Université PSL



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Signal enhancement in electronic detection of DNA hybridization
Laboratoire Nanobiophysiques - C. Gentil, G. Philippin, and U. Bockelmann
Phys. Rev. E - 75(1) :011926 - DOI:10.1103/PhysRevE.75.011926 - 2007
Electronic detection of the specific recognition between complementary DNA sequences is investigated. DNA probes are immobilized at different lateral positions on a Poly(L-lysine)-coated surface of an integrated silicon transistor array. Hybridization and field effect detection are done with the solid surface immersed in electrolyte solutions. Differential measurements are performed, where DNA hybridization leads to surface potential shifts between the transistors of the array. We experimentally show that these differential signals of hybridization can be enhanced significantly by changing the salt concentration between hybridization and detection.
Collective migration of an epithelial monolayer in response to a model wound
Laboratoire Physico-biologie aux méso-échelles - Poujade M., Grasland-Mongrain E., Hertzog A., Jouanneau J., Chavrier P., Ladoux B., Buguin A., Silberzan P.
Proc. Nat. Acad. Sci. USA - 104(41) :15988-93 - DOI:10.1073/pnas.0705062104 - 2007
Using an original microfabrication-based technique, we experimentally study situations in which a virgin surface is presented to a confluent epithelium with no damage made to the cells. Although inspired by wound-healing experiments, the situation is markedly different from classical scratch wounding because it focuses on the influence of the free surface and uncouples it from the other possible contributions such as cell damage and/or permeabilization. Dealing with Madin–Darby canine kidney cells on various surfaces, we found that a sudden release of the available surface is sufficient to trigger collective motility. This migration is independent of the proliferation of the cells that mainly takes place on the fraction of the surface initially covered. We find that this motility is characterized by a duality between collective and individual behaviors. On the one hand, the velocity fields within the monolayer are very long range and involve many cells in a coordinated way. On the other hand, we have identified very active “leader cells” that precede a small cohort and destabilize the border by a fingering instability. The sides of the fingers reveal a pluricellular actin “belt” that may be at the origin of a mechanical signaling between the leader and the followers. Experiments performed with autocrine cells constitutively expressing hepatocyte growth factor (HGF) or in the presence of exogenous HGF show a higher average velocity of the border and no leader.
Rigidity-driven growth and migration of epithelial cells on microstructured anisotropic substrates
Laboratoire Physico-biologie aux méso-échelles - Saez A., Ghibaudo M., Buguin A., Silberzan P., Ladoux B.
Proc. Nat. Acad. Sci. USA - 104(20) :8281-6 - DOI:10.1073/pnas.0702259104 - 2007
The physical properties of the cellular environment are involved in regulating the formation and maintenance of tissues. In particular, substrate rigidity appears to be a key factor dictating cell response on culture surfaces. Here we study the behavior of epithelial cells cultured on microfabricated substrates engineered to exhibit an anisotropic stiffness. The substrate consists of a dense array of micropillars of oval cross-section, so that one direction is made stiffer than the other. We demonstrate how such an anisotropic rigidity can induce directional epithelial growth and guide cell migration along the direction of greatest rigidity. Regions of high tractional stress and large cellular deformations within the sheets of cells are concentrated at the edges, in particular at the two poles of the islands along their long axis, in correlation with the orientation of actin stress fibers and focal adhesions. By inducing scattering activity of epithelial cells, we show that isolated cells also migrate along the direction of greatest stiffness. Taken together, these findings show that the mechanical interactions of cells with their microenvironment can be tuned to engineer particular tissue properties.
Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism
Laboratoire Physique des biomolécules - T. Lionnet, M. M. Spiering, S. J. Benkovic, D. Bensimon and V. Croquette
Proc. Nat. Acad. Sci. USA - 104(50) :19790–95 - DOI:10.1073/pnas.0709793104 - 2007
Helicases are enzymes that couple ATP hydrolysis to the unwinding of double-stranded (ds) nucleic acids. The bacteriophage T4 helicase (gp41) is a hexameric helicase that promotes DNA replication within a highly coordinated protein complex termed the replisome. Despite recent progress, the gp41 unwinding mechanism and regulatory interactions within the replisome remain unclear. Here we use a single tethered DNA hairpin as a real-time reporter of gp41-mediated dsDNA unwinding and single-stranded (ss) DNA translocation with 3-base pair (bp) resolution. Although gp41 translocates on ssDNA as fast as the in vivo replication fork (approximate to 400 bp/s), its unwinding rate extrapolated to zero force is much slower (approximate to 30 bp/s). Together, our results have two implications: first, gp41 unwinds DNA through a passive mechanism; second, this weak helicase cannot efficiently unwind the T4 genome alone. Our results suggest that important regulations occur within the replisome to achieve rapid and processive replication.
Highly Parallel Mix-and-Match Fabrication of Nanopillar Arrays Integrated in Microfluidic Channels for Long DNA Molecule Separation
Laboratoire Pôle Microfluidique - J. Shi, A. P. Fang, L. Malaquin, A. Pepin, D. Decanini, J. L. Viovy and Y. Chen
Applied Physics Letters - 91(15) :153114 - DOI:153114 10.1063/1.2793616 - 2007
We report on a mix-and-match method based on a combination of soft UV nanoimprint lithography, contact optical lithography, and reactive-ion-etch techniques, which is applicable for high throughput manufacturing of nanostructure integrated microfluidic devices. We demonstrate the integration of high density and high aspect ratio nanopillars into microfluidic channels as electrophoresis sieving matrices. As a result, ? DNA and T4 DNA can be separated within a few minutes. By changing the pattern design, the device could be used for separation of other types of molecules.
Microcontact Printing of Living Bacteria Arrays with Cellular Resolution
Laboratoire Pôle Microfluidique - L. P. Xu, L. Robert, O. Y. Qi, F. Taddei, Y. Chen, A.B. Lindner, D. Baigl
Nano Lett. - 7(7) :2068-72 - DOI:10.1021/nl070983z - 2007
Arrays of living bacteria were printed on agarose substrate with cellular resolution using elastomeric stamps with a high aspect ratio generated by reverse in situ lithography (RISL). The printed bacteria reproduced the original stamp patterns with high fidelity and continued growing as in bulk culture. This methodology provides a simple route to any desired bacterial spatial 2D distribution and may be applied to screening as well as to studies of bacteria phenotypic variability, population dynamics, and ecosystem evolution.
Online preconcentration using monoliths in electrochromatography capillary format and microchips
Laboratoire Sciences Analytiques, Bioanalytiques et Miniaturisation - V. Augustin, G. Proczek, J. Dugay, S. Descroix, M.C. Hennion
J. Sep. Sci. - 30(17) :2858-65 - PMID:17973277 - 2007
Online preconcentration and separation of analytes using an in situ photopolymerized hexyl acrylate-based monolith stationary phase was evaluated using electrochromatography in capillary format and microchip. The band broadening occurring during the preconcentration process by frontal electrochromatography and during the desorption process by elution electrochromatography was studied. The hexyl acrylate-based monolith provides high retention for neutral analytes allowing the handling of large sample volumes and its structure allows rapid mass transfer, thus reducing the band broadening. For moderately polar analytes such as mono-chlorophenols that are slightly retained in water, it was shown that enrichment factors up to 3500 can be obtained by a hydrodynamic injection of several bed volumes for 120 min under 0.8 MPa with a decrease in efficiency of 50% and a decrease of 30% for the resolution between 2- and 3-chlorophenol. An 8 min preconcentration time allows enrichment factors above 100 for polyaromatic hydrocarbons. The interest of these monoliths when synthesized in microchip is also demonstrated. A 200-fold enrichment was easily obtained for PAHs with only 1 min as preconcentration time, without decrease in efficiency.
Time-course analysis of mouse serum proteome changes following exposure of the skin to ionizing radiation
Laboratoire Spectrométrie de masse biologique et protéomique - Guipaud O, Holler V, Buard V, Tarlet G, Royer N, Vinh J, Benderitter M.
Proteomics - 7(21) :3992-4002 - PMID:17960731 - 2007
Radiation-induced lesion outcomes of normal tissues are difficult to predict. In particular, radiotherapy or local exposure to a radioactive source by accident can trigger strong injury to the skin. The finding of biomarkers is of fundamental relevance for the prediction of lesion apparition and its evolution, and for the settlement of therapeutic strategies. In order to study radiation-induced cutaneous lesions, we developed a mouse model in which the dorsal skin was selectively exposed to ionizing radiation (IR). 2-D difference gel electrophoresis (2-D DIGE) coupled with MS was used to investigate proteins altered in expression and/or PTM in serum. Proteome changes were monitored from 1 day to 1 month postirradiation, at a dose of 40 Gy, in this specific model developing reproducible clinical symptoms ranging from erythema to skin ulceration with wound healing. About 60 proteins (including some isoforms and likely post-translational variants), representing 20 different proteins, that exhibited significant and reproducible kinetic expression changes, were identified using MS and database searches. Several proteins, down- or up-regulated from day one, could prove to be good candidates to prognosticate the evolution of a skin lesion such as necrosis. In addition, we observed shifts in pI of several spot trains, revealing potential PTM changes, which could also serve as indicators of irradiation or as predictors of lesion severity.
Polyadenylation of a functional mRNA controls gene expression in Escherichia coli
Laboratoire Spectrométrie de masse biologique et protéomique - Joanny G, Le Derout J, Bréchemier-Baey D, Labas V, Vinh J, Régnier P, Hajnsdorf E.
Nucleic Acids Res. - 35(8) :2494-502 - PMID:17395638 - 2007
Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5' monophosphate and a 3' stable hairpin is highly susceptible to poly(A)-dependent degradation.
In vivo electrochemical detection of nitric oxide in tumor-bearing mice
Laboratoire Synthèse, Electrochimie, Imagerie et Systèmes Analytiques... - Griveau S., Dumezy C, Seguin J., Chabot GG. Scherman D., Bedioui F.
Anal. Chem. - 79(3) :1030-3 - PMID:17263331 - 2007
Interest in elucidating the mechanisms of action of various classes of anticancer agents and exploring the pathways of the induced-nitric oxide (NO) release provides an impetus to conceive a better designed approach to locally detect NO in tumors, in vivo. We report here on the first use of an electrochemical sensor that allows the in vivo detection of NO in tumor-bearing mice. In a first step, we performed the electrochemical characterization of a stable electroactive probe, K4Fe(CN)6, directly injected into the liquid microenvironment especially created around the electrode in the tumor. Second, the ability of the inserted electrode system to detect the presence of NO itself in the tumoral tissue was achieved by using the chemically modified Pt/Ir electrode as NO sensor and two NO donor molecules: diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium 1,2-diolate (DEA-NONOate) and (Z)-1-[N-(2-aminopropyl)-N-(2-ammonio propyl)amino]diazen-1-ium 1,2-diolate (PAPA-NONOate). These two NO donor molecules allowed proving the electrochemical detection of (i) directly injected exogenous NO phosphate buffer solution into the tumor (decomposed DEA-NONOate) and (ii) biomimetically induced endogeneous release of NO in the tumoral tissue, upon injection of PAPA-NONOate into the tumor. This approach could be applied to the in vivo study of candidate anticancer drugs acting on the NO pathways.

410 publications.