existing between standard wet laboratories and bioinformatics, the ENFIN Network runs integrative research projects to bring the latest computational techniques to bear directly on questions dedicated to systems biology in the wet laboratory environment. The Network maintains internally close collaboration between experimental and computational research, enabling a permanent cycling of experimental validation and improvement of computational prediction methods. The computational work includes the development of a database infrastructure (EnCORE), bioinformatics analysis methods and a novel platform for protein function analysis FuncNet.

Concentrative nucleoside transporter 1 (hCNT1) promotes phenotypic changes relevant to tumor biology in a translocation independent manner

Nucleoside transporters (NTs) mediate the uptake of nucleosides and nucleobases across the plasma membrane, mostly for salvage purposes. The canonical NTs belong to two gene families, SLC29 and SLC28. The former encode equilibrative nucleoside transporter proteins (ENTs), which mediate the facilitative diffusion of natural nucleosides with broad selectivity, whereas the latter encode concentrative nucleoside transporters (CNTs), which are sodium-coupled and show high affinity for substrates with variable selectivity. These proteins are expressed in most cell types, exhibiting apparent functional redundancy. This might indicate that CNTs play specific roles in the physiology of the cell beyond nucleoside salvage. Here, we addressed this possibility using adenoviral vectors to restore tumor cell expression of hCNT1 or a polymorphic variant (hCNT1S546P) lacking nucleoside translocation ability. We found that hCNT1 restoration in pancreatic cancer cells significantly altered cell-cycle progression and phosphorylation status of key signal-transducing kinases, promoted poly-(ADP ribose) polymerase hyperactivation and cell death, and reduced tumor growth and cell migration. Importantly, the translocation-defective transporter triggered these same effects on cell physiology. These data predict a novel and totally unexpected biological role for the nucleoside transporter protein hCNT1 that appears to be independent of its role as mediator of nucleoside uptake by cells, thereby suggesting a transceptor function. Cell Death & Disease Anastasis Stephanou Receiving Editor Cell Death & Disease 19th Apr 2013 Dr Perez-Torras Av/ Diagonal 643. Edif. Prevosti, Pl -1 Barcelona 08028 Spain RE: Manuscript CDDIS-13-0136R, 'CDDIS-13-0136R' Dear Dr Perez-Torras, It is a pleasure to inform you that your manuscript has been evaluated at the editorial level and has now been officially accepted for publication in Cell Death & Disease, pending you meet the following editorial requirements: 1) the list of the abbreviations is missing please include Could you send us the revised text as word file via e-mail and we will proceed and transfer the paper onto our typesetters. Please download, print, sign, and return the Licence to Publish Form using the link below. This must be returned via FAX to ++ 39 06 7259 6977 before your manuscript can be published:

The planarian flatworm: an in vivo model for stem cell biology and nervous system regeneration

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.

Animal modelling for inherited central vision loss.

Disease-causing variants of a large number of genes trigger inherited retinal degeneration leading to photoreceptor loss. Because cones are essential for daylight and central vision such as reading, mobility, and face recognition, this review focuses on a variety of animal models for cone diseases. The pertinence of using these models to reveal genotype/phenotype correlations and to evaluate new therapeutic strategies is discussed. Interestingly, several large animal models recapitulate human diseases and can serve as a strong base from which to study the biology of disease and to assess the scale-up of new therapies. Examples of innovative approaches will be presented such as lentiviral-based transgenesis in pigs and adeno-associated virus (AAV)-gene transfer into the monkey eye to investigate the neural circuitry plasticity of the visual system. The models reported herein permit the exploration of common mechanisms that exist between different species and the identification and highlighting of pathways that may be specific to primates, including humans.

The maspin expression in canine mammary tumors: an immunohistochemical and molecular study

The serpin maspin, a tumor suppressor in breast cancer was described as an inhibitor of cell migration and inducer of cell adhesion between the basement membrane and extracellular matrix resulting in inhibition of tumor metastasis. In contrast, overexpression of maspin is correlated with poor prognosis in other types of cancer. Little is known about expression, regulation and function of maspin in canine mammary tumors. It was demonstrated in this study, a loss of maspin expression in malignant canine mammary cells compared with a pool of normal canine mammary tissue, analyzed by quantitative real-time PCR; weak maspin expression in malignant canine mammary tumors were observed by immunohistochemistry. It was also demonstrated that a correlation with nuclear maspin expression and a good prognosis. It is suggested that maspin could be used as a prognostic marker in canine mammary neoplasia.

Biology and clinical utilization of mesenchymal progenitor cells

Within the complex cellular arrangement found in the bone marrow stroma there exists a subset of nonhematopoietic cells referred to as mesenchymal progenitor cells (MPC). These cells can be expanded ex vivo and induced, either in vitro or in vivo, to terminally differentiate into at least seven types of cells: osteocytes, chondrocytes, adipocytes, tenocytes, myotubes, astrocytes and hematopoietic-supporting stroma. This broad multipotentiality, the feasibility to obtain MPC from bone marrow, cord and peripheral blood and their transplantability support the impact that the use of MPC will have in clinical settings. However, a number of fundamental questions about the cellular and molecular biology of MPC still need to be resolved before these cells can be used for safe and effective cell and gene therapies intended to replace, repair or enhance the physiological function of the mesenchymal and/or hematopoietic systems.

Protein folding: a perspective for biology, medicine and biotechnology

At the present time, protein folding is an extremely active field of research including aspects of biology, chemistry, biochemistry, computer science and physics. The fundamental principles have practical applications in the exploitation of the advances in genome research, in the understanding of different pathologies and in the design of novel proteins with special functions. Although the detailed mechanisms of folding are not completely known, significant advances have been made in the understanding of this complex process through both experimental and theoretical approaches. In this review, the evolution of concepts from Anfinsen's postulate to the "new view" emphasizing the concept of the energy landscape of folding is presented. The main rules of protein folding have been established from in vitro experiments. It has been long accepted that the in vitro refolding process is a good model for understanding the mechanisms by which a nascent polypeptide chain reaches its native conformation in the cellular environment. Indeed, many denatured proteins, even those whose disulfide bridges have been disrupted, are able to refold spontaneously. Although this assumption was challenged by the discovery of molecular chaperones, from the amount of both structural and functional information now available, it has been clearly established that the main rules of protein folding deduced from in vitro experiments are also valid in the cellular environment. This modern view of protein folding permits a better understanding of the aggregation processes that play a role in several pathologies, including those induced by prions and Alzheimer's disease. Drug design and de novo protein design with the aim of creating proteins with novel functions by application of protein folding rules are making significant progress and offer perspectives for practical applications in the development of pharmaceuticals and medical diagnostics.

Molecular Mechanisms of Sexual Dimorphism in Threespine Stickleback

Sexual dimorphism is commonly understood as differences in external features, such as morphological features or coloration. However, it can more broadly encompass behavior and physiology and at the core of these differences is the genetic mechanism – mRNA and protein expression. How, and which, molecular mechanisms influence sexually dimorphic features is not well understood thus far. DNA, RNA and proteins are the template required to create the phenotype of an individual, and they are connected to each other via processes of transcription and translation. As the genome of males and females are almost identical with the exception of the few genes on the sex chromosome or the sex-determining alleles (in the case of organisms without sex chromosomes), it is likely that many of the downstream processes resulting in sexual dimorphism are produced by changes in gene regulation and result from a regulatory cascade and not from a vastly different gene composition. Thus, in this thesis a systems biology approach is used to understand sexual dimorphism at all molecular levels and how different genomic features, e.g. sex chromosome evolution, can affect the interplay of these molecules. The threespine stickleback, Gasterosteus aculeatus, is used as the model to investigate molecular mechanisms of sexual dimorphism. It has well-characterized ecology and behavior, especially in the breeding season when sexual dimorphism is high. Moreover, threespine stickleback has a recently evolved Y chromosome in the early stages of sex chromosome evolution, characterized by a lack of recombination leading to degeneration (i.e. gene loss). The aim of my thesis is to investigate how the genotype links to the molecular phenotype and relates to differences in molecular expression between males and females. Based on previous research on sex differences in mRNA expression, I investigated sex-biased protein expression in adult fish outside the breeding season to see if differences persisted after translation. As sex-biased expression also prevailed in the proteome and previous transcription expression seemed to be related to the sex chromosomes, I investigated the genome level with a particular focus on the sex-chromosomes. I characterized the status of Y chromosome degeneration in the threespine stickleback and its effects on gene function. Furthermore, since the degeneration process leaves genes in a single copy in males, I examined whether the resulting dosage difference of messenger RNA for hemizygous genes is compensated as it is in other organisms. In addition, threespine sticklebacks have wellcharacterized behavioral differences related to the male’s social status during the breeding season. To understand the connection between the genotype and behavior, I examined gene expression patterns related to breeding behavior using dominant and subordinate males as well as female

Targeting oncogenic signaling proteins : new insights using a FRET-based chemical biology approach

Cancer affects more than 20 million people each year and this rate is increasing globally. The Ras/MAPK-pathway is one of the best-studied cancer signaling pathways. Ras proteins are mutated in almost 20% of all human cancers and despite numerous efforts, no effective therapy that specifically targets Ras is available to date. It is now well established that Ras proteins laterally segregate on the plasma membrane into transient nanoscale signaling complexes called nanoclusters. These Ras nanoclusters are essential for the high-fidelity signal transmission. Disruption of nanoclustering leads to reduction in Ras activity and signaling, therefore targeting nanoclusters opens up important new therapeutic possibilities in cancer. This work describes three different studies exploring the idea of membrane protein nanoclusters as novel anti-cancer drug targets. It is focused on the design and implementation of a simple, cell-based Förster Resonance Energy Transfer (FRET)-biosensor screening platform to identify compounds that affect Ras membrane organization and nanoclustering. Chemical libraries from different sources were tested and a number of potential hit molecules were validated on full-length oncogenic proteins using a combination of imaging, biochemical and transformation assays. In the first study, a small chemical library was screened using H-ras derived FRET-biosensors. Surprisingly from this screen, commonly used protein synthesis inhibitors (PSIs) were found to specifically increase H-ras nanoclustering and downstream signalling in a H-ras dependent manner. Using a representative PSI, increase in H-ras activity was shown to induce cancer stem cell (CSC)-enriched mammosphere formation and tumor growth of breast cancer cells. Moreover, PSIs do not increase K-ras nanoclustering, making this screening approach suitable for identifying Ras isoform-specific inhibitors. In the second study, a nanoncluster-directed screen using both H- and K-ras derived FRET biosensors identified CSC inhibitor salinomycin to specifically inhibit K-ras nanocluster organization and downstream signaling. A K-ras nanoclusteringassociated gene signature was established that predicts the drug sensitivity of cancer cells to CSC inhibitors. Interestingly, almost 8% of patient tumor samples in the The Cancer Genome Atlas (TCGA) database had the above gene signature and were associated with a significantly higher mortality. From this mechanistic insight, an additional microbial metabolite screen on H- and K-ras biosensors identified ophiobolin A and conglobatin A to specifically affect K-ras nanoclustering and to act as potential breast CSC inhibitors. In the third study, the Ras FRET-biosensor principle was used to investigate membrane anchorage and nanoclustering of myristoylated proteins such as heterotrimeric G-proteins, Yes- and Src-kinases. Furthermore, Yes-biosensor was validated to be a suitable platform for performing chemical and genetic screens to identify myristoylation inhibitors. The results of this thesis demonstrate the potential of the Ras-derived FRETbiosensor platform to differentiate and identify Ras-isoform specfic inhibitors. The results also highlight that most of the inhibitors identified predominantly perturb Ras subcellular distribution and membrane organization through some novel and yet unknown mechanisms. The results give new insights into the role of Ras nanoclusters as promising new molecular targets in cancer and in stem cells.

Systems biology of the human MHC class I immunopeptidome

Le système de différenciation entre le « soi » et le « non-soi » des vertébrés permet la détection et le rejet de pathogènes et de cellules allogéniques. Il requiert la surveillance de petits peptides présentés à la surface cellulaire par les molécules du complexe majeur d’histocompatibilité de classe I (CMH I). Les molécules du CMH I sont des hétérodimères composés par une chaîne lourde encodée par des gènes du CMH et une chaîne légère encodée par le gène β2-microglobuline. L’ensemble des peptides est appelé l’immunopeptidome du CMH I. Nous avons utilisé des approches en biologie de systèmes pour définir la composition et l’origine cellulaire de l’immunopeptidome du CMH I présenté par des cellules B lymphoblastoïdes dérivés de deux pairs de fratries avec un CMH I identique. Nous avons découvert que l’immunopeptidome du CMH I est spécifique à l’individu et au type cellulaire, qu’il dérive préférentiellement de transcrits abondants, est enrichi en transcrits possédant d’éléments de reconnaissance par les petits ARNs, mais qu’il ne montre aucun biais ni vers les régions génétiques invariables ni vers les régions polymorphiques. Nous avons également développé une nouvelle méthode qui combine la spectrométrie de masse, le séquençage de nouvelle génération et la bioinformatique pour l’identification à grand échelle de peptides du CMH I, dont ceux résultants de polymorphismes nucléotidiques simples non-synonymes (PNS-ns), appelés antigènes mineurs d’histocompatibilité (AMHs), qui sont les cibles de réponses allo-immunitaires. La comparaison de l’origine génomique de l’immunopeptidome de soeurs avec un CMH I identique a révélé que 0,5% des PNS-ns étaient représentés dans l’immunopeptidome et que 0,3% des peptides du CMH I seraient immunogéniques envers une des deux soeurs. En résumé, nous avons découvert des nouveaux facteurs qui modèlent l’immunopeptidome du CMH I et nous présentons une nouvelle stratégie pour l’indentification de ces peptides, laquelle pourrait accélérer énormément le développement d’immunothérapies ciblant les AMHs.

Molecular determinants of congenital hypothyroidism due to thyroid dysgenesis

L’hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT) est la condition endocrinienne néonatale la plus fréquemment rencontrée, avec une incidence d’un cas sur 4000 naissances vivantes. L’HCDT comprend toutes les anomalies du développement de la thyroïde. Parmi ces anomalies, le diagnostic le plus fréquent est l’ectopie thyroïdienne (~ 50% des cas). L’HCDT est fréquemment associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdisme) pouvant conduire à un retard mental sévère si non traitée. Le programme de dépistage néonatal assure un diagnostic et un traitement précoce par hormones thyroïdiennes. Cependant, même avec un traitement précoce (en moyenne à 9 jours de vie), un retard de développement est toujours observé, surtout dans les cas les plus sévères (c.-à-d., perte de 10 points de QI). Bien que des cas familiaux soient rapportés (2% des cas), l’HCTD est essentiellement considérée comme une entité sporadique. De plus, plus de 92% des jumeaux monozygotiques sont discordants pour les dysgénésies thyroïdiennes et une prédominance féminine est rapportée (spécialement dans le cas d’ectopies thyroïdiennes), ces deux observations étant clairement incompatible avec un mode de transmission héréditaire mendélien. Il est donc cohérent de constater que des mutations germinales dans les facteurs de transcription thyroïdiens connus (NKX2.1, PAX8, FOXE1, and NKX2.5) ont été identifiées dans seulement 3% des cas sporadiques testés et furent, de plus, exclues lors d’analyse d’association dans certaines familles multiplex. Collectivement, ces données suggèrent que des mécanismes non mendéliens sont à l’origine de la majorité des cas de dysgénésie thyroïdienne. Parmi ces mécanismes, nous devons considérer des modifications épigénétiques, des mutations somatiques précoces (au stade du bourgeon thyroïdien lors des premiers stades de l’embryogenèse) ou des défauts développementaux stochastiques (c.-à-d., accumulation aléatoire de mutations germinales ou somatiques). Voilà pourquoi nous proposons un modèle «2 hits » combinant des mutations (épi)génétiques germinales et somatiques; ce modèle étant compatible avec le manque de transmission familial observé dans la majorité des cas d’HCDT. Dans cette thèse, nous avons déterminé si des variations somatiques (épi)génétiques sont associées à l’HCTD via une approche génomique et une approche gène candidat. Notre approche génomique a révélé que les thyroïdes ectopiques ont un profil d’expression différent des thyroïdes eutopiques (contrôles) et que ce profil d’expression est enrichi en gènes de la voie de signalisation Wnt. La voie des Wnt est cruciale pour la migration cellulaire et pour le développement de plusieurs organes dérivés de l’endoderme (p.ex. le pancréas). De plus, le rôle de la voie des Wnt dans la morphogénèse thyroïdienne est supporté par de récentes études sur le poisson-zèbre qui montrent des anomalies du développement thyroïdien lors de la perturbation de la voie des Wnt durant différentes étapes de l’organogénèse. Par conséquent, l’implication de la voie des Wnt dans l’étiologie de la dysgénésie thyroïdienne est biologiquement plausible. Une trouvaille inattendue de notre approche génomique fut de constater que la calcitonine était exprimée autant dans les thyroïdes ectopiques que dans les thyroïdes eutopiques (contrôles). Cette trouvaille remet en doute un dogme de l’embryologie de la thyroïde voulant que les cellules sécrétant la calcitonine (cellules C) proviennent exclusivement d’une structure extrathyroïdienne (les corps ultimobranchiaux) fusionnant seulement avec la thyroïde en fin de développement, lorsque la thyroïde a atteint son emplacement anatomique définitif. Notre approche gène candidat ne démontra aucune différence épigénétique (c.-à-d. de profil de méthylation) entre thyroïdes ectopiques et eutopiques, mais elle révéla la présence d’une région différentiellement méthylée (RDM) entre thyroïdes et leucocytes dans le promoteur de FOXE1. Le rôle crucial de FOXE1 dans la migration thyroïdienne lors du développement est connu et démontré dans le modèle murin. Nous avons démontré in vivo et in vitro que le statut de méthylation de cette RDM est corrélé avec l’expression de FOXE1 dans les tissus non tumoraux (c.-à-d., thyroïdes et leucocytes). Fort de ces résultats et sachant que les RDMs sont de potentiels points chauds de variations (épi)génétiques, nous avons lancé une étude cas-contrôles afin de déterminer si des variants génétiques rares localisés dans cette RDM sont associés à la dysgénésie thyroïdienne. Tous ces résultats générés lors de mes études doctorales ont dévoilé de nouveaux mécanismes pouvant expliquer la pathogenèse de la dysgénésie thyroïdienne, condition dont l’étiologie reste toujours une énigme. Ces résultats ouvrent aussi plusieurs champs de recherche prometteurs et vont aider à mieux comprendre tant les causes des dysgénésies thyroïdiennes que le développement embryonnaire normal de la thyroïde chez l’homme.