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An integrated microfluidic chip for immunocapture, preconcentration and separation of beta-amyloid peptides
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Mohamadi RM; Svobodova Z, Bilkova Z, Otto M, Taverna M, Descroix S,Viovy JL
Biomicrofluidics - 9 054117 - - 2015
Combining microfluidics, optogenetics and calcium imaging to study neuronal communication in vitro
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Renault R, Sukenik N, Descroix S, Malaquin L , Viovy JL, Peyrin JM, Bottani S, Monceau P, Moses E, Vignes M
PLoS One - 10(4) 0120680 - - 2015
Selective handling of droplets in a microfluidic device using magnetic rails
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Teste, B; Jamond, N; Ferraro, D; Viovy, JL; Malaquin, L
Microfluidics and Nanofluidics - 19 (1) 141-153 - - 2015
FISH in chips: turning microfluidic fluorescence in situ hybridization into a quantitative and clinically reliable molecular diagnosis tool
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - 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 - 2015
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.
Human Upf1 is a highly processive RNA helicase and translocase with RNP remodelling activities
Laboratoire Physique des biomolécules - Francesca Fiorini, Debjani Bagchi, Hervé Le Hir and Vincent Croquette
Nature Communications - Volume 6 (2015) Article number: 7581 - doi:10.1038/ncomms8581 - 2015
RNA helicases are implicated in most cellular RNA-dependent events. In eukaryotes however, only few have been functionally characterized. Upf1 is a RNA helicase essential for nonsense-mediated mRNA decay (NMD). Here, using magnetic tweezers and bulk assays, we observe that human Upf1 is able to translocate slowly over long single-stranded nucleic acids with a processivity >10 kb. Upf1 efficiently translocates through double-stranded structures and protein-bound sequences, demonstrating that Upf1 is an efficient ribonucleoprotein complex remodeler. Our observation of processive unwinding by an eukaryotic RNA helicase reveals that Upf1, once recruited onto NMD mRNA targets, can scan the entire transcript to irreversibly remodel the mRNP, facilitating its degradation by the NMD machinery.
Two-step local functionalization of fluoropolymer Dyneon THV microfluidic materials by scanning electrochemical microscopy combined to click reaction
Laboratoire Sciences Analytiques Bioanalytiques et Miniaturisation - Cyrine Slim, Eva Ratajovà, Sophie Griveau, Frédéric Kanoufi, David Ferraro, Camille Perréard, Fanny d’Orlyé, Anne Varenne and Fethi Bedioui
Electrochemistry Communications - 60 (5–8) - doi:10.1016/j.elecom.2015.07.019 - 2015
We propose an original two-step strategy combining the use of scanning electrochemical microscopy (SECM) and molecular chemistry via a “click” reaction (copper (I)-catalyzed azide alkyne cycloaddition (CuAAC)) to locally functionalize Dyneon THV surfaces, an attractive fluoropolymer for microfluidic applications. The first step consists in the local reduction of THV using a SECM tip to activate the surface by the creation of a locally carbonized zone and notably the formation of surface alkyne functions. This is then followed by a direct CuAAC reaction with an azide-bearing ligand for its local immobilization. The proof of concept is demonstrated by efficient local functionalization of the substrate with a fluorescent dye stable up to 6 months. Surface modifications were characterized by IR-ATR, XPS, and fluorescence microscopy.
Enhanced chemical synthesis at soft interfaces: a universal reaction-adsorption mechanism in microcompartments
Laboratoire Biochimie - 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 - 2014
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.
Droplet-based microfluidic platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms.
Laboratoire Biochimie - Najah M, Calbrix R, Mahendra-Wijaya IP, Beneyton T, Griffiths AD, Drevelle A.
Chem. Biol. - 21(12) 1722-32 - doi: 10.1016/j.chembiol.2014.10.020 - 2014
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.
Droplet-based microfluidics at the femtolitre scale
Laboratoire Biochimie - Leman M1, Abouakil F, Griffiths AD, Tabeling P.
Lab. Chip - 15(3) 753-65 - doi: 10.1039/c4lc01122h - 2014
We have built a toolbox of modules for droplet-based microfluidic operations on femtolitre volume droplets. We have demonstrated monodisperse production, sorting, coalescence, splitting, mixing, off-chip incubation and re-injection at high frequencies (up to 3 kHz). We describe the constraints and limitations under which satisfactory performances are obtained, and discuss the physics that controls each operation. For some operations, such as internal mixing, we obtained outstanding performances: for instance, in 75 fL droplets the mixing time was 45 μs, 35-fold faster than previously reported for a droplet microreactor. In practice, in all cases, a level of control comparable to nanolitre or picolitre droplet manipulation was obtained despite the 3 to 6 order of magnitude reduction in droplet volume. Remarkably, all the operations were performed using devices made using standard soft-lithography techniques and PDMS rapid prototyping. We show that femtolitre droplets can be used as microreactors for molecular biology with volumes one billion times smaller than conventional microtitre plate wells: in particular, the Polymerase Chain Reaction (PCR) was shown to work efficiently in 20 fL droplets.
An evolutionarily significant unicellular strategy in response to starvation in Dictyostelium social amoebae
Laboratoire Biochimie - Dubravcic D, van Baalen M, Nizak C
- 0.125 133 - doi: 10.12688/f1000research.4218.2 - 2014
The social amoeba Dictyostelium discoideum is widely studied for its multicellular development program as a response to starvation. Aggregates of up to 10 (6) cells form fruiting bodies containing (i) dormant spores (~80%) that can persist for months in the absence of nutrients, and (ii) dead stalk cells (~20%) that promote the dispersion of the spores towards nutrient-rich areas. It is often overlooked that not all cells aggregate upon starvation. Using a new quantitative approach based on time-lapse fluorescence microscopy and a low ratio of reporting cells, we have quantified this fraction of non-aggregating cells. In realistic starvation conditions, up to 15% of cells do not aggregate, which makes this third cell fate a significant component of the population-level response of social amoebae to starvation. Non-aggregating cells have an advantage over cells in aggregates since they resume growth earlier upon arrival of new nutrients, but have a shorter lifespan under prolonged starvation. We find that phenotypic heterogeneities linked to cell nutritional state bias the representation of cells in the aggregating vs. non-aggregating fractions, and thus affect population partitioning. Next, we report that the fraction of non-aggregating cells depends on genetic factors that regulate the timing of starvation, signal sensing efficiency and aggregation efficiency. In addition, interactions between clones in mixtures of non-isogenic cells affect the partitioning of each clone into both fractions. We further build a numerical model to test the evolutionary significance of the non-aggregating cell fraction. The partitioning of cells into aggregating and non-aggregating fractions is optimal in fluctuating environments with an unpredictable duration of starvation periods. Our study highlights the unicellular component of the response of social amoebae to starvation, and thus extends its evolutionary and ecological framework.
CotA laccase: high-throughput manipulation and analysis of recombinant enzyme libraries expressed in E. coli using droplet-based microfluidics
Laboratoire Biochimie - Beneyton T, Coldren F, Baret JC, Griffiths AD, Taly V.
Analyst - 139(13): 3314-23 - doi: 10.1039/c4an00228h. - 2014
We present a high-throughput droplet-based microfluidic analysis/screening platform for directed evolution of CotA laccase: droplet-based microfluidic modules were combined to develop an efficient system that allows cell detection and sorting based on the enzymatic activity. This platform was run on two different operating modes: the "analysis" mode allowing the analysis of the enzymatic activity in droplets at very high rates (>1000 Hz) and the "screening" mode allowing sorting of active droplets at 400 Hz. The screening mode was validated for the directed evolution of the cytoplasmic CotA laccase from B. subtilis, a potential interesting thermophilic cathodic catalyst for biofuel cells. Single E. coli cells expressing either the active CotA laccase (E. coli CotA) or an inactive frameshifted variant (E. coli ΔCotA) were compartmentalized in aqueous droplets containing expression medium. After cell growth and protein expression within the droplets, a fluorogenic substrate was "picoinjected" in each droplet. Fluorescence-activated droplet sorting was then used to sort the droplets containing the desired activity and the corresponding cells were then recultivated and identified using colorimetric assays. We demonstrated that E. coli CotA cells were enriched 191-fold from a 1 : 9 initial ratio of E. coli CotA to E. coli ΔCotA cells (or 437-fold from a 1 : 99 initial ratio) using a sorting rate of 400 droplets per s. This system allows screening of 10(6) cells in only 4 h, compared to 11 days for screening using microtitre plate-based systems. Besides this low error rate sorting mode, the system can also be used at higher throughputs in "enrichment" screening mode to make an initial purification of a library before further steps of selection. Analysis mode, without sorting, was used to rapidly quantify the activity of a CotA library constructed using error-prone PCR. This mode allows analysis of 10(6) cells in only 1.5 h.
ESCRT Machinery Is Required for Plasma Membrane Repair
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Ana Joaquina Jimenez, Paolo Maiuri, Julie Lafaurie-Janvore, Séverine Divoux, Matthieu Piel and Franck Perez
Science - Vol.343(n°6174) 1247136 - DOI: 10.1126/science.1247136 - 2014
Plasma membrane damage can be triggered by numerous phenomena, and efficient repair is essential for cell survival. Endocytosis, membrane patching, or extracellular budding can be used for plasma membrane repair. We found that endosomal sorting complex required for transport (ESCRT), involved previously in membrane budding and fission, plays a critical role in plasma membrane repair. ESCRT proteins were recruited within seconds to plasma membrane wounds. Quantitative analysis of wound closure kinetics coupled to mathematical modeling suggested that ESCRTs are involved in the repair of small wounds. Real-time imaging and correlative scanning electron microscopy (SEM) identified extracellular buds and shedding at the site of ESCRT recruitment. Thus, the repair of certain wounds is ensured by ESCRT-mediated extracellular shedding of wounded portions.
Exploring the function of cell shape and size during mitosis
Laboratoire Biologie cellulaire systémique de la polarité et de la division - Clotilde Cadart, Ewa Zlotek-Zlotkiewicz, Maël Le Berre, Matthieu Piel and Helen K. Matthews
Dev Cell - Vol.29(2) 159–169 - DOI: - 2014
Dividing cells almost always adopt a spherical shape. This is true of most eukaryotic cells lacking a rigid cell wall and is observed in tissue culture and single-celled organisms, as well as in cells dividing inside tissues. While the mechanisms underlying this shape change are now well described, the functional importance of the spherical mitotic cell for the success of cell division has been thus far scarcely addressed. Here we discuss how mitotic rounding contributes to spindle assembly and positioning, as well as the potential consequences of abnormal mitotic cell shape and size on chromosome segregation, tissue growth, and cancer.
High Spatiotemporal Control of Spontaneous Reactions Using Ultrasound-Triggered Composite Droplets
Laboratoire Biophysique et Evolution - M. Bezagu, C. Errico, V. Chaulot-Talmon, F. Monti, M. Tanter, . Tabeling, J. Cossy, T. Arseniyadis and O. Couture
JACS - 136 (20) 7205–7208 - DOI: 10.1021/ja5019354 - 2014
Achieving high spatial and temporal control over a spontaneous reaction is a particularly challenging task with potential breakthroughs in various fields of research including surface patterning and drug delivery. We report here an exceptionally effective method that allows attaining such control. This method relies on a remotely triggered ultrasound-induced release of a reactant encapsulated in a composite microdroplet of liquid perfluorohexane. More specifically, the demonstration was achieved by locally applying a focused 2.25 MHz transducer onto a microfluidic channel in which were injected composite microdroplets containing a solution of an azidocoumarin and an external flow containing a reactive alkyne.
Recent progress in the physics of microfluidics and related biotechnological applications
Laboratoire Biophysique et Evolution - Patrick Tabeling
Curr. Opin. Biotechnol. - -25 129-34 - 10.1016/j.copbio.2013.11.009 - 2014
Since the mid-nineties, the physical understanding of microfluidic flows has reached a level sufficiently elaborate for envisaging applications in all sorts of domains. As the domain expanded, the existence of new situations where fluid dynamics at small or moderate Reynolds numbers combines with confinement, interfaces, transport, particles along with disordered substrates raised new challenges. The present review is restricted to three domains in which progress in the physical description has been made recently (droplet-based, inertial and paper-based microfluidics) and for which biotechnological applications are foreseeable.
Physics and technological aspects of nanofluidics
Laboratoire Biophysique et Evolution - Lydéric Bocquet et Patrick Tabeling
Lab. Chip - 14 3143–3158 - DOI: 10.1039/c4lc00325j - 2014
From a physical perspective, nanofluidics represents an extremely rich domain. It hosts many mechanisms acting on the nanoscale, which combine together or interact with the confinement to generate new phenomena. Superfast flows in carbon nanotubes, nonlinear electrokinetic transport, slippage over smooth surfaces, nanobubble stability, etc. are the most striking phenomena that have been unveiled over the past few years, and some of them are still awaiting an explanation. One may anticipate that new nanofluidic effects will be discovered in the future, but at the moment, the technological barrier is high. Fabrication of nanochannels is most often a tour de force, slow and costly. However, with the accumulation of technological skills along with the use of new nanofluidic materials (like nanotubes), nanofluidics is becoming increasingly accessible to experimentalists. Among the technological challenges faced by the field, fabricating devices mimicking natural nanometric systems, such as aquaporins, ionic pumps or kidney osmotic filtering, seems the most demanding in terms of groundbreaking ideas. Nanoflow characterization remains delicate, although considerable progress has been achieved over the past years. The targeted application of nanofluidics is not only in the field of genomics and membrane science - with disruptive developments to be expected for water purification, desalination, and energy harvesting - but also for oil and gas production from unconventional reservoirs. Today, in view of the markets that are targeted, nanofluidics may well impact the industry more than microfluidics; this would represent an unexpected paradox. These successes rely on using a variety of materials and technologies, using state-of-the-art nanofabrication, or low-tech inexpensive approaches. As a whole, nanofluidics is a fascinating field that is facing considerable challenges today. It possesses a formidable potential and offers much space for creative groundbreaking ideas.
Apparition de vitiligo sous biothérapie: une série de 12 cas
Laboratoire Biophysique et Evolution - L Bocquet, P Tabeling
Lab. Chip - 14 3143–3158 - - 2014
Power transduction of actin filaments ratcheting in vitro against a load
Laboratoire Colloïdes et Matériaux Divisés - Démoulin D., Carlier M.F, Bibette J. and Baudry J.
Proc. Nat. Acad. Sci. USA - Vol.111(n°50) 17845-50 - DOI: 10.1073/pnas.1414184111 - 2014
The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.
ß-amyloid induces a dying-back process and remote trans-synaptic alterations in a microfluidic-based reconstructed neuronal network
Laboratoire Macromolécules et Microsystèmes en Biologie et Médecine - Deleglise B, Magnifico S, Duplus E, Vaur P, Soubeyre V, Belle M, Vignes M, Viovy JL, Jacotot E, Peyrin JM and Brugg B
Acta Neuropathologica - 2 145 - DOI: 10.1186/s40478-014-0145-3 - 2014
INTRODUCTION: Recent histopathological studies have shown that neurodegenerative processes in Alzheimer's and Parkinson's Disease develop along neuronal networks and that hallmarks could propagate trans-synaptically through neuronal pathways. The underlying molecular mechanisms are still unknown, and investigations have been impeded by the complexity of brain connectivity and the need for experimental models allowing a fine manipulation of the local microenvironment at the subcellular level.

RESULTS: In this study, we have grown primary cortical mouse neurons in microfluidic (μFD) devices to separate soma from axonal projections in fluidically isolated microenvironments, and applied β-amyloid (Aβ) peptides locally to the different cellular compartments. We observed that Aβ application to the somato-dendritic compartment triggers a "dying-back" process, involving caspase and NAD(+) signalling pathways, whereas exposure of the axonal/distal compartment to Aβ deposits did not induce axonal degeneration. In contrast, co-treatment with somatic sub-toxic glutamate and axonal Aβ peptide triggered axonal degeneration. To study the consequences of such subcellular/local Aβ stress at the network level we developed new μFD multi-chamber devices containing funnel-shaped micro-channels which force unidirectional axon growth and used them to recreate in vitro an oriented cortico-hippocampal pathway. Aβ application to the cortical somato-dendritic chamber leads to a rapid cortical pre-synaptic loss. This happens concomitantly with a post-synaptic hippocampal tau-phosphorylation which could be prevented by the NMDA-receptor antagonist, MK-801, before any sign of axonal and somato-dendritic cortical alteration.

CONCLUSION: Thanks to μFD-based reconstructed neuronal networks we evaluated the distant effects of local Aβ stress on neuronal subcompartments and networks. Our data indicates that distant neurotransmission modifications actively take part in the early steps of the abnormal mechanisms leading to pathology progression independently of local Aβ production. This offers new tools to decipher mechanisms underlying Braak's staging. Our data suggests that local Aβ can play a role in remote tauopathy by distant disturbance of neurotransmission, providing a putative mechanism underlying the spatiotemporal appearance of pretangles.
Photosensitive Polyamines for High-Performance Photocontrol of DNA Higher-Order Structure Venancio-Marques
Laboratoire Nanobioscience et Microsystèmes - Venancio-Marques, Anna, Bergen Anna, Rossi-Gendron Caroline, Rudiuk Sergii, and Baigl Damien
American Chemical Society Nano (ACS Nano) - Volume 8 (4) 3654–3663 - DOI: 10.1021/nn500266b - 2014
Polyamines are small, ubiquitous, positively charged molecules that play an essential role in numerous biological processes such as DNA packaging, gene regulation, neuron activity, and cell proliferation. Here, we synthesize the first series of photosensitive polyamines (PPAs) and demonstrate their ability to photoreversibly control nanoscale DNA higher-order structure with high efficiency. We show with fluorescence microscopy imaging that the efficiency of the PPAs as DNA-compacting agents is directly correlated to their molecular charge. Micromolar concentration of the most efficient molecule described here, a PPA containing three charges at neutral pH, compacts DNA molecules from a few kilobase pairs to a few hundred kilobase pairs, while subsequent 3 min UV illuminations at 365 nm triggers complete unfolding of DNA molecules. Additional application of blue light (440 nm for 3 min) induces the refolding of DNA into the compact state. Atomic force microscopy reveals that the compaction involves a global folding of the whole DNA molecule, whereas UV-induced unfolding is a modification initiated from the periphery of the compacted DNA, resulting in the occurrence of intermediate flower-like structures prior to the fully unfolded state.

Keywords: polyamines; DNA compaction; photocontrol; DNA; AFM; light


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