Université PSL



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Analysis of gene expression at a single cell level in microdroplets
Laboratoire Microfluidique MEMS et nanostructures - P. Mary, L. Dauphinot, N. Bois, M.C Potier, V. Studer, P. Tabeling
Biomicrofluidics - 5(2) :24109 - DOI:10.1063/1.3596394 - 2011
In the present work, we have measured the messenger RNA expression of specific genes both from total RNA and cells encapsulated in droplets. The microfluidic chip introduced includes the following functionalities: RNA/cell encapsulation, lysis, reverse transcription and real-time polymerase chain reaction. We have shown that simplex and duplex gene expression measurements can be carried out over a population of 100 purified RNA samples encapsulated simultaneously in 2 nl droplets in less than 2 h. An analysis of 100 samples containing one to three cells has shown excellent consistency with standard techniques regarding average values. The cell-to-cell distributions of the E-cadherin expression suggest fluctuations on the order of 80% in the number of transcripts, which is highly consistent with the general findings from the literature. A mathematical model has also been introduced to strengthen the interpretation of our results. The present work paves the way for the systematic acquisition of such information in biological and biomedical studies.
Ultrasound Internal Tattooing
Laboratoire Microfluidique MEMS et nanostructures - O. Couture, M. Faivre, N. Pannacci, A. Babataheri, V. Servois, P. Tabeling, M. Tanter
Med. Phys - 38(2) :1116-23 - PMID:21452748 - 2011
The ability of remotely tagging tissues in a controlled and three-dimensional manner during preoperative imaging could greatly help surgeons to identify targets for resection. The authors' objective is to selectively and noninvasively deposit markers under image guidance for such internal tattooing.
This study describes the production of new ultrasound-inducible droplets carrying large payloads of fluorescent markers and the in vivo proof of concept of their remote and controlled deposition via focused ultrasound. The droplets are monodispersed multiple emulsions produced in a microfluidic system, consisting of aqueous fluorescein in perfluorocarbon in water. Their conversion (either by vaporization or cavitation) is performed remotely using a clinical ultrasonic imaging probe.
When submitted to 5 MHz imaging pulses, the droplets vaporize in vitro at 1.4 MPa peak-negative pressure and eject their content. After several seconds, a brightly fluorescent spot (0.5 mm diameter) is observed at the focus of the transducer. Experiments in the chorioallantoique membrane of chicken eggs and chicken embryo demonstrate that the spot is stable and is easily seen by naked eye.
These ultrasound-inducible multiple emulsions could be used to deliver large amounts of contrast agents, chemotherapy, and genetic materials in vivo using a conventional ultrasound scanner.
Probing ribosomal protein–RNA interactions with an external force
Laboratoire Nanobiophysiques - Pierre Mangeol, Thierry Bizebard, Claude Chiaruttini, Marc Dreyfus, Mathias Springer, and Ulrich Bockelmann
Proc. Nat. Acad. Sci. USA - 108(45) :18272-6 - DOI:10.1073/pnas.1107121108 - 2011
Ribosomal (r-) RNA adopts a well-defined structure within the ribosome, but the role of r-proteins in stabilizing this structure is poorly understood. To address this issue, we use optical tweezers to unfold RNA fragments in the presence or absence of r-proteins. Here, we focus on Escherichia coli r-protein L20, whose globular C-terminal domain (L20C) recognizes an irregular stem in domain II of 23S rRNA. L20C also binds its own mRNA and represses its translation; binding occurs at two different sites—i.e., a pseudoknot and an irregular stem. We find that L20C makes rRNA and mRNA fragments encompassing its binding sites more resistant to mechanical unfolding. The regions of increased resistance correspond within two base pairs to the binding sites identified by conventional methods. While stabilizing specific RNA structures, L20C does not accelerate their formation from alternate conformations—i.e., it acts as a clamp but not as a chaperone. In the ribosome, L20C contacts only one side of its target stem but interacts with both strands, explaining its clamping effect. Other r-proteins bind rRNA similarly, suggesting that several rRNA structures are stabilized by “one-side” clamping.
Quantifying how DNA stretches, melts and changes twist under tension
Laboratoire Nanobiophysiques - Peter Gross, Niels Laurens, Lene B. Oddershede, Ulrich Bockelmann, Erwin J.G. Peterman, and Gijs J. L. Wuite
Nature Physics - 7 :731-6 - DOI:10.1038/nphys2002 - 2011
In cells, DNA is constantly twisted, bent and stretched by numerous proteins mediating genome transactions. Understanding these essential biological processes requires in-depth knowledge of how DNA complies to mechanical stress. Two important physical features of DNA, helical structure and sequence, are not incorporated in current descriptions of DNA elasticity. Here we connect well-defined force–extension measurements with a new model for DNA elasticity: the twistable worm-like chain, in which DNA is considered a helical, elastic entity that complies to tension by extending and twisting. In addition, we reveal hitherto unnoticed stick–slip dynamics during DNA overstretching at ~65?pN, caused by the loss of base-pairing interactions. An equilibrium thermodynamic model solely based on DNA sequence and elasticity is presented, which captures the full complexity of this transition. These results offer deep quantitative insight in the physical properties of DNA and present a new standard description of DNA mechanics.
Rectification of the current in alpha-hemolysin pore depends on the cation type : the alkali series probed by MD simulations and experiments
Laboratoire Nanobiophysiques - S. Bhattacharya, L. Muzard, L. Payet, J. Mathe, U. Bockelmann, A. Aksimentiev, and V. Viasnoff
J Phys Chem C - 115(10) :4255-64 - PMID:21860669 - 2011
A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel's end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.
Orientation and polarity in collectively migrating cell structures: statics and dynamics
Laboratoire Physico-biologie aux méso-échelles - Reffay M., Petitjean L., Coscoy S., Grasland-Mongrain E., Amblard F., Buguin A., Silberzan P.
Biophys. J. - 100(11) :2566-75 - DOI:10.1016/j.bpj.2011.04.047. - 2011
Collective cell migration is often characterized by the spontaneous onset of multicellular protrusions (known as fingers) led by a single leader cell. Working with epithelial Madin-Darby canine kidney monolayers we show that cells within the fingers, as compared with the epithelium, are well oriented and polarized along the main finger direction, which suggests that these cells actively migrate. The cell orientation and polarity decrease continuously from the tip toward the epithelium over a penetration distance of typically two finger lengths. Furthermore, laser photoablation experiments at various locations along these fingers demonstrate that the cells in the fingers are submitted to a tensile stress whose value is larger close to the tip. From a dynamical point of view, cells entering a finger gradually polarize on timescales that depend upon their particular initial position. Selective laser nanosurgery of the leader lamellipodium shows not only that these structures need a leader to progress, but that this leader itself is the consequence of a prior self-organization of the cells forming the finger. These results highlight the complex interplay between the collective orientation within the fingers and the mechanical action of the leader.
Directional persistence of chemotactic bacteria in a traveling concentration wave
Laboratoire Physico-biologie aux méso-échelles - Saragosti J., Calvez V., Bournaveas N., Perthame B., Buguin A., Silberzan P.
Proc. Nat. Acad. Sci. USA - 108 :16235 - DOI:10.1073/pnas.1101996108 - 2011
Chemotactic bacteria are known to collectively migrate towards sources of attractants. In confined convectionless geometries, concentration “waves” of swimming Escherichia coli can form and propagate through a self-organized process involving hundreds of thousands of these microorganisms. These waves are observed in particular in microcapillaries or microchannels; they result from the interaction between individual chemotactic bacteria and the macroscopic chemical gradients dynamically generated by the migrating population. By studying individual trajectories within the propagating wave, we show that, not only the mean run length is longer in the direction of propagation, but also that the directional persistence is larger compared to the opposite direction. This modulation of the reorientations significantly improves the efficiency of the collective migration. Moreover, these two quantities are spatially modulated along the concentration profile. We recover quantitatively these microscopic and macroscopic observations with a dedicated kinetic model.
Integrated and Diffusion-Based Micro-Injectors for Open Access Cell Assays
Laboratoire Pôle Microfluidique - X. Li, L. Liu, L. Wang, K. Kamei, Q. H. Yuan, F. Zhang, J. Shi, A. Kusumi, M. Xie, Z. J. Zhao and Y. Chen
Lab. Chip - 11 :2612-7 - DOI:10.1039/c1lc20258h - 2011
Currently, most microfluidic devices are fabricated with embedded micro-channels and other elements in a close form with outward connections. Although much functionality has been demonstrated and a large number of applications have been developed, they are not easy for routine operation in biology laboratories where most in vitro cell processing still relies on the use of culture dishes, glass slides, multi-well plates, tubes, pipettes, etc. We report here an open access device which consists of an array of isolated micro-channels plated on a large culture surface, each of them having tiny nozzles for localized drug delivery. In a diffusion dominant regime, steady gradients of molecule concentration could be obtained and varied by changing the flow rate inside the micro-channels. As assay examples, cell staining and drug-induced cell apoptosis were demonstrated, showing fast cell responses in close proximity of the nozzles.
Photoreversible Fragmentation of a Liquid Interface for Micro-Droplet Generation by Light Actuation
Laboratoire Pôle Microfluidique - A. Diguet, H. Li, N. Queyriaux, Y. Chen and D. Baigl
Lab. Chip - 11 :2666-9 - DOI:10.1039/c1lc20328b - 2011
We describe a method to induce by light a reversible switch from a continuous two-phase laminar flow to a droplet generating regime, in microfluidic devices with a usual water-in-oil flow focusing geometry. It consists in adding a photosensitive surfactant to the aqueous phase to modulate using light the interfacial energy between flowing liquids and the microfluidic substrate. We show that UV irradiation induces liquid fragmentation into monodisperse water microdroplets and that many cycles of reversible and rapid switches (<2 s) between continuous laminar flows and stable droplet regimes can be realized. By spatially controlling the application of the light stimulus, we also demonstrate the first spatially resolved remote induction of droplet generation.
Fluidized bed plasma for pre-treatment of Co-ferrierite catalysts: An approach to NOx abatement
Laboratoire Procédés - Plasmas - Microsystèmes - R. Bartolomeu, M. Foix, A. Fernandes, M. Tatoulian, M.F. Ribeiro, C. Henriques, P. Da Costa
CATALYSIS TODAY - 176(1) :234-8 - DOI:10.1016/j.cattod.2010.12.051 - 2011
Replacement of calcination procedures used during catalyst preparation, by a plasma treatment, was studied over a Co-ferrierite (Co-FER) catalyst. The catalyst was tested in the NOx selective catalytic reduction reaction. A combination of UV–Vis spectroscopy and TG analysis revealed the presence of ammonium ions on the untreated and plasma Co-FER samples but not on the calcined one. Therefore, it can be concluded that the plasma treatment was not able to replace the thermal calcination step. The evaluation of catalyst behaviour was performed both under temperature programmed surface reaction (TPSR) and under steady-state conditions at different temperatures. NO oxidation tests showed that, during TPSR runs, calcined catalyst produces more NO2 than plasma catalyst. NOx consumption during TPSR of plasma catalyst confirms that precursors used on the ion-exchange procedure are still present on the catalyst even after the plasma treatment, reacting with NO to produce R-NOx, N2O and N2. Concerning deNOx tests using ethanol as reducing agent, TPSR tests showed higher NOx conversions over untreated and plasma catalysts due to the presence of ammonium and acetate precursors on these catalysts. Untreated, plasma and calcined catalysts present the same NOx and COx conversions in isothermal tests.

410 publications.