A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

International audienceMiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues M icroRNAs are a class of endogenous, small (19–25 nucleotides), single-stranded non-coding RNAs that regulate gene expression in all eukaryotic organisms. In metazoans, microRNAs most commonly bind to the 39 untranslated region (39UTR) of their mRNA target transcript and cause translational repression and/or mRNA degradation. Every microRNA is predicted to regulate from a dozen to thousands of genes, including transcription factors. This fine-tuning of protein expression is known to be involved in many physiological processes, such as development, apoptosis, signal transduction and even cancer progression 1,2. More than 2,000 mature human microRNAs are listed in the 20 th release of miRBase: http://www.mirbase.org (2014) (Date of access:19/08/2013), and some authors hypothesise that the majority of human genes are regulated by microRNAs 3. Since their discovery in 1993 4 , a fair understanding of their role in animal development and in the onset and progression of diseases 2 , as well as of their potential use in therapies 5 , has been gathered. However, the cooperative behaviour of microRNAs is still under investigation. A growing body of experimental evidence suggests that microRNAs can regulate genes through complementarity, meaning that microRNAs can act together to regulate individual genes or groups of genes involved in similar processes 6. For example, Hu and co-workers demonstrated that transducing a cocktail of precursor microRNAs (miR-21, miR-24 and miR-221) can result in more effective engraftment of transplanted cardiac progenitor cells 7. Consistent with these discoveries, Zhu et al. demonstrated that miR-21 and miR-221 coregulate 56 gene ontology (GO) processes 8. In the same study, the authors also showed that cotransfection of miR-1 and miR-21 increases H 2 O 2-induced myocardial apoptosis and oxidative stress. These recent findings support the idea of microRNA-mediated cooperative regulation but also argue for the use of systemic approaches, notably based on graph theory, to decipher individual and complementary roles of microRNAs. Some work has been conducted to use recent high-throughput experiment-derived data sets to infer microRNA synergistic relationships 9–12. Herein, we present a microRNA network based on target similarities among microRNAs to infer clusters of microRNAs. Clusters are defined as groups of microRNAs sharing a set of common targets, predicted by either DIANA-microT v3 13 or TargetScan v6.2 14. Some authors have used GO enrichment analysis as a confirmatory tool for their clustering approach 11. In our case, GO enrichment is not used to infer networks but as a way to estimate the probable metabolic pathway(s) a cluster of microRNAs could co-regulate. Moreover, the novelty of our approach is to consider not only clusters of microRNAs but also OPE

Efficient multipoles modeling for linear magnetized beads manipulations

International audienceMany Magnetic MEMS have been recently developed to manipulate spherical diamagnetic or superparamagnetic particles. Magnetic forces can be computed using the dipole method or the semi-numerical integral. However, these methods appear inaccurate or time consuming, respectively. This paper proposes a multipolar approach which provides a satisfying compromise between precision and rapidity to compute the magnetic force on linear magnetized beads. Gain in precision and CPU time will be detailed for water droplets levitating above micromagnets

Lévitation diamagnétique sur micro-aimants (applications à la microfluidique digitale et à la biologie)

La lévitation diamagnétique est un des rares phénomènes permettant de soustraire des corps à la gravité. Si la répulsion diamagnétique est négligeable à nos échelles, elle devient significative aux petites dimensions, allant jusqu'à entraîner, sous certaines conditions, la mise en lévitation de micro-objets diamagnétiques. A travers le développement de microaimants, de modèles numériques et analytiques ainsi que de réalisations expérimentales, ces travaux explorent les potentialités de la lévitation diamagnétique de microgouttes et du piégeage de cellules en milieu paramagnétique. Ils démontrent notamment la possibilité de mesurer précisément l'interaction entre gouttes chargées en lévitation. Ces travaux analysent par ailleurs, le comportement des cellules piégées dans un environnement paramagnétique. Cette étude ouvre de nouvelles perspectives pour le tri de cellules basé sur leur taille, leur susceptibilité voire même leur propriété endocytotique. Enfin, la possibilité d'actionner sans contact des microgouttes dans l'air est explorée par la modélisation d'un système innovant couplant la diélectrophorèse à la lévitation diamagnétique.Diamagnetic levitation is one of the rare way to compensate action of gravity. This kind of repulsion is negligible at our scale. However, at microscale, this effect becomes significant and can achieve levitation of diamagnetic objects. Through the development of micromagnets, analytical and numerical models, and experiments, applications of diamagnetic levitation of microdroplets and trapping of cells in paramagnetic media is explored. It is shown that diamagnetic levitation allows quantifying interactions between levitating charged droplets. The behaviour of cells trapped in paramagnetic medium is also analysed. This study opens attractive alternatives to cells sorting, based on size, magnetic susceptibilities and endocytoticity. Finally, remote handling of levitating droplets is proposed and simulated by coupling diamagnetic levitation and dielectrophoresis.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Experimental investigation of the evaporation of droplets in diamagnetic levitation

International audienceThe time evolution of levitating pure water and water-ethanol droplets is investigated. Droplets are trapped in stable diamagnetic levitation in the magnetic well produced by a cylindrical permanent magnet. Radius of evaporating droplets and their temperature differences with air are monitored over time using a thermographic camera. Experimental setup A cylindrical permanent magnet made by 8 NdFeB sectors with radial magnetization and covered by a pole piece (Figure 1(a)) is used to trap diamagnetic droplets in levitation. A screwed brass base maintains all the NdFeB sectors together. In addition, a cylindrical hole of 1.6 mm diameter is realized along the symmetry axis of this set up. The gap between the sectors and the pole piece is adjusted in order to produce a potential well where diamagnetic levitation can be achieved [1,2]. Droplets are generated by a piezoelectric jetting device. It consists of a tiny glass tube covered with a piezoelectric ceramic (Figure 1(b)). A capillary transports the liquid solution from a 15 mL test tube to the glass tube. The applied potential in the piezoelectric ceramic is computer-controlled. As the ceramic shrinks, pressure builds up in the glass tube and a jet of microdroplets (i.e. droplets with a radius of few tens of micrometers) is ejected from the tube nozzle (Figure 1(b)). The ejection energy depends upon the applied potential and therefore determines the properties of the jet. The main difficulty in this procedure consists in the requirement for microdroplets to fall into the magnetic well. As the jet is extremely sensitive to disturbances of environment, it tends to behave randomly. A confinement facility is used to limit this drawback. Once microdroplets are trapped in the magnetic well, they spontaneously coalesce after few seconds and form larger droplets. When millimeter-sized droplets are produced (i.e., with a radius larger than 200 m), the jetting is stopped. Figure 1. (a) Top view of the permanent magnet. (b) Experimental setup made of a piezoelectric nozzle, the magnet and a thermographic camera. The microdroplet jet from the piezoelectric nozzle to the magnetic levitation site is schematically plotted with a full blue line. A thermographic camera placed above the magnetic levitation site captures infrared (IR) radiation emitted by the droplet in the spectral wavelength range from 7 m to 10 m. It records a field of view (FOV) of ~2.0 mm x 1.6 mm and allows measuring the droplet size as well as the intensity of emitted IR radiation over time. Images are grabbed on a 160 x 128 matrix of sensors and post treated with the ImageJ software. The spatial resolution is about 12.8 m. Thermography measurements depend on the IR emissivity and observation angles. Accessing to the temperature field in the full FOV is therefore not straightforward (although sensitivity of the camera is less than 20 mK), especially for ethanol that is transparent in the considered spectral range. Used liquid solutions comprise pure water and water/ethanol mixtures (20%, 40%, 60% and 80%, 96% v/v). Water is deionized. In order to achieve a better reproducibility, experiments were carried out several times for each solution. Room temperature T and humidity H are 203 °C and 305%, respectively

Quantitative imaging of cell dynamics Parallelized contact imaging and automated analysis of cell migration dynamics

International audienceWe show the capacity to carry out screens for cell migrations using 96 wound healing assays achieved in 96-well microtiter plates based on a new optical technique developed by our group. Dynamics of the wound closures were obtained by parallelized time lapse contact imaging microscopy and dedicated automated image analysis. Our plate reader relies on an array of 96 image sensors, namely the planar arrangement of 12×8 image sensors, placed under the transparent flat-bottomed 96-well microtiter plate so that each well can be imaged by the image sensor placed underneath. Week-long monitoring of live cell populations showed long-term imaging position stability and no focus drift in any image series, which makes our time-lapse plate reader very competitive in comparison to conventional video microscopy equipment. The 96 wound closure dynamics were extracted from the images using a specifically developed automated segmentation method. Robust localization of the wound edges in low contrast images was achieved by global segmentation algorithms based on Markov random fields and active contours even with non-uniform illumination conditions. A parallel double snake was used to model the approximate parallelism between the two edges of a wound. The performance of global segmentation was validated on a set of images showing wounds in confluent epithelial cell cultures. Automated wound localization was compared with manual segmentation performed by seven cell biology experts by determining the root-mean-square error between the segmented interfaces and region-oriented analysis. Evaluations of intra and inter-biologist variabilities showed that automated segmentations are as accurate and robust as the cell biologist's ones. Wound closure dynamics was applied to measure and compare the motility of four affiliated prostate cell lines representing various grades of prostate cancer development

Device for dispensing microfluidic droplets, particularly for cytometry

The invention relates to a device for dispensing droplets comprising a first channel (8, 10), known as the main channel, for circulating a first liquid flow, a second channel (12, 13) for circulating fluid, forming an intersection area (27) with the first channel and being terminated by an ejection opening (20), means (4) for measuring a physical property of particles or cells in the first channel (18), and means for producing a pressure wave in the second channel (12,13)

High-throughput strain identification and production of fungal enzymatic cocktails for the valorisation of lignocellulosic biomass

International audienc

Magnetophoretic and Dielectrophoretic Actuations Coupled with Diamagnetic Trapping in Air and Liquids

International audienc

Permanent magnetic structure(s) for trapping and/or channelling and/or separating and/or filtering particles

Internationa