A new generation of pPRIG-based retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviral vectors are valuable tools for gene transfer. Particularly convenient are IRES-containing retroviral vectors expressing both the protein of interest and a marker protein from a single bicistronic mRNA. This coupled expression increases the relevance of tracking and/or selection of transduced cells based on the detection of a marker protein. pAP2 is a retroviral vector containing eGFP downstream of a modified IRES element of EMCV origin, and a CMV enhancer-promoter instead of the U3 region of the 5'LTR, which increases its efficiency in transient transfection. However, pAP2 contains a limited multicloning site (MCS) and shows weak eGFP expression, which previously led us to engineer an improved version, termed pPRIG, harboring: i) the wild-type ECMV IRES sequence, thereby restoring its full activity; ii) an optimized MCS flanked by T7 and SP6 sequences; and iii) a HA tag encoding sequence 5' of the MCS (pPRIG HAa/b/c).</p> <p>Results</p> <p>The convenience of pPRIG makes it a good basic vector to generate additional derivatives for an extended range of use. Here we present several novel pPRIG-based vectors (collectively referred to as PRIGs) in which : i) the HA tag sequence was inserted in the three reading frames 3' of the MCS (3'HA PRIGs); ii) a functional domain (ER, VP16 or KRAB) was inserted either 5' or 3' of the MCS (« modular » PRIGs); iii) eGFP was replaced by either eCFP, eYFP, mCherry or puro-R (« single color/resistance » PRIGs); and iv) mCherry, eYFP or eGFP was inserted 5' of the MCS of the IRES-eGFP, IRES-eCFP or IRES-Puro-R containing PRIGs, respectively (« dual color/selection » PRIGs). Additionally, some of these PRIGs were also constructed in a pMigR MSCV background which has been widely used in pluripotent cells.</p> <p>Conclusion</p> <p>These novel vectors allow for straightforward detection of any expressed protein (3'HA PRIGs), for functional studies of chimeric proteins (« modular » PRIGs), for multiple transductions and fluorescence analyses of transduced cells (« single color/resistance » PRIGs), or for quantitative detection of studied proteins in independently identified/selected transduced cells (« dual color/selection » PRIGs). They maintain the original advantages of pPRIG and provide suitable tools for either transient or stable expression and functional studies in a large range of experimental settings.</p

Region-based active contours for computer-aided analysis of carotid Phase Contrast MRI

International audienc

Stratégies volitionnelles, sentiment d’autoefficacité et accompagnement étudiant : quelles relations dans un dispositif hybride?

La recherche à l’origine de cet article aborde l’autonomie d’étudiantes et étudiants infirmiers inscrits dans un dispositif hybride de formation sous l’angle des stratégies d’autorégulation qu’ils mettent en oeuvre afin de contrôler leurs apprentissages, que ce soit lors des périodes de formation en présentiel ou en distanciel. Elle porte sur un type spécifique de stratégies d’autorégulation : celles qualifiées de « volitionnelles ». Cette recherche étudie en particulier les liens existants entre ces stratégies et deux autres dimensions : d’une part, la perception qu’ont ces étudiants et étudiantes de l’accompagnement apporté par leur formateur référent ou formatrice référente et d’autre part, leur sentiment d’autoefficacité à persister en formation. Elle met donc en relation ces trois dimensions spécifiques et peu étudiées conjointement, a fortiori en contexte de formation hybride dans l’enseignement supérieur.The research behind this article deals with the autonomy of nursing students enrolled in a hybrid learning system from the perspective of the self-regulation strategies they use to control their learning, during both in-person and distance learning sessions. It focuses on a specific type of self-regulation strategy known as “volitional”. In particular, this research studies the links between these volitional strategies and two other elements: on the one hand, the students’ perception of the support provided by their referent trainer and, on the other hand, their sense of self-efficacy regarding their persistence in training. This, it puts together these three specific elements that are rarely studied as a group, and all the less when it comes to hybrid training in higher education

Gene transfer to pre-hematopoietic and committed hematopoietic precursors in the early mouse Yolk Sac: a comparative study between in situ electroporation and retroviral transduction

<p>Abstract</p> <p>Background</p> <p>Hematopoietic development in vertebrate embryos results from the sequential contribution of two pools of precursors independently generated. While intra-embryonic precursors harbour the features of hematopoietic stem cells (HSC), precursors formed earlier in the yolk sac (YS) display limited differentiation and self-renewal potentials. The mechanisms leading to the generation of the precursors in both sites are still largely unknown, as are the molecular basis underlying their different potential. A possible approach to assess the role of candidate genes is to transfer or modulate their expression/activity in both sites. We thus designed and compared transduction protocols to target either native extra-embryonic precursors, or hematopoietic precursors.</p> <p>Results</p> <p>One transduction protocol involves transient modification of gene expression through <it>in situ </it>electroporation of the prospective blood islands, which allows the evolution of transfected mesodermal cells in their "normal" environment, upon organ culture. Following <it>in situ </it>electroporation of a GFP reporter construct into the YS cavity of embryos at post-streak (mesodermal/pre-hematopoietic precursors) or early somite (hematopoietic precursors) stages, high GFP expression levels as well as a good preservation of cell viability is observed in YS explants. Moreover, the erythro-myeloid progeny typical of the YS arises from GFP⁺mesodermal cells or hematopoietic precursors, even if the number of targeted precursors is low. The second approach, based on retroviral transduction allows a very efficient transduction of large precursor numbers, but may only be used to target 8 dpc YS hematopoietic precursors. Again, transduced cells generate a progeny quantitatively and qualitatively similar to that of control YS.</p> <p>Conclusion</p> <p>We thus provide two protocols whose combination may allow a thorough study of both early and late events of hematopoietic development in the murine YS. <it>In situ </it>electroporation constitutes the only possible gene transfer method to transduce mesodermal/pre-hematopoietic precursors and analyze the earliest steps of hematopoietic development. Both <it>in situ </it>electroporation and retroviral transduction may be used to target early hematopoietic precursors, but the latter appears more convenient if a large pool of stably transduced cells is required. We discuss the assets and limitation of both methods, which may be alternatively chosen depending on scientific constraints.</p

A semi-automated method for measuring the evolution of both lumen area and blood flow in carotid from Phase Contrast MRI

Phase-Contrast (PC) velocimetry Magnetic Resonance Imaging (MRI) is a useful modality to explore cardiovascular pathologies, but requires the automatic segmentation of vessels and the measurement of both lumen area and blood flow evolutions. In this paper, we propose a semi-automated method for extracting lumen boundaries of the carotid artery and compute both lumen area and blood flow evolutions over the cardiac cycle. This method uses narrow band region-based active contours in order to correctly capture the lumen boundary without being corrupted by surrounding structures. This approach is compared to traditional edge-based active contours, considered in related works, which significantly underestimate lumen area and blood flow. Experiments are performed using both a sequence of a homemade phantom and sequences of 20 real carotids, including a comparison with manual segmentation performed by a radiologist expert. Results obtained on the phantom sequence show that the edge-based approach leads to an underestimate of carotid lumen area and related flows of respectively 18.68% and 4.95%. This appears significantly larger than weak errors obtained using the region-based approach (respectively 2.73% and 1.23%). Benefits appear even better on the real sequences. The edge-based approach leads to underestimates of 40.88% for areas and 13.39% for blood flows, compared to limited errors of 7.41% and 4.6% with our method. Experiments also illustrate the high variability and therefore the lack of reliability of manual segmentation

NK Cells Infiltrating a MHC Class I-Deficient Lung Adenocarcinoma Display Impaired Cytotoxic Activity toward Autologous Tumor Cells Associated with Altered NK Cell-Triggering Receptors

Abstract NK cells are able to discriminate between normal cells and cells that have lost MHC class I (MHC-I) molecule expression as a result of tumor transformation. This function is the outcome of the capacity of inhibitory NK receptors to block cytotoxicity upon interaction with their MHC-I ligands expressed on target cells. To investigate the role of human NK cells and their various receptors in the control of MHC-I-deficient tumors, we have isolated several NK cell clones from lymphocytes infiltrating an adenocarcinoma lacking β2-microglobulin expression. Unexpectedly, although these clones expressed NKG2D and mediated a strong cytolytic activity toward K562, Daudi and allogeneic MHC-class I+ carcinoma cells, they were unable to lyse the autologous MHC-I− tumor cell line. This defect was associated with alterations in the expression of natural cytotoxicity receptor (NCR) by NK cells and the NKG2D ligands, MHC-I-related chain A, MHC-I-related chain B, and UL16 binding protein 1, and the ICAM-1 by tumor cells. In contrast, the carcinoma cell line was partially sensitive to allogeneic healthy donor NK cells expressing high levels of NCR. Indeed, this lysis was inhibited by anti-NCR and anti-NKG2D mAbs, suggesting that both receptors are required for the induced killing. The present study indicates that the MHC-I-deficient lung adenocarcinoma had developed mechanisms of escape from the innate immune response based on down-regulation of NCR and ligands required for target cell recognition

Lyl-1 regulates primitive macrophages and microglia development

During ontogeny, macrophage populations emerge in the Yolk Sac (YS) via two distinct progenitor waves, prior to hematopoietic stem cell development. Macrophage progenitors from the primitive/ early EMP and transient-definitive/ late EMP waves both contribute to various resident primitive macrophage populations in the developing embryonic organs. Identifying factors that modulates early stages of macrophage progenitor development may lead to a better understanding of defective function of specific resident macrophage subsets. Here we show that YS primitive macrophage progenitors express Lyl-1, a bHLH transcription factor related to SCL/Tal-1. Transcriptomic analysis of YS macrophage progenitors indicate that primitive macrophage progenitors present at embryonic day 9 are clearly distinct from those present at later stages. Disruption of Lyl-1 basic helix-loop-helix domain leads initially to an increased emergence of primitive macrophage progenitors, and later to their defective differentiation. These defects are associated with a disrupted expression of gene sets related to embryonic patterning and neurodevelopment. Lyl-1-deficiency also induce a reduced production of mature macrophages/microglia in the early brain, as well as a transient reduction of the microglia pool at midgestation and in the newborn. We thus identify Lyl-1 as a critical regulator of primitive macrophages and microglia development, which disruption may impair resident-macrophage function during organogenesis

Constraints from deuterium on the formation of icy bodies in the Jovian system and beyond

We consider the role of deuterium as a potential marker of location and ambient conditions during the formation of small bodies in our Solar system. We concentrate in particular on the formation of the regular icy satellites of Jupiter and the other giant planets, but include a discussion of the implications for the Trojan asteroids and the irregular satellites. We examine in detail the formation of regular planetary satellites within the paradigm of a circum-Jovian subnebula. Particular attention is paid to the two extreme potential subnebulae - "hot" and "cold". In particular, we show that, for the case of the "hot" subnebula model, the D:H ratio in water ice measured from the regular satellites would be expected to be near-Solar. In contrast, satellites which formed in a "cold" subnebula would be expected to display a D:H ratio that is distinctly over-Solar. We then compare the results obtained with the enrichment regimes which could be expected for other families of icy small bodies in the outer Solar system - the Trojan asteroids and the irregular satellites. In doing so, we demonstrate how measurements by Laplace, the James Webb Space Telescope, HERSCHEL and ALMA will play an important role in determining the true formation locations and mechanisms of these objects.Comment: Accepted and shortly to appear in Planetary and Space Science; 11 pages with 5 figure

Making the Earth: Combining Dynamics and Chemistry in the Solar System

No terrestrial planet formation simulation completed to date has considered the detailed chemical composition of the planets produced. While many have considered possible water contents and late veneer compositions, none have examined the bulk elemental abundances of the planets produced as an important check of formation models. Here we report on the first study of this type. Bulk elemental abundances based on disk equilibrium studies have been determined for the simulated terrestrial planets of O'Brien et al. (2006). These abundances are in excellent agreement with observed planetary values, indicating that the models of O'Brien et al. (2006) are successfully producing planets comparable to those of the Solar System in terms of both their dynamical and chemical properties. Significant amounts of water are accreted in the present simulations, implying that the terrestrial planets form "wet" and do not need significant water delivery from other sources. Under the assumption of equilibrium controlled chemistry, the biogenic species N and C still need to be delivered to the Earth as they are not accreted in significant proportions during the formation process. Negligible solar photospheric pollution is produced by the planetary formation process. Assuming similar levels of pollution in other planetary systems, this in turn implies that the high metallicity trend observed in extrasolar planetary systems is in fact primordial.Comment: 61 pages (including online material), 12 figures (7 in paper, 5 online). Accepted to Icaru

Origin of the Ocean on the Earth: Early Evolution of Water D/H in a Hydrogen-rich Atmosphere

The origin of the Earth's ocean has been discussed on the basis of deuterium/hydrogen ratios (D/H) of several sources of water in the solar system. The average D/H of carbonaceous chondrites (CC's) is known to be close to the current D/H of the Earth's ocean, while those of comets and the solar nebula are larger by about a factor of two and smaller by about a factor of seven, respectively, than that of the Earth's ocean. Thus, the main source of the Earth's ocean has been thought to be CC's or adequate mixing of comets and the solar nebula. However, those conclusions are correct only if D/H of water on the Earth has remained unchanged for the past 4.5 Gyr. In this paper, we investigate evolution of D/H in the ocean in the case that the early Earth had a hydrogen-rich atmosphere, the existence of which is predicted by recent theories of planet formation no matter whether the nebula remains or not. Then we show that D/H in the ocean increases by a factor of 2-9, which is caused by the mass fractionation during atmospheric hydrogen loss, followed by deuterium exchange between hydrogen gas and water vapor during ocean formation. This result suggests that the apparent similarity in D/H of water between CC's and the current Earth's ocean does not necessarily support the CC's origin of water and that the apparent discrepancy in D/H is not a good reason for excluding the nebular origin of water.Comment: Accepted to Icaru