Université PSL

Publications

RECHERCHER

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RACK1 controls IRES-mediated translation of viruses
Majzoub K., Hafirassou M.L, Meignin C., Goto A., Marzi S., Fedorova A., Verdier Y., Vinh J., Hoffmann J.A, Martin F., Baumert T.F, Schuster C., Imler J.L
Cell - 159(5) 1086-95 - DOI: 10.1016/j.cell.2014.10.041 - 2014
Fighting viral infections is hampered by the scarcity of viral targets and their variability, resulting in development of resistance. Viruses depend on cellular molecules-which are attractive alternative targets-for their life cycle, provided that they are dispensable for normal cell functions. Using the model organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by internal ribosome entry site (IRES)-containing viruses. We further show that RACK1 is an essential determinant for hepatitis C virus translation and infection, indicating that its function is conserved for distantly related human and fly viruses. Inhibition of RACK1 does not affect Drosophila or human cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for RACK1 in selective mRNA translation and uncover a target for the development of broad antiviral intervention.
Single-cell analysis and sorting using droplet-based microfluidics
Linas Mazutis, John Gilbert, W Lloyd Ung, David A Weitz, Andrew D Griffiths and John A Heyman
Nature Protocols - -8 870–891 - DOI: 10.1038/nprot.2013.046 - 2013
We present a droplet-based microfluidics protocol for high-throughput analysis and sorting of single cells. Compartmentalization of single cells in droplets enables the analysis of proteins released from or secreted by cells, thereby overcoming one of the major limitations of traditional flow cytometry and fluorescence-activated cell sorting. As an example of this approach, we detail a binding assay for detecting antibodies secreted from single mouse hybridoma cells. Secreted antibodies are detected after only 15 min by co-compartmentalizing single mouse hybridoma cells, a fluorescent probe and single beads coated with anti-mouse IgG antibodies in 50-pl droplets. The beads capture the secreted antibodies and, when the captured antibodies bind to the probe, the fluorescence becomes localized on the beads, generating a clearly distinguishable fluorescence signal that enables droplet sorting at ∼200 Hz as well as cell enrichment. The microfluidic system described is easily adapted for screening other intracellular, cell-surface or secreted proteins and for quantifying catalytic or regulatory activities. In order to screen ∼1 million cells, the microfluidic operations require 2–6 h; the entire process, including preparation of microfluidic devices and mammalian cells, requires 5–7 d.
Multiplex Picodroplet Digital PCR to Detect KRAS Mutations in Circulating DNA from the Plasma of Colorectal Cancer Patients
Valerie Taly, Deniz Pekin, Leonor Benhaim, Steve K. Kotsopoulos, Delphine Le Corre, Xinyu Li, Ivan Atochin, Darren R. Link, Andrew D. Griffiths, Karine Pallier, Hélène Blons, Olivier Bouche, Bruno Landi, J. Brian Hutchison, and Pierre Laurent-Puig
Clin. Chem. - 59(12) 1722-31 - DOI: 10.1373/clinchem.2013.206359 - 2013
BACKGROUND: Multiplex digital PCR (dPCR) enables noninvasive and sensitive detection of circulating tumor DNA with performance unachievable by current molecular-detection approaches. Furthermore, picodroplet dPCR facilitates simultaneous screening for multiple mutations from the same sample.

METHODS: We investigated the utility of multiplex dPCR to screen for the 7 most common mutations in codons 12 and 13 of the KRAS (Kirsten rat sarcoma viral oncogene homolog) oncogene from plasma samples of patients with metastatic colorectal cancer. Fifty plasma samples were tested from patients for whom the primary tumor biopsy tissue DNA had been characterized by quantitative PCR.

RESULTS: Tumor characterization revealed that 19 patient tumors had KRAS mutations. Multiplex dPCR analysis of the plasma DNA prepared from these samples identified 14 samples that matched the mutation identified in the tumor, 1 sample contained a different KRAS mutation, and 4 samples had no detectable mutation. Among the tumor samples that were wild type for KRAS, 2 KRAS mutations were identified in the corresponding plasma samples. Duplex dPCR (i.e., wild-type and single-mutation assay) was also used to analyze plasma samples from patients with KRAS-mutated tumors and 5 samples expected to contain the BRAF (v-raf murine sarcoma viral oncogene homolog B) V600E mutation. The results for the duplex analysis matched those for the multiplex analysis for KRAS-mutated samples and, owing to its higher sensitivity, enabled detection of 2 additional samples with low levels of KRAS-mutated DNA. All 5 samples with BRAF mutations were detected.

CONCLUSIONS: This work demonstrates the clinical utility of multiplex dPCR to screen for multiple mutations simultaneously with a sensitivity sufficient to detect mutations in circulating DNA obtained by noninvasive blood collection.
Enhanced imine synthesis in water: from surfactantmediated catalysis to host–guest mechanisms
Kamel Meguellati, Ali Fallah-Araghi, Jean-Christophe Baret, Abdeslam El Harrak, Thomas Mangeat, Carlos M. Marques, Andrew D. Griffiths and Sylvain Ladame
Chem. Comm. - 49 11332-34 - DOI: 10.1039/C3CC46461J - 2013
An environment-responsive and fluorogenic reaction is reported and used as a model system to demonstrate experimentally three mechanisms of enhanced imine synthesis in water using either surfactants (below and above their CMC) or double-stranded DNA (acting as a reaction host).
New Glycosidase Substrates for Droplet-Based Microfluidic Screening
Majdi Najah, Estelle Mayot, Putu Mahendra-Wijaya, Andrew D. Griffiths, Sylvain Ladame, and Antoine Drevelle
Anal. Chem. - 85 (20) 9807–14 - DOI: 10.1021/ac4022709 - 2013
Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30 °C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-d-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.
Droplet-based microfluidic platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms
Najah M, Calbrix R, Mahendra-Wijaya IP, Beneyton T, Griffiths A.D and Drevelle A
Chem. Biol. - 21(12) 1722-32 - DOI: 10.1016/j.chembiol.2014.10.020. - 2013
Discovery of microorganisms producing enzymes that can efficiently hydrolyze cellulosic biomass is of great importance for biofuel production. To date, however, only a miniscule fraction of natural biodiversity has been tested because of the relatively low throughput of screening systems and their limitation to screening only culturable microorganisms. Here, we describe an ultra-high-throughput droplet-based microfluidic system that allowed the screening of over 100,000 cells in less than 20 min. Uncultured bacteria from a wheat stubble field were screened directly by compartmentalization of single bacteria in 20 pl droplets containing a fluorogenic cellobiohydrolase substrate. Sorting of droplets based on cellobiohydrolase activity resulted in a bacterial population with 17- and 7-fold higher cellobiohydrolase and endogluconase activity, respectively, and very different taxonomic diversity than when selected for growth on medium containing starch and carboxymethylcellulose as carbon source.
Enhanced chemical synthesis at soft interfaces: a universal reaction-adsorption mechanism in microcompartments
A. Fallah-Araghi, K. Meguellati, J.-C. Baret, A. El Harrak, T. Mangeat, M. Karplus, S. Ladame, C. M. Marques and A.D. Griffiths
Phys. Rev. Lett. - 112 28301 - DOI: 10.1103/PhysRevLett.112.028301 - 2013
A bimolecular synthetic reaction (imine synthesis) was performed compartmentalized in micrometer-diameter emulsion droplets. The apparent equilibrium constant (Keq) and apparent forward rate constant (k1) were both inversely proportional to the droplet radius. The results are explained by a noncatalytic reaction-adsorption model in which reactants adsorb to the droplet interface with relatively low binding energies of a few kBT, react and diffuse back to the bulk. Reaction thermodynamics is therefore modified by compartmentalization at the mesoscale—without confinement on the molecular scale—leading to a universal mechanism for improving unfavorable reactions.
Cellular capsules as a tool for multicellular spheroid production and for investigating the mechanics of tumor progression in vitro
K. Alessandri, Bibhu Ranjan Sarangi, V. Valérïévitch Gurchenkov, B. Sinha, T. Kissling, L. Fetler, F. Rico, Simon Scheuring, Christophe Lamaze, S. Geraldo, D. Vignjević, H. Doméjean, L. Rolland, A. Funfak, Jérôme Bibette, Nicolas Bremond, Pierre Nas
Proc. Nat. Acad. Sci. USA - vol.110(n°37) 14843–48 - DOI: 10.1073/pnas.1309482110 - 2013
Deciphering the multifactorial determinants of tumor progression requires standardized high-throughput preparation of 3D in vitro cellular assays. We present a simple microfluidic method based on the encapsulation and growth of cells inside permeable, elastic, hollow microspheres. We show that this approach enables mass production of size-controlled multicellular spheroids. Due to their geometry and elasticity, these microcapsules can uniquely serve as quantitative mechanical sensors to measure the pressure exerted by the expanding spheroid. By monitoring the growth of individual encapsulated spheroids after confluence, we dissect the dynamics of pressure buildup toward a steady-state value, consistent with the concept of homeostatic pressure. In turn, these confining conditions are observed to increase the cellular density and affect the cellular organization of the spheroid. Postconfluent spheroids exhibit a necrotic core cemented by a blend of extracellular material and surrounded by a rim of proliferating hypermotile cells. By performing invasion assays in a collagen matrix, we report that peripheral cells readily escape preconfined spheroids and cell–cell cohesivity is maintained for freely growing spheroids, suggesting that mechanical cues from the surrounding microenvironment may trigger cell invasion from a growing tumor. Overall, our technology offers a unique avenue to produce in vitro cell-based assays useful for developing new anticancer therapies and to investigate the interplay between mechanics and growth in tumor evolution.
Influence of Size, Surface Coating and Fine Chemical Composition on the In Vitro Reactivity and In Vivo Biodistribution of Lipid Nanocapsules Versus Lipid Nanoemulsions in Cancer Models
Samuli Hirsjärvi, Sandrine Dufort, Julien Gravier, Isabelle Texier, Qiao Yan, Jérome Bibette, Lucie Sancey, Véronique Josserand, Catherine Passirani, Jean-Pierre Benoit and Jean-Luc Coll
Nanomed. Nanotech. Biol. and Med. - 9(3) 375-87 - DOI: 10.1016/j.nano.2012.08.005 - 2013
Lipid nanocapsules (LNCs) and lipid nanoemulsions (LNEs) are biomimetic synthetic nanocarriers. Their in vitro and in vivo performance was evaluated as a function of their size (25, 50 and 100 nm) and the surface PEG chain length. Analysis methods included complement activation test, particle uptake in macrophage and HEK293(β3) cells and biodistribution studies with tumor-grafted mice by fluorescence imaging. A particular attention was paid to keep the concentration of each nanocarrier and to the amount of fluorescent dye in comparable conditions between the in vitro and in vivo studies. Under these conditions, no significant differences were found among the three tested particle sizes and the two nanocarrier types. Longer PEG chains on the LNE surface provided better stealth properties, whereas PEG modification on the LNC formulations inhibited the production of stable nanocarriers. Passive accumulation of LNCs and LNEs in different tumor types depended on the degree of tumor vascularization.
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.

278 publications.