Université PSL



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Live Imaging of Bicoid-Dependent Transcription in Drosophila Embryos
Tanguy Lucas, Teresa Ferraro, Baptiste Roelens, Jose De Las Heras Chanes, Aleksandra M. Walczak, Mathieu Coppey, and Nathalie Dostatni
Curr Biol. - Vol. 23(21) 2135–39 - DOI: 10.1016/j.cub.2013.08.053 - 2013
The early Drosophila embryo is an ideal model to understand the transcriptional regulation of well-defined patterns of gene expression in a developing organism [ 1 ]. In this system, snapshots of transcription measurements obtained by RNA FISH on fixed samples [ 2, 3 ] cannot provide the temporal resolution needed to distinguish spatial heterogeneity [ 3 ] from inherent noise [ 4, 5 ]. Here, we used the MS2-MCP system [ 6, 7 ] to visualize in living embryos nascent transcripts expressed from the canonical hunchback (hb) promoter [ 8, 9 ] under the control of Bicoid (Bcd) [ 10 ]. The hb-MS2 reporter is expressed as synchronously as endogenous hb in the anterior half of the embryo, but unlike hb it is also active in the posterior, though more heterogeneously and more transiently than in the anterior. The length and intensity of active transcription periods in the anterior are strongly reduced in absence of Bcd, whereas posterior ones are mostly Bcd independent. This posterior noisy signal decreases progressively through nuclear divisions, so that the MS2 reporter expression mimics the known anterior hb pattern at cellularization. We propose that the establishment of the hb pattern relies on Bcd-dependent lengthening of transcriptional activity periods in the anterior and may require two distinct repression mechanisms in the posterior.
Foam Drainage Control Using Thermocapillary Stress in a Two-Dimensional Microchamber
V. Miralles, B. Selva, I. Cantat, and M.-C. Jullien
Phys. Rev. Lett. - Vol.112(23) 238302 - DOI: 10.1103/PhysRevLett.112.238302 - 2013
We investigate the drainage of a 2D microfoam in a vertical Hele-Shaw cell, and show that the Marangoni stress at the air-water interface generated by a constant temperature gradient applied in situ can be tuned to control the drainage. The temperature gradient is applied in such a way that thermocapillarity and gravity have an antagonistic effect. We characterize the drainage over time by measuring the liquid volume fraction in the cell and find that thermocapillarity can overcome the effect of gravity, effectively draining the foam towards the top of the cell, or exactly compensate it, maintaining the liquid fraction at its initial value over at least 60 s. We quantify these results by solving the mass balance in the cell, and provide insight into the interplay between gravity, thermocapillarity, and capillary pressure governing the drainage dynamics.
A low cost and high throughput magnetic bead-based immuno-agglutination assay in confined droplets
Teste B, Ali-Cherif A, Viovy JL and Malaquin L
Lab. Chip - 13(12) 2344-9 - DOI: 10.1039/c3lc50353d - 2013
Although passive immuno-agglutination assays consist of one step and simple procedures, they are usually not adapted for high throughput analyses and they require expensive and bulky equipment for quantitation steps. Here we demonstrate a low cost, multimodal and high throughput immuno-agglutination assay that relies on a combination of magnetic beads (MBs), droplets microfluidics and magnetic tweezers. Antibody coated MBs were used as a capture support in the homogeneous phase. Following the immune interaction, water in oil droplets containing MBs and analytes were generated and transported in Teflon tubing. When passing in between magnetic tweezers, the MBs contained in the droplets were magnetically confined in order to enhance the agglutination rate and kinetics. When releasing the magnetic field, the internal recirculation flows in the droplet induce shear forces that favor MBs redispersion. In the presence of the analyte, the system preserves specific interactions and MBs stay in the aggregated state while in the case of a non-specific analyte, redispersion of particles occurs. The analyte quantitation procedure relies on the MBs redispersion rate within the droplet. The influence of different parameters such as magnetic field intensity, flow rate and MBs concentration on the agglutination performances have been investigated and optimized. Although the immuno-agglutination assay described in this work may not compete with enzyme linked immunosorbent assay (ELISA) in terms of sensitivity, it offers major advantages regarding the reagents consumption (analysis is performed in sub microliter droplet) and the platform cost that yields to very cheap analyses. Moreover the fully automated analysis procedure provides reproducible analyses with throughput well above those of existing technologies. We demonstrated the detection of biotinylated phosphatase alkaline in 100 nL sample volumes with an analysis rate of 300 assays per hour and a limit of detection of 100 pM.
New non-covalent strategies for stable surface treatment of thermoplastic chips
Karla Perez-Toralla, Jérôme Champ, Mohamad Reza Mohamadi, Olivier Braun, Laurent Malaquin, Jean-Louis Viovy and Stéphanie Descroix
Lab. Chip - 13(22) 4409-18 - DOI: 10.1039/c3lc50888a - 2013
In order to be more extensively used outside of research laboratories, lab-on-chip technologies must be mass-produced using low-cost materials such as thermoplastics. Thermoplastics, however, are generally hydrophobic in their native state, which makes them unsuitable for direct use with biological samples in aqueous solution, and thus require surface coating. This coating should be robust, inexpensive and simple to implement, in order not to hinder the industrial advantage of thermoplastic chips. Cyclic Olefin Copolymer (COC) is a particularly appealing polymer, but it is also difficult to functionalize due to its chemical inertness. Here we introduce and compare the performance of two new approaches for COC coating. One relies on the use of a commercial triblock copolymer, Pluronic® F127. The second approach uses new copolymers synthesized by radical polymerization, and consisting of a dimethylacrylamide (DMA) backbone carrying aliphatic side chains (C22). Two DMA-C22 copolymers were synthesized with various C22/DMA ratios: DMA-S at 0.175% and DMA-M at 0.35%. Different physicochemical properties of the polymers such as critical micellar concentration (CMC), water contact angle, electroosmosis were investigated. Coated COC chips were then tested for their ability to reduce the adsorption of proteins, microparticles, and for protein electrophoresis. For each application we found an optimal treatment protocol to considerably improve the performance of the thermoplastic chip. These treatments use physisorption in situ which requires no photografting or chemical reaction and can be performed by a simple incubation either after chip production, or just prior to use.
FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool
Perez-Toralla Karla, Mottet Guillaume, Guneri Ezgi Tulukcuoglu, Champ Jérôme, Bidard François-Clément, Jean-Yves Pierga, Jerzy Klijanienko, Irena Draskovic, Laurent Malaquin, Jean-Louis Viovya and Stéphanie Descroix*
Lab. Chip - 15 (2015) 811-22 - DOI: 10.1039/c4lc01059k - 2013
Microfluidic systems bear promise to provide new powerful tools for the molecular characterization of cancer cells, in particular for the routine detection of multiple cancer biomarkers using a minute amount of the sample. However, taking miniaturized cell-based assays into the clinics requires the implementation and validation of complex biological protocols on chip, as well as the development of disposable microdevices produced at a low cost. Based on a recently developed microfluidic chip made of Cyclic Olefin Copolymer for cell immobilization with minimal dead volume and controlled shear stress, we developed a protocol performed entirely in the liquid phase, allowing the immobilization and fixation of cells and their quantitative characterization by fluorescence in situ hybridization. We demonstrated first in cell lines and then in two clinical case studies the potential of this method to perform quantitative copy number measurement and clinical scoring of the amplification of the ERBB2 gene, a decisive biomarker for the prescription of HER2+ related targeted therapies. This validation was performed in a blind protocol in two clinical case studies, in reference to the gold standard and clinically used method based on glass slides. We obtained a comparable reproducibility and a minor difference in apparent amplification, which can be corrected by internal calibration. The method thus reaches the standard of robustness needed for clinical use. The protocol can be fully automated, and its consumption of samples and DNA probes is reduced as compared to glass slide protocols by a factor of at least 10. The total duration of the assay is divided by two.
Supershear Rayleigh Waves at a Soft Interface
Anne Le Goff, Pablo Cobelli, and Guillaume Lagubeau
Phys. Rev. Lett. - Vol.110 236101 - DOI: http://dx.doi.org/10.1103/PhysRevLett.110.236101 - 2013
We report on the experimental observation of waves at a liquid foam surface propagating faster than the bulk shear waves. The existence of such waves has long been debated, but the recent observation of supershear events in a geophysical context has inspired us to search for their existence in a model viscoelastic system. An optimized fast profilometry technique allows us to observe on a liquid foam surface the waves triggered by the impact of a projectile. At high impact velocity, we show that the expected subshear Rayleigh waves are accompanied by faster surface waves that can be identified as supershear Rayleigh waves.
Electronic hybridization detection in microarray format and DNA genotyping
Antoine Blin, Ismaïl Cissé and Ulrich Bockelmann
Scientific Reports - 4(n°4194) - DOI: 10.1038/srep04194 - 2013
We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.
Revealing the competition between peeled DNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy
Graeme A. Kinga, Peter Grossa, Ulrich Bockelmannb, Mauro Modestic, Gijs J. L. Wuitea, and Erwin J. G. Petermana
Proc. Nat. Acad. Sci. USA - vol.110 (n°10) 3859–64 - DOI: 10.1073/pnas.1213676110 - 2013
Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.
Cyclic Olefin Copolymer Plasma millireactors
Schelcher G, Guyon C, Ognier S, Cavadias S, Martinez E, Taniga V, Malaquin L, Tabeling P and Tatoulian M
Lab. Chip - 14(16) 3037-42 - DOI: 10.1039/c4lc00423j - 2013
The novelty of this paper lies in the development of a multistep process for the manufacturing of plasma millireactors operating at atmospheric pressure. The fabrication process relies on the integration of metallic electrodes over a cyclic olefin copolymer chip by a combination of photopatterning and sputtering. The developed plasma millireactors were successfully tested by creating air discharges in the gas volume of the millichannel. A sputtered silica layer was deposited on the channel walls to provide a barrier between the plasma and the polymer in order to prevent the alteration of polymer surfaces during the plasma treatment. Interest in this process of employing plasma millireactor as a high reactive environment is demonstrated here by the degradation of a volatile organic compound (acetaldehyde) in ambient air. In this miniaturized device, we obtained a high acetaldehyde conversion (98%) for a specific input energy lower than 200 J L(-1).
Red blood cells decorated with functionalized core–shell magnetic nanoparticles: elucidation of the adsorption mechanism
Thanh Duc Mai, Fanny d’Orlye, Christine Ménager, Anne Varenne and Jean-Michel Siaugue
Chem. Comm. - -49 5393—95 - DOI: 10.1039/C3CC41513A - 2013

326 publications.