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17515
07/02/2018

Lab-on-Chip for DNA Analysis With Resolution Length of 3 bp and Concentration of 10 nM

B.CHAMI, M.SOCOL, R.MALBEC, A.BANCAUD

MILE, MICA

Affiche/Poster : MICROFLUIDICS2017 - Ecole thématique ( ) 2017 du 25 juin au 30 juin 2017, Carcans Maubuisson (France), Février 2018, 1p. , N° 17515

Lien : https://hal.archives-ouvertes.fr/hal-01686185

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142319
18007
01/02/2018

Ciguatoxins activate the Calcineurin signalling pathway in Yeasts: Potential for development of an alternative detection tool?

H.MARTIN-YKEN, C.GIRONDE, S.DERICK, H.TAIANA DARIUS, C.FURGER, D.LAURENT, M.CHINAIN

LISBP, MH2F, ILM, Papeete, Toulouse III

Revue Scientifique : Environmental Research, Vol.162, pp.144-151, Février 2018 , N° 18007

Lien : https://hal.archives-ouvertes.fr/hal-01680541

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Ciguatoxins (CTXs) are lipid-soluble polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp. typically found in tropical and subtropical zones. This endemic area is however rapidly expanding due to environmental perturbations, and both toxic Gambierdiscus spp. and ciguatoxic fishes have been recently identified in the North Atlantic Ocean (Madeira and Canary islands) and Mediterranean Sea. Ciguatoxins bind to Voltage Gated Sodium Channels on the membranes of sensory neurons, causing Ciguatera Fish Poisoning (CFP) in humans, a disease characterized by a complex array of gastrointestinal, neurological, neuropsychological, and cardiovascular symptoms. Although CFP is the most frequently reported non bacterial food-borne poisoning worldwide, there is still no simple and quick way of detecting CTXs in contaminated samples. In the prospect to engineer rapid and easy-to-use CTXs live cells-based tests, we have studied the effects of CTXs on the yeast Saccharomyces cerevisiae, a unicellular model which displays a remarkable conservation of cellular signalling pathways with higher eukaryotes. Taking advantage of this high level of conservation, yeast strains have been genetically modified to encode specific transcriptional reporters responding to CTXs exposure. These yeast strains were further exposed to different concentrations of either purified CTX or micro-algal extracts containing CTXs. Our data establish that CTXs are not cytotoxic to yeast cells even at concentrations as high as 1μM, and cause an increase in the level of free intracellular calcium in yeast cells. Concomitantly, a dose-dependent activation of the calcineurin signalling pathway is observed, as assessed by measuring the activity of specific transcriptional reporters in the engineered yeast strains. These findings offer promising prospects regarding the potential development of a yeast cells-based test that could supplement or, in some instances, replace current methods for the routine detection of CTXs in seafood products

142177
18012
30/01/2018

Accelerated transport of particles in confined channels with high roughness amplitude

H.RANCHON, J.CACHEUX, B.REIG, O.LIOT, P.TEERAPANICH, T.LEICHLE, P.JOSEPH, A.BANCAUD

MILE, MEMS, TEAM

Revue Scientifique : Langmuir, Vol.34, N°4, pp.1394-1399, Janvier 2018 , N° 18012

Lien : https://hal.archives-ouvertes.fr/hal-01682628

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We investigate the pressure-driven transport of particles 200 or 300 nm in diameter in shallow microfluidic channels ∼1 μm in height with a bottom wall characterized by a high roughness amplitude of ∼100 nm. This study starts with the description of an assay to generate cracks in hydrophilic thin polymer films together with a structural characterization of these corrugations. Microfluidic chips of variable height are then assembled on top of these rough surfaces, and the transport of particles is assessed by measuring the velocity distribution function for a set of pressure drops. We specifically detect anomalous transport properties for rough surfaces. The maximum particle velocity at the centerline of the channel is comparable to that obtained with smooth surfaces, but the average particle velocity increases nonlinearly with the flow rate. We suggest that the change in the boundary condition at the rough wall is not sufficient to account for our data and that the occurrence of contacts between the particle and the surface transports the particle away from the wall and speeds up its motion. We finally draw perspectives for the separation by field-flow fractionation.

142202
18008
29/01/2018

On-chip conductometric detection of short DNA sequences via electro-hydrodynamic aggregation

B.VENZAC, M.DIAKITE, D.HERTHNEK, I.CISSE, U.BOCKELMANN, S.DESCROIX, L.MALAQUIN, J.L.VIOVY

UPMC, Univ of Stockholm, ESPCI, ELIA

Revue Scientifique : The Analyst, Vol.143, N°1, pp.190-199, Janvier 2018 , N° 18008

Lien : http://hal.upmc.fr/hal-01679664

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Fluorescence measurement is the main technology for post-amplification DNA detection in automated systems. Direct electrical reading of DNA concentration in solution could be an interesting alternative to go toward more miniaturized or less expensive devices, in particular in the pathogen detection field. Here we present the detection of short bacterial biomarkers with a direct impedancemetric measurement, within solutions of amplified and elongated DNA sequences in a microchannel. This technology relies on the electrohydrodynamic instability occurring in solutions of long charged macromolecules in a strong electric field. This instability specifically induces the aggregation of long DNAs and triggers conductivity variations that can be monitored by on-contact conductometry. An innovative isothermal amplification and elongation strategy was developed, combining SDA and HRCA reactions, in order to yield long DNAs suitable to be detected by the above principle, from a dilute initial DNA target. In contrast with previous label-free detection methods, this new strategy is very robust to matrix effects, thanks to the unique molecular weight dependence of the instability, coupled with this specific DNA amplification strategy. We demonstrate the detection of a 1 pM gene sequence specific to Staphylococcus aureus, in a portable system.

142183
18015
11/01/2018

Système microfluidique µLAS pour l’analyse de l’ADN résiduel : Application au diagnostic de la maladie de Huntington et à l’analyse de l’ADN circulant

R.MALBEC

MICA

Doctorat : INSA de Toulouse, 11 Janvier 2018, 238p., Président: P.CORDELIER, Rapporteurs: X.GIDROL, V.TALY, Examinateurs: P.JOSEPH, J.SAMITIER, Directeurs de thèse: A.BANCAUD , N° 18015

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Abstract

DNA fragments are circulating in the bloodstream. Circulating DNA fragments size, concentration or sequence are analytical information for the clinician or the molecular biology specialists. For instance, circulating DNA, stem from tumoral cells, can serve as biomarkers for cancer detection and follow up. Collecting those information may be difficult for samples presenting minute amount of DNA. In practice, detecting and analyzing residual DNA, with the relevant level of sensitivity, ask for the development and the association of analytical technologies, such as electrophoresis, to molecular biology techniques, such as PCR amplification. In the prospect of simplifying and speeding up the processes, we have developed and optimized µLAS, a microfluidic system for the simultaneous concentration, separation and detection of residual DNA. Furthermore, µLAS has been applied to the diagnostic of Huntington’s disease and the analysis of residual DNA circulating in the bloodstream. Huntington’s disease, is caused by the expansion of CAG/CTG repeats on the Huntingtin gene, and provoke neurological degeneration. The diagnostic of Huntington’s disease consist in amplifying and measuring this expansion. As the amplification of trinucleotide repeat is far from reliable, we benefit from µLAS sensitivity to reduce the number of amplification cycles, and the time to result. Additionally, for the sensitive analysis of circulating DNA by µLAS, we have proposed an original approach, aiming to reduce the blood sample pre-analytical steps to a simple enzymatic digestion followed by a centrifugation step. Finally, the development of a function for the detection of specific sequences has been made by the selective concentration of a target of interest hybridized to a probe. This approach which use some probes fluorescently labelled in volume, has been patented.

Résumé

La distribution en taille, la concentration, ou la séquence des fragments d’ADN circulants dans le sang sont autant d’informations analytiques exploitables pour les cliniciens ou les spécialistes de la biologie moléculaire. Par exemple, l’ADN circulant, issu de cellules tumorales, peut servir de biomarqueur pour la détection et le suivi du cancer. L’accès à ces informations est d’autant plus difficile que la quantité d’ADN dans l’échantillon est faible. En pratique, pour atteindre des niveaux de sensibilité adaptés, la détection et l’analyse de résidus d’ADN requiert le développement et l’association de technologies d’analyses de type électrophorèse aux techniques de la biologie moléculaire telles que l’amplification PCR. Dans la perspective de simplifier et d’accélérer les procédures, nous avons développé et optimisé µLAS, un système microfluidique pour la concentration, la séparation et la détection simultanée de l’ADN résiduel. µLAS a ensuite été appliqué au diagnostic de la maladie de Huntington et à l’analyse de l’ADN résiduel circulant dans le sang. La maladie de Huntington, causée par l’expansion de répétitions CAG/CTG sur le gène Huntingtin, est à l’origine d’une dégénérescence neurologique. Le diagnostic de la maladie de Huntington consiste à amplifier et à mesurer la taille de cette expansion. L’amplification de répétitions trinucléotidiques étant peu fiable, nous profitons de la sensibilité de µLAS, pour réduire le nombre de cycles d’amplification, et donc le temps d’analyse. Par ailleurs, pour l’analyse sensible de l’ADN circulant par µLAS, nous avons proposé une approche originale, visant à réduire les manipulations pré-analytiques de l’échantillon sanguin à une simple digestion enzymatique suivie d’une centrifugation. Enfin le développement d’une fonction de détection spécifique de séquence a été réalisée par concentration sélective d’une cible d’intérêt hybridée à une sonde. Cette approche qui utilise des sondes marquées en fluorescence en volume, a notamment fait l’objet d’un brevet.

Mots-Clés / Keywords
ADN; Analyse; Microfluidique; Diagnostic; Cancer; Maladie de Huntington; DNA; Analysis; Microfluidic; Huntington’s disease;

142313
15044
11/12/2017

Microwave biosensors for non-invasive molecular and cellular investigations

K.GRENIER, D.DUBUC

MH2F

Ouvrage (contribution) : Medical and Biological Microwave Sensors and Systems, Cambridge University Press, N°ISBN 9781107297302, Décembre 2017, 25p. , N° 15044

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Abstract

In this comprehensive work, experts in the field detail recent advances in medical and biological microwave sensors and systems, with chapters on topics such as implantable sensors, wearable microwave tags, and UWB technology. Each chapter explores the theory behind the technology, as well as its design and implementation. This is supported by practical examples and details of experimental results, along with discussion of system design, design trade-offs, and possible constraints and manufacturing issues. Applications described include intracranial pressure monitoring, vital signs monitoring, and non-invasive molecular and cellular investigations. Presenting new research and advances in the field, and focusing on the state of the art in medical and biological microwave sensors, this work is an invaluable resource for enthusiastic researchers and practicing engineers in the fields of electrical engineering, biomedical engineering, and medical physics.

141743
17437
08/12/2017

Cell biology of microbes and pharmacology of antimicrobial drugs explored by Atomic Force Microscopy

C.FORMOSA, R.DUVAL, E.DAGUE

SRSMC, ELIA

Revue Scientifique : Seminars in Cell & Developmental Biology, 12p., Décembre 2017 , N° 17437

Lien : https://hal.univ-lorraine.fr/hal-01651186

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Abstract

Antimicrobial molecules have been used for more than 50 years now and are the basis of modern medicine. No surgery can nowdays be imagined to be performed without antibiotics; dreadful diseases like tuberculosis, leprosis, siphilys, and more broadly all microbial induced diseases, can be cured only through the use of antimicrobial treatments. However, the situation is becoming more and more complex because of the ability of microbes to adapt, develop, acquire, and share mechanisms of resistance to antimicrobial agents. We choose to introduce this review by briefly drawing the panorama of antimicrobial discovery and development, but also of the emergence of microbial resistance. Then we describe how Atomic Force Microscopy (AFM) can be used to provide a better understanding of the mechanisms of action of these drugs at the nanoscale level on microbial interfaces. In this section, we will address these questions: (1) how does drug treatment affect the morphology of single microbes?; (2) do antimicrobial molecules modify the nanomechanical properties of microbes, or do the nanomechanical properties of microbes play a role in antimicrobial activity and efficiency?; and (3) how are the adhesive abilitites of microbes affected by antimicrobial drugs treatment? Finally, in a second part of this review we focus on recent studies aimed at changing the paradigm of the single molecule/cell technology that AFM typically represents. Recent work dealing with the creation of a microbe array which can be explored by AFM will be presented, as these developments constitute the first steps toward transforming AFM into a higher throughput technology. We also discuss papers using AFM as NanoMechnanicalSensors (NEMS), and demonstrate the interest of such approaches in clinical microbiology to detect quickly and with high accuracy microbial resistance.

141700
17385
05/12/2017

Regenerative potential of primary adult human neural stem cells on micropatterned bioimplants boosts motor recovery

C.DAVOUST, B.PLAS, A.BEDUER, B.DEMAIN, ASALABERT, J.C.SOL, C.VIEU, L.VAYSSE, I.LOUBINOUX

INSERM, EXT, UPS, ELIA

Revue Scientifique : Stem Cell Research & Therapy, art ID 253, Vol.8, Décembre 2017 , N° 17385

Lien : https://hal.laas.fr/hal-01618948

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Background: The adult brain is unable to regenerate itself sufficiently after large injuries . Therefore, hopes rely o n therapies using neural stem cell or biomaterial transplantation to sustain brain reconstruction. The aim of the present study was to evaluate the improvement in sensorimotor recovery brought about by human p rimary adult NSCs in combination with bio implant s . Methods: hNSCs were pre - seeded on implants micropatterned for neurite guidance and inserted intracerebrally 2 weeks after a primary motor cortex lesion in rats . Long - term behavio u r was significantly improved after hNSC - Implants versus cell engraf tment in the grip strength test. MRI and immunohistological studies were conducted to elucidate the underlying mechanisms of neuro implants integration . Results: hNSC - I mplants promoted tissue reconstruction and limited hemispheric atrophy and glial scar expansion. After 3 months , grafted hNSCs were detected on implants and expressed mature neuron al markers (NeuN, MAP2, and SMI 312). They also migrated over a short distance to the reconstructed and to the perilesional tissue s , where 26% integrated as mature neurons. Newly formed host neural progenitors ( n e stin, DCX) colonized the implants, notably in the presence of hNSCs, and participated to tissue reconstruction. The microstructured bioimplants sustain ed the guided maturation of both grafted hNSCs and endo genous progenitors. Conclusions: t hese immunohistological results are coherent with and could explain the late improvement observed in sensorimotor recovery. These findings provide novel insight s into the regenerative potential of primary adult hNSCs combined with microstructure d implants .

141634
17484
01/12/2017

Colon cancer cells adhesion on polymeric nanostructured surfaces

A.ACCARDO, V.SHALABAEVA, R.LA ROCCA

ELIA, LCC

Revue Scientifique : MRS Communications, Décembre 2017 , N° 17484

Lien : https://hal.laas.fr/hal-01675463

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n this work, we report on the adhesion of HCT116 (human colon carcinoma cells) cultured on nanofibrillar polymethylmethacrylate (PMMA) and SU-8 micropillars substrates. Both surfaces enabled a good cell proliferation and promoted the formation of adherent interconnections with the fabricated nano- and microstructures. The three-dimensional immunofluorescence confocal characterization of the cells on nanotextured PMMA highlighted the expression of well-spread F-actin cytoskeletal networks as well as the presence of focal adhesions. This study provides thus interesting perspectives for further investigations on the force/adhesion mechanisms related to cancer cell growth and proliferation.

142075
17238
01/12/2017

Evaluating depth distribution of excimer laser induced defects in silicon using micro-photoluminescence spectroscopy

R.MONFLIER, T.TABATA, M.TURPIN, A.BENYOUCEF, F.CRISTIANO, K.HUET, F.MAZZAMUTO, A.LA MAGNA, S.LOMBARDO, J.ROUL, E.BEDEL-PEREIRA

MPN, SCREEN-LASSE, CNR-IMM, Catania, I2C

Manifestation avec acte : MRS Fall Meeting 2017 du 26 novembre au 01 décembre 2017, Boston (USA), Décembre 2017, 1p. , N° 17238

Lien : https://hal.archives-ouvertes.fr/hal-01555348

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Laser Thermal Annealing (LTA) has been demonstrated to be an effective method to create heavily doped regions required for ultra-shallow junctions, in which dopants are typically introduced by ion implantation. More generally, laser annealing is very attractive due to the localised nature of the annealing process (both on the wafer surface and in depth), allowing dopants to be activated while preserving the integrity of the surrounding areas. Similarly, it is generally accepted that the laser induced damage, if any, is also localised and is reduced when using ultrashort pulses. However, the depth distribution of the laser induced damage has been rarely investigated in detail, with few works reporting on the subsurface doping and damage in laser-doped Si solar cells [1, 2].

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