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Accueil > Publications > Recherche par années > Années 2010 > 2013

2013

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A comparison of reduced coordinate sets for describing protein structure

In all-atom molecular simulation studies of proteins, each atom in the protein is represented by a point mass and interactions are defined in terms of the atomic positions. In recent years, various simplified approaches have been proposed. These approaches aim to improve computational effi- ciency and to provide a better physical insight. The simplified models can differ widely in their description of the geometry and the interactions inside the protein. This study explores the most fun- damental choice in the simplified protein models : the choice of a coordinate set defining the protein structure. A simplified model can use fewer point masses than the all-atom model and/or eliminate some of the internal coordinates of the molecule by setting them to an average or ideal value. We look at the implications of such choices for the overall protein structure. We find that care must be taken for angular coordinates, where even very small variations can lead to significant changes in the positions of far away atoms. In particular, we show that the φ/ψ torsion angles are not a sufficient coordinate set, whereas another coordinate set with two degrees of freedom per residue, virtual Cα backbone bond, and torsion angles performs satisfactorily.

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A Postsynthetic Modification of II-VI Nanoparticles to Create Tb3+ and Eu3+ Luminophores

We describe a novel method for creating luminescent lanthanide-containing nanoparticles in which the lanthanide cations are sensitized by the semiconductor nanoparticle’s electronic excitation. In contrast to previous strategies, this new approach creates such materials by addition of external salt to a solution of fully formed nanoparticles. We demonstrate this post-synthetic modification for the lanthanide luminescence sensitization of two visible emitting lanthanides (Ln), Tb3+ and Eu3+ ions, through ZnS nanoparticles in which the cations were added post-synthetically as external Ln(NO3)3.xH2O salt to solutions of ZnS nanoparticles. The post-synthetically treated ZnS nanoparticle systems display Tb3+ and Eu3+ luminescence intensities that are comparable to those of doped Zn(Ln)S nanoparticles, which we reported previously (J. Phys. Chem. A, 2011, 115, 4031-4041). A comparison with the synthetically doped systems is used to contrast the spatial distribution of the lanthanide ions, bulk versus surface localized. The post-synthetic strategy described in this work is fundamentally different from the synthetic incorporation (doping) approach and offers a rapid and less synthetically demanding protocol for Tb3+:ZnS and Eu3+:ZnS luminophores, thereby facilitating their use in a broad range of applications.

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Ability of plasmid DNA complexed with histidinylated lPEI and lPEI to cross in vitro lung and muscle vascular endothelial barriers

DNA complexes made with cationic polymers (polyplexes) developed as nonviral vectors for gene therapy must be enabled to cross through vascular endothelium to transfect underlying tissues upon their administration in the blood circulation. Here, we evaluated the transendothelial passage (TEP) of DNA complexes made with histidinylated linear polyethylenimine (His-lPEI) or linear polyethylenimine (lPEI). In vitro studies were performed by using established transwell lung and skeletal muscle vascular endothelial barriers. The models were composed of a monolayer of human lung microvascular endothelial (HMVEC-L) cells and mouse cardiac endothelial (MCEC) cells formed on a PET insert and immortalized human tracheal epithelial (ΣCFTE29o-) cells and mouse myoblasts (C2C12) as target cells cultured in the lower chamber, respectively. When the vascular endothelium monolayer was established and characterized, the transfection efficiency of target (ΣCFTE29o- and C2C12) cells with plasmid DNA encoding luciferase was used to evaluate TEP of polyplexes. The luciferase activities with His-lPEI and lPEI polyplexes compared to those obtained in the absence of endothelial cell monolayer were 6.5% and 4.3% into ΣCFTE29o- cells, and 18.5% and 0.23% into C2C12 cells, respectively. The estimated rate for His-lPEI polyplexes was 0.135 μg/cm(2).h and 0.385 μg/cm(2).h through the HMVEC-L and MCEC monolayers, respectively. These results indicate that His-lPEI polyplexes can pass through the lung and skeletal muscle vascular endothelium and can transfect underlying cells.

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Absence of thermalization for systems with long-range interactions coupled to a thermal bath

We investigate the dynamics of a small long-range interacting system, in contact with a large long-range thermal bath. Our analysis reveals the existence of striking anomalies in the energy flux between the bath and the system. In particular, we find that the evolution of the system is not influenced by the kinetic temperature of the bath, as opposed to what happens for short-range collisional systems. As a consequence, the system may get hotter also when its initial temperature is larger than the bath temperature. This observation is explained quantitatively in the framework of the collisionless Vlasov description of toy models with long-range interactions and shown to be valid whenever the Vlasov picture applies, from cosmology to plasma physics.

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Amide conjugates of the DO3A-N-(α-amino)propionate ligand : leads for stable, high relaxivity contrast agents for MRI ?

A novel synthetic methodology for preparing amide conjugates of the DO3A-N-(α-amino)propionate chelator is described, using the synthesis of the DO3A-N-(α-benzoylamido)propionate chelator as an illustrative example. The model Gd[DO3A-N-(α-benzoylamido)propionate] chelate displays accelerated water exchange, stability in a wide pH range and inertness towards transmetallation by Zn(2+). The Gd[DO3A-N-(α-benzoylamido)propionate] complex is mainly excreted via the kidneys, producing a significant increase in the kidney medulla/cortex enhancement ratio in MR images of Wistar rats, reflecting probably its higher lipophilicity compared with Gd(DTPA). The results presented suggest that Gd[DO3A-N-(α-amido)propionate] chelates can be valuable leads for preparing potentially safe high relaxivity MRI contrast agents.

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An E3-14.7K peptide that promotes microtubules-mediated transport of plasmid DNA increases polyplexes transfection efficiency

Chemical vectors as cationic polymers and cationic lipids are promising alternatives to viral vectors for gene therapy. Beside endosome escape and nuclear import, plasmid DNA (pDNA) migration in the cytosol toward the nuclear envelope is also regarded as a limiting step for efficient DNA transfection with non-viral vectors. Here, the interaction between E3-14.7K and FIP-1 to favor migration of pDNA along microtubules is exploited. E3-14.7K is an early protein of human adenoviruses that interacts via FIP-1 (Fourteen.7K Interacting Protein 1) protein with the light-chain components of the human microtubule motor protein dynein (TCTEL1). This peptide is conjugated with pDNA and mediates interaction of pDNA in vitro with isolated microtubules as well as with microtubules in cellulo. Videomicroscopy and tracking treatment of images clearly demonstrate that P79-98/pDNA conjugate exhibits a linear transport with large amplitude along microtubules upon 2 h transfection with polyplexes whereas control pDNA conjugate exhibits small non-directional movements in the cytoplasm. Remarkably, P79-98/peGFP polyplexes enhance by a factor 2.5 (up to 76%) the number of transfected cells. The results demonstrate, for the first time, that the transfection efficiency of polyplexes can be drastically increased when the microtubules migration of pDNA is facilitated by a peptide allowing pDNA docking to TCTEL1. This is a real breakthrough in the non viral gene delivery field that opens hope to build artificial viruses.

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Analysis of the scientific capabilities of the ExoMars Raman Laser Spectrometer instrument

The Raman Laser Spectrometer (RLS) is part of the payload of the 2018 ExoMars rover. The Sample Preparation and Distribution System (SPDS) of the rover will crush samples acquired from down to two meters depth under the Martian surface, and provide them to the RLS instrument in the form of flattened powdered samples. The RLS instrument will acquire a minimum of 20 points on the flattened surface of the samples. To be able to obtain the maximum scientific return from the instrument once on Mars, a simulator of the SPDS system has been built to perform a series of experiments in a representative scenario. The crushing process implies the loss of rock structure and texture and, hence, the geological context of the samples. However, qualitative analysis with the RLS simulator on powdered natural samples and rocks showed that the RLS is capable of detecting carbonaceous material occurring in trace amounts in one of the rock samples (a silicified volcanic sand), more easily than with the same analysis on bulk. Furthermore, it is shown that minor phases in carbonate cements that cannot be detected by Raman in the bulk sample can be detected in the powder, thus allowing the identification of all the carbonate phases present in the cement crust.

In order to quantify the detection threshold of the instrument, further analysis on controlled samples were performed. The results with the RLS SPDS simulator showed that the instrument can reach detection thresholds down to 1 % on powdered samples. Furthermore, analysis of controlled mixtures showed that performing a very simple intensity-based statistical analysis of the spectra can provide semi-quantification of the abundance of the mineral species with quite linear calibration curves.

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Characterization of histidine-aspartate kinase HK1 and identification of histidine phosphotransfer proteins as potential partners in a Populus multistep phosphorelay

In poplar, we identified proteins homologous to yeast proteins involved in osmosensing multistep phosphorelay Sln1p-Ypd1p-Ssk1p. This finding led us to speculate that Populus cells could sense osmotic stress by a similar mechanism. This study focuses on first and second protagonists of this possible pathway : a histidine-aspartate kinase (HK1), putative osmosensor and histidine phosphotransfer proteins (HPt1 to 10), potential partners of this HK. Characterization of HK1 showed its ability to homodimerize in two-hybrid tests and to act as an osmosensor with a kinase activity in yeast, by functional complementation of sln1Δ sho1Δ strain. Moreover, in plant cells, plasma membrane localization of HK1 is shown. Further analysis on HPts allowed us to isolate seven new cDNAs, leading to a total of 10 different HPts identified in poplar. Interaction tests showed that almost all HPts can interact with HK1, but two of them exhibit stronger interactions, suggesting a preferential partnership in poplar. The importance of the phosphorylation status in these interactions has been investigated with two-hybrid tests carried out with mutated HK1 forms. Finally, in planta co-expression analysis of genes encoding these potential partners revealed that only three HPts are co-expressed with HK1 in different poplar organs. This result reinforces the hypothesis of a partnership between HK1 and these three preferential HPts in planta. Taken together, these results shed some light on proteins partnerships that could be involved in the osmosensing pathway in Populus.

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Co-transcriptional recruitment of the RNA exosome cofactors Rrp47p, Mpp6p and two distinct TRAMP complexes assists the exonuclease Rrp6p in the targeting and degradation of an aberrant mRNP in yeast

The co-transcriptional mRNA processing and packaging reactions that lead to the formation of export-competent mRNPs are under the surveillance of quality control steps. Aberrant mRNPs resulting from faulty events are retained in the nucleus with ensuing elimination of their mRNA component. The molecular mechanisms by which the surveillance system recognizes defective mRNPs and stimulates their destruction by the RNA degradation machinery are still not completely elucidated. Using an experimental approach in which mRNP formation in yeast is disturbed by the action of the bacterial Rho helicase, we have shown previously that the targeting of Rho-induced aberrant mRNPs is mediated by Rrp6p which is recruited co-transcriptionally in association with Nrd1p following Rho action. Here, we investigated the specific involvement in this quality control process of different cofactors associated with the nuclear RNA degradation machinery. We show that, in addition to the main hydrolytic action of the exonuclease Rrp6p, the cofactors Rrp47p and Mpp6p as well as the TRAMP components Trf4p, Trf5p and Air2p contribute significantly by stimulating the degradation process upon their co-transcriptional recruitment. Trf4p and Trf5p are apparently recruited in two distinct TRAMP complexes that both contain Air2p as component. Surprisingly, Rrp47p appears to play an important role in mutual protein stabilization with Rrp6p which highlights a close association between the two partners. Together, our results provide an integrated view of how different cofactors of the RNA degradation machinery cooperate to target and eliminate aberrant mRNPs.

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Constitutive expression of ligand for natural killer cell NKp44 receptor (NKp44L) by normal human articular chondrocytes

Normal chondrocytes display susceptibility to lysis by natural killer (NK) cells and this phenomenon may play a role in some inflammatory cartilage disorders. The mechanisms of chondrocyte recognition and killing by NK cells remain unclear. Using flow cytometry and immunohistochemical staining we found that normal human articular chondrocytes constitutively express a ligand for NKp44, one of stimulatory NK cell receptors involved in recognition and killing of target cells. Expression of NKp44 ligand by normal articular chondrocytes is not involved in their killing by unstimulated NK cells ; however, it is responsible for anti-chondrocyte cytotoxicity mediated by long-term activated NK cells. Thus, expression of NKp44 ligand may play a role in chondrocyte destruction in course of chronic inflammatory cartilage disorders.

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