Heat-induced and spontaneous expression of Hsp70.1Luciferase transgene copies localized on Xp22 in female bovine cells

<p>Abstract</p> <p>Background</p> <p>Expression of several copies of the heat-inducible <it>Hsp70.1Luciferase </it>(<it>LUC</it>) transgene inserted at a single X chromosome locus of a bull (<it>Bos taurus</it>) was assessed in females after X-chromosome inactivation (XCI). Furthermore, impact of the chromosomal environment on the spontaneous expression of these transgene copies before XCI was studied during early development in embryos obtained after in vitro fertilization (IVF), when the locus was carried by the X chromosome inherited from the bull, and after somatic cell nuclear transfer (SCNT) cloning, when the locus could be carried by the inactive Xi or the active Xa chromosome in a female donor cell, or by the (active) X in a male donor cell.</p> <p>Findings</p> <p>Transgene copies were mapped to bovine Xp22. In XX^{<it>LUC </it>}female fibroblasts, i.e. after random XCI, the proportions of late-replicating inactive and early-replicating active X^{<it>LUC </it>}chromosomes were not biased and the proportion of cells displaying an increase in the level of immunostained luciferase protein after heat-shock induction was similar to that in male fibroblasts. Spontaneous transgene expression occurred at the 8-16-cell stage both in transgenic (female) embryos obtained after IVF and in male and female embryos obtained after SCNT.</p> <p>Conclusions</p> <p>The X^{<it>LUC </it>}chromosome is normally inactivated but at least part of the inactivated X-linked <it>Hsp70.1Luciferase </it>transgene copies remains heat-inducible after random XCI in somatic cells. Before XCI, the profile of the transgenes' spontaneous expression is independent of the epigenetic origin of the X^{<it>LUC </it>}chromosome since it is similar in IVF female, SCNT male and SCNT female embryos.</p

Atlas-Based Reduced Models of Blood Flows for Fast Patient-Specific Simulations

The original publication is available at www.springerlink.com.International audienceModel-based interpretation of the complex clinical data now available (shape, motion, flow) can provide quantitative information for diagnosis as well as predictions. However such models can be extremely time consuming, which does not always fit with the clinical time constraints. The aim of this work is to propose a model reduction technique to perform faster patient-specific simulations with prior knowledge built from simulations on an average anatomy. Rather than simulating a full fluid problem on individual patients, we create a representative `template' of the artery shape. A full flow simulation is carried out only on this template, and a reduced model is built from the results. Then this reduced model can be transported to the individual geometries, allowing faster computational analysis. % Here we propose a preliminary validation of this idea. A well-posed framework based on currents representation of shapes is used to create an unbiased template of the pulmonary artery for 4 patients with Tetralogy of Fallot. Then, a reduced computational fluid dynamics model is built on this template. Finally, we demonstrate that this reduced model can represent a specific patient simulation

Increase of mitochondrial DNA content and transcripts in early bovine embryogenesis associated with upregulation of mtTFA and NRF1 transcription factors

BACKGROUND: Recent work has shown that mitochondrial biogenesis and mitochondrial functions are critical determinants of embryonic development. However, the expression of the factors controlling mitochondrial biogenesis in early embryogenesis has received little attention so far. METHODS: We used real-time quantitative PCR to quantify mitochondrial DNA (mtDNA) in bovine oocytes and in various stages of in vitro produced embryos. To investigate the molecular mechanisms responsible for the replication and the transcriptional activation of mtDNA, we quantified the mRNA corresponding to the mtDNA-encoded cytochrome oxidase 1 (COX1), and two nuclear-encoded factors, i.e. the Nuclear Respiratory Factor 1 (NRF1), and the nuclear-encoded Mitochondrial Transcription Factor A (mtTFA). RESULTS: Unlike findings reported in mouse embryos, the mtDNA content was not constant during early bovine embryogenesis. We found a sharp, 60% decrease in mtDNA content between the 2-cell and the 4/8-cell stages. COX1 mRNA was constant until the morula stage after which it increased dramatically. mtTFA mRNA was undetectable in oocytes and remained so until the 8/16-cell stage; it began to appear only at the morula stage, suggesting de novo synthesis. In contrast, NRF1 mRNA was detectable in oocytes and the quantity remained constant until the morula stage. CONCLUSION: Our results revealed a reduction of mtDNA content in early bovine embryos suggesting an active process of mitochondrial DNA degradation. In addition, de novo mtTFA expression associated with mitochondrial biogenesis activation and high levels of NRF1 mRNA from the oocyte stage onwards argue for the essential function of these factors during the first steps of bovine embryogenesis

Group-wise Construction of Reduced Models for Understanding and Characterization of Pulmonary Blood Flows from Medical Images

International audience3D computational fluid dynamics (CFD) in patient-specific geometries provides complementary insights to clinical imaging, to better understand how heart disease, and the side effects of treating heart disease, affect and are affected by hemodynamics. This information can be useful in treatment planning for designing artificial devices that are subject to stress and pressure from blood flow. Yet, these simulations remain relatively costly within a clinical context. The aim of this work is to reduce the complexity of patient-specific simulations by combining image analysis, computational fluid dynamics and model order reduction techniques. The proposed method makes use of a reference geometry estimated as an average of the population, within an efficient statistical framework based on the currents representation of shapes. Snapshots of blood flow simulations performed in the reference geometry are used to build a POD (Proper Orthogonal Decomposition) basis, which can then be mapped on new patients to perform reduced order blood flow simulations with patient specific boundary conditions. This approach is applied to a data-set of 17 tetralogy of Fallot patients to simulate blood flow through the pulmonary artery under normal (healthy or synthetic valves with almost no backflow) and pathological (leaky or absent valve with backflow) conditions to better understand the impact of regurgitated blood on pressure and velocity at the outflow tracts. The model reduction approach is further tested by performing patient simulations under exercise and varying degrees of pathophysiological conditions based on reduction of reference solutions (rest and medium backflow conditions respectively)

Ring-Like Distribution of Constitutive Heterochromatin in Bovine Senescent Cells

Background: Cells that reach ‘‘Hayflick limit’ ’ of proliferation, known as senescent cells, possess a particular type of nuclear architecture. Human senescent cells are characterized by the presence of highly condensed senescent associated heterochromatin foci (SAHF) that can be detected both by immunostaining for histone H3 three-methylated at lysine 9 (H3K9me3) and by DAPI counterstaining. Methods: We have studied nuclear architecture in bovine senescent cells using a combination of immunofluorescence and 3D fluorescent in-situ hybridization (FISH). Results: Analysis of heterochromatin distribution in bovine senescent cells using fluorescent in situ hybridization for pericentric chromosomal regions, immunostaining of H3K9me3, centromeric proteins CENP A/B and DNA methylation showed a lower level of heterochromatin condensation as compared to young cells. No SAHF foci were observed. Instead, we observed fibrous ring-like or ribbon-like heterochromatin patterns that were undetectable with DAPI counterstaining. These heterochromatin fibers were associated with nucleoli

Uncoupled Embryonic and Extra-Embryonic Tissues Compromise Blastocyst Development after Somatic Cell Nuclear Transfer

Somatic cell nuclear transfer (SCNT) is the most efficient cell reprogramming technique available, especially when working with bovine species. Although SCNT blastocysts performed equally well or better than controls in the weeks following embryo transfer at Day 7, elongation and gastrulation defects were observed prior to implantation. To understand the developmental implications of embryonic/extra-embryonic interactions, the morphological and molecular features of elongating and gastrulating tissues were analysed. At Day 18, 30 SCNT conceptuses were compared to 20 controls (AI and IVP: 10 conceptuses each); one-half of the SCNT conceptuses appeared normal while the other half showed signs of atypical elongation and gastrulation. SCNT was also associated with a high incidence of discordance in embryonic and extra-embryonic patterns, as evidenced by morphological and molecular “uncoupling”. Elongation appeared to be secondarily affected; only 3 of 30 conceptuses had abnormally elongated shapes and there were very few differences in gene expression when they were compared to the controls. However, some of these differences could be linked to defects in microvilli formation or extracellular matrix composition and could thus impact extra-embryonic functions. In contrast to elongation, gastrulation stages included embryonic defects that likely affected the hypoblast, the epiblast, or the early stages of their differentiation. When taking into account SCNT conceptus somatic origin, i.e. the reprogramming efficiency of each bovine ear fibroblast (Low: 0029, Med: 7711, High: 5538), we found that embryonic abnormalities or severe embryonic/extra-embryonic uncoupling were more tightly correlated to embryo loss at implantation than were elongation defects. Alternatively, extra-embryonic differences between SCNT and control conceptuses at Day 18 were related to molecular plasticity (high efficiency/high plasticity) and subsequent pregnancy loss. Finally, because it alters re-differentiation processes in vivo, SCNT reprogramming highlights temporally and spatially restricted interactions among cells and tissues in a unique way

Exome sequencing identifies germline variants in DIS3 in familial multiple myeloma

[Excerpt] Multiple myeloma (MM) is the third most common hematological malignancy, after Non-Hodgkin Lymphoma and Leukemia. MM is generally preceded by Monoclonal Gammopathy of Undetermined Significance (MGUS) [1], and epidemiological studies have identified older age, male gender, family history, and MGUS as risk factors for developing MM [2]. The somatic mutational landscape of sporadic MM has been increasingly investigated, aiming to identify recurrent genetic events involved in myelomagenesis. Whole exome and whole genome sequencing studies have shown that MM is a genetically heterogeneous disease that evolves through accumulation of both clonal and subclonal driver mutations [3] and identified recurrently somatically mutated genes, including KRAS, NRAS, FAM46C, TP53, DIS3, BRAF, TRAF3, CYLD, RB1 and PRDM1 [3,4,5]. Despite the fact that family-based studies have provided data consistent with an inherited genetic susceptibility to MM compatible with Mendelian transmission [6], the molecular basis of inherited MM predisposition is only partly understood. Genome-Wide Association (GWAS) studies have identified and validated 23 loci significantly associated with an increased risk of developing MM that explain ~16% of heritability [7] and only a subset of familial cases are thought to have a polygenic background [8]. Recent studies have identified rare germline variants predisposing to MM in KDM1A [9], ARID1A and USP45 [10], and the implementation of next-generation sequencing technology will allow the characterization of more such rare variants. [...]French National Cancer Institute (INCA) and the Fondation Française pour la Recherche contre le Myélome et les Gammapathies (FFMRG), the Intergroupe Francophone du Myélome (IFM), NCI R01 NCI CA167824 and a generous donation from Matthew Bell. This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. Research reported in this paper was supported by the Office of Research Infrastructure of the National Institutes of Health under award number S10OD018522. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The authors thank the Association des Malades du Myélome Multiple (AF3M) for their continued support and participation. Where authors are identified as personnel of the International Agency for Research on Cancer / World Health Organization, the authors alone are responsible for the views expressed in this article and they do not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer / World Health Organizatio

Evolution des composants structuraux de la barriere hemato-testiculaire chez le Hamster au cours de la periode postnatale

SIGLECNRS T 57093 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Evolution des composants structuraux de la barriere hemato-testiculaire chez le Hamster au cours de la periode postnatale

SIGLECNRS T 57093 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

L'architecture nucléaire chez les embryons bovins produits par fécondation in vitro et clonage (la dynamique de l' hétérochromatine)

Developmental reprogramming during mammalian fertilization and pre-implantation development in in vitro fertilized (IVF) embryos is a complex process that allows the highly differentiated gametes to revert to undifferentiated cell types following syngamy and then gradually differentiate into individual cell lineages. These processes involve changes in chromatin structure, in global epigenetic modifications and in nuclear architecture of embryonic nuclei. The objective of the present study was to develop approaches for understanding these series of phenomena in early embryos. We used the possibility to compare normal (IVF) and nuclear tranfer (NT) embryos in bovine species where the pre-implantation development is long enough to assess the nuclear dynamics of reprogramming events before major embryonic genome activation. In a first approach, we studied the establishment of heterochromatin pattern specific for embryo during the reprogramming period in IVF and NT bovine embryos. We applied two markers of pericentric heterochromatin: heterochromatin protein 1 (HP1beta) and tri methylated histone H3 (H3K9me3); and a marker of centromeres (CENPA/B), in order to characterize structural parameters of constitutive heterochromatin in early stages of development. Using immunofluorescence technique, we were able to observe dynamic changes in heterochromatin organization in connection with embryos origin. In IVF and some NT embryos, heterochromatin was observed in dispersed state up to the 8-cell stage, and then in a condensed pattern corresponding to the well characterized chromocenters constituted by blocks of HP1beta and H3K9me3 associated with centromeres. However, a significant part of NT embryos underwent an altered dynamics of heterochromatinization characterized by a precocious heterochromatin condensation as soon as the 2-cell stage. In a second approach, we used senescent somatic cells as donors for nuclear transfer experiments in order to assess the influence of structural organization of donor cell nucleus on structural organization of nuclei in cloned embryos. Surprisingly, the large-scale three-dimensional arrangement of heterochromatin within the senescent-NT nucleus recapitulated the dynamics observed in IVF or somatic non-senescent NT embryos, with comparable percentages of embryos with dispersed and precociously condensed heterochromatin similar to those observed in the previous investigation. These results suggest that a robust process of epigenetic reprogramming is piloted by bovine oocyte in early embryos. The altered kinetics in genome restructuring in some NT embryos might be linked to the precocious transcriptional activation and precocious increase of DNA methylation level reported in other studies. The present work allowed us to point out the distinction between NT embryos with correct developmental reprogramming and abnormal, at least temporally, epigenetic reprogramming, as well as to demonstrate that the impact of nuclear organization of heterochromatin in the donor cell on structural organization of nucleus in NT embryos is relatively low. This may account for the relatively high development efficiency in bovine cloning as compared to other species.La reprogrammation au cours de la fécondation et du développement pré-implantatoire de l embryon fécondé in vitro (IVF) chez les mammifères est un processus complexe qui permet le retour à un état indifférencié des cellules hautement spécialisées que sont les gamètes lors de la syngamie, puis leur différenciation progressive en différent lignages cellulaires dans l embryon. Ces processus comprennent des changements dans la structure de la chromatine, dans les modifications épigénétiques et dans l architecture nucléaire des noyaux embryonnaires. L objectif de cette étude était de mettre en place des approches pour la compréhension de ces processus dans les embryons précoces. Nous avons tiré parti de la comparaison entre embryons normaux (IVF) et issus de transfert de noyau (NT) dans l espèce bovine où le développement pré-implantatoire s étant sur une période suffisamment longue pour caractériser la dynamique des événements de reprogrammation nucléaire avant la mise en route de l activation majeure du génome embryonnaire. Dans une première approche, nous avons étudié la mise en place de l hétérochromatine spécifique à l embryon au cours de la période de reprogrammation dans les embryons bovins issus de fécondation et de NT. Nous avons utilisé deux marqueurs de l hétérochromatine péricentrique : la protéine hétérochromatine 1 (HP1-beta) et l histone H3 tri-méthylée (H3K9me3) ; et un marqueur des centromères (CENP A/B), de façon à caractériser les paramètres structuraux de l hétérochromatine constitutive dans les premiers stades de développement embryonnaire. En utilisant des techniques d immunofluorescence, nous avons pu observer les changements dynamiques dans l organisation de l hétrochromatine en relation avec l origine des embryons. Dans les embryons IVF et une partie des embryons NT, l hétérochromatine apparaît dans un état dispersé, et ceci jusqu au stade 8-cellule ; puis sous une forme condensée qui correspond à l association bien caractéristique de blocs d hétérochromatine et d H3K9me3 avec les centromères, connues sous le nom de chromocentres. Cependant, une partie significative des embryons NT montrent une altération de la dynamique d hétérochromatinisation révélée par une condensation précoce, dès le stade 2-cellule, de HP1 et H3K9-me3. Dans une seconde approche, nous avons utilisé des cellules somatiques au stade de la sénescence comm cellules donneuses de noyau de façon à tester l influence de l organisation structurale du noyau donneur sur l organisation structurale des noyaux dans les embryons clonés. Les arrangements tridimensionnels à large échelle de l hétérochromatine dans les noyaux sénescents NT récapitulent la dynamique observée dans les embryons IVF ou NT issus de noyau de cellules non sénescentes. Notamment, les pourcentages d embryons avec une hétérochromatine dispersée ou condensée prématurément sont comparables avec les études précédentes.Ces résultats suggèrent l existence d un processus de reprogrammation épigénétique robuste piloté par l ovocyte dans les embryons précoces de bovins. Les cinétiques de restructuration du génome altérées dans une partie des embryons NT pourraient être liées à l activation précoce de la transcription et l augmentation du taux de méthylation qui ont été rapportées dans d autres études. Le présent travail nous a permis de mettre en évidence une distinction entre des embryons NT avec une reprogrammation normale et des embryons NT avec une anomalie, au moins temporelle, de la reprogrammtion épigénétique. Egalement, nous avons démontré que l impact de l organisation nucléaire de la cellule donneuse au niveau de l hétérochromatine sur l organisation structurale du noyau dans l embryon NT est relativement modéré.VERSAILLES-BU Sciences et IUT (786462101) / SudocSudocFranceF