Puces à cellules et génomique fonctionnelle

À l’interface du vivant et de l’inerte, se développe un ensemble de nouvelles technologies regroupées sous le terme générique de biopuces. Grâce à la miniaturisation, nous pouvons imaginer que, demain, de nombreuses études biologiques et médicales se feront avec des biopuces qui permettront d’accroître de plusieurs ordres de grandeur le parallélisme des analyses, les vitesses de réaction des tests et leur débit, tout en réduisant les coûts. Cette évolution a démarré avec l’apparition des puces à ADN et se poursuit aujourd’hui avec, entre autres, les puces à cellules qui permettent d’accélérer considérablement l’étude des gènes de fonctions inconnues et leurs implications potentielles dans différentes maladies. Bien que la technologie en soit encore à ses prémices, il est vraisemblable que les puces à cellules feront évoluer la biologie et la médecine de manière significative.With the complete sequencing of the human genome, research priorities have shifted from the identification of genes to the elucidation of their function. Methods currently used by scientists to characterize gene function, such as knock-out mice, are based upon loss of protein function and analysis of the resulting phenotypes to infer a potential role for the protein under scrutiny. Until now, these methods have been successful but time consuming and only a few genes at a time could be analyzed. Cell microarrays allow to simultaneously transfect thousands of different nucleic acid molecules, RNA or DNA, into adherent cells. It is then possible to analyze a large pallet of resulting phenotypes in clusters of transfected cells. We are currently manufacturing cell microarrays with collections of full-length cDNA cloned in expression vectors (gain of function analyses) or siRNA (loss of function studies) to unravel function of genes involved in differentiation and proliferation of human cells. Although there are still some technological difficulties to overcome, the potential for cell microarrays to speed up functional exploration of genomes is very promising

Prospective comparison of speckle tracking longitudinal bidimensional strain between two vendors

SummaryBackgroundSpeckle tracking is a relatively new, largely angle-independent technique used for the evaluation of myocardial longitudinal strain (LS). However, significant differences have been reported between LS values obtained by speckle tracking with the first generation of software products.AimsTo compare LS values obtained with the most recently released equipment from two manufacturers.MethodsSystematic scanning with head-to-head acquisition with no modification of the patient's position was performed in 64 patients with equipment from two different manufacturers, with subsequent off-line post-processing for speckle tracking LS assessment (Philips QLAB 9.0 and General Electric [GE] EchoPAC BT12). The interobserver variability of each software product was tested on a randomly selected set of 20 echocardiograms from the study population.ResultsGE and Philips interobserver coefficients of variation (CVs) for global LS (GLS) were 6.63% and 5.87%, respectively, indicating good reproducibility. Reproducibility was very variable for regional and segmental LS values, with CVs ranging from 7.58% to 49.21% with both software products. The concordance correlation coefficient (CCC) between GLS values was high at 0.95, indicating substantial agreement between the two methods. While good agreement was observed between midwall and apical regional strains with the two software products, basal regional strains were poorly correlated. The agreement between the two software products at a segmental level was very variable; the highest correlation was obtained for the apical cap (CCC 0.90) and the poorest for basal segments (CCC range 0.31–0.56).ConclusionsA high level of agreement and reproducibility for global but not for basal regional or segmental LS was found with two vendor-dependent software products. This finding may help to reinforce clinical acceptance of GLS in everyday clinical practice

DOMINO : une pratique collaboratie durable ? : rapport final du projet de recherche DOMINO

International audienc

Quantitative analysis of highly parallel transfection in cell microarrays

As more genomes are sequenced, we are facing the challenge of rapidly unraveling the functions of genes. To that end, cell microarrays have recently been described that transfect thousands of nucleic acids in parallel and can be used to analyze the phenotypic consequences of such perturbations. As many parameters can influence the efficacy of transfection in such a format, we describe some important features in manufacturing cell microarrays that may improve reliability and efficiency of both plasmid DNA and siRNA transfection. We have also developed image analysis software that allows automatic detection of cell clusters, quantification of transfection efficiency and levels of expression/extinction of genes. Along with cell microarrays, this bioinformatic tool should expedite functional exploration of the human genome

Holographic laser Doppler imaging of microvascular blood flow

International audienceWe report on local superficial blood flow monitoring in biological tissue from laser Doppler holographic imaging. In time-averaging recording conditions, holography acts as a narrowband bandpass filter, which, combined with a frequency-shifted reference beam, permits frequency-selective imaging in the radiofrequency range. These Doppler images are acquired with an off-axis Mach–Zehnder interferometer. Microvascular hemodynamic components mapping is performed in the cerebral cortex of the mouse and the eye fundus of the rat with near-infrared laser light without any exogenous marker. These measures are made from a basic inverse-method analysis of local first-order optical fluctuation spectra at low radiofrequencies, from 0 Hz to 100 kHz. Local quadratic velocity is derived from Doppler broadenings induced by fluid flows, with elementary diffusing wave spectroscopy formalism in backscattering configuration. We demonstrate quadratic mean velocity assessment in the 0.1-10 mm/s range in vitro and imaging of superficial blood perfusion with a spatial resolution of about 10 micrometers in rodent models of cortical and retinal blood flow

Prebiotics Supplementation Impact on the Reinforcing and Motivational Aspect of Feeding.

Energy homeostasis is tightly regulated by the central nervous system which responds to nervous and circulating inputs to adapt food intake and energy expenditure. However, the rewarding and motivational aspect of food is tightly dependent of dopamine (DA) release in mesocorticolimbic (MCL) system and could be operant in uncontrolled caloric intake and obesity. Accumulating evidence indicate that manipulating the microbiota-gut-brain axis through prebiotic supplementation can have beneficial impact of the host appetite and body weight. However, the consequences of manipulating the implication of the microbiota-gut-brain axis in the control motivational and hedonic/reinforcing aspects of food are still underexplored. In this study, we investigate whether and how dietary prebiotic fructo-oligosaccharides (FOS) could oppose, or revert, the change in hedonic and homeostatic control of feeding occurring after a 2-months exposure to high-fat high-sugar (HFHS) diet. The reinforcing and motivational components of food reward were assessed using a two-food choice paradigm and a food operant behavioral test in mice exposed to FOS either during or after HFHS exposure. We also performed mRNA expression analysis for key genes involved in limbic and hypothalamic control of feeding. We show in a preventive-like approach, FOS addition of HFHS diet had beneficial impact of hypothalamic neuropeptides, and decreased the operant performance for food but only after an overnight fast while it did not prevent the imbalance in mesolimbic markers for DA signaling induced by palatable diet exposure nor the spontaneous tropism for palatable food when given the choice. However, when FOS was added to control diet after chronic HFHS exposure, although it did not significantly alter body weight loss, it greatly decreased palatable food tropism and consumption and was associated with normalization of MCL markers for DA signaling. We conclude that the nature of the diet (regular chow or HFHS) as well as the timing at which prebiotic supplementation is introduced (preventive or curative) greatly influence the efficacy of the gut-microbiota-brain axis. This crosstalk selectively alters the hedonic or motivational drive to eat and triggers molecular changes in neural substrates involved in the homeostatic and non-homeostatic control of body weight

Validation d'un questionnaire spécifique des troubles anorectaux dans la sclérose en plaques: STAR-Q

International audienceBackground: Bowel symptoms are commonly experienced by patients with Multiple sclerosis (PwMS), but no specific questionnaire validated in this population allows a rigorous assessment. Objective: Validation of a multidimensional questionnaire assessing bowel disorders in PwMS. Methods: A prospective, multicenter study was conducted between April 2020 and April 2021. The STAR-Q (Symptoms’ assessmenT of AnoRectal dysfunction Questionnaire), was built in 3 steps. First, literature review and qualitative interviews were performed to create the first version, discussed with a panel of experts. Then, a pilot study assessed comprehension, acceptation and pertinence of items. Finally, the validation study was designed to measure content validity, internal consistency reliability (alpha coefficient of Cronbach) and test–retest reliability [intraclass correlation coefficient (ICC)]. The primary outcome was good psychometric properties with Cronbach's α > 0.7 and ICC > 0.7. Results: We included 231 PwMS. Comprehension, acceptation and pertinence were good. STAR-Q showed a very good internal consistency reliability (Cronbach's α = 0.84) and test-retest reliability (ICC = 0.89). Final version of STAR-Q was composed of 3 domains corresponding in symptoms (Q1–Q14), treatment and constraints (Q15–Q18) and impact on quality of life (Q19). Three categories of severity were determined (STAR-Q ≤ 16: minor, between 17 and 20: moderate, and ≥ 21: severe). Conclusions: STAR-Q presents very good psychometric properties and allows a multidimensional assessment of bowel disorders in PwMS. Level of evidence:

Relationship Between the Ratio of Acceleration Time/Ejection Time and Mortality in Patients With High-Gradient Severe Aortic Stenosis

International audienceBackground The ratio of acceleration time/ejection time (AT/ET) is a simple and reproducible echocardiographic parameter that integrates aortic stenosis severity and its consequences on the left ventricle. No study has specifically assessed the prognostic impact of AT/ET on outcome in patients with high-gradient severe aortic stenosis (SAS) and no or mild symptoms. We sought to evaluate the relationship between AT/ET and mortality and determine the best predictive AT/ET cutoff value in these patients. Methods and Results A total of 353 patients (median age, 79 years; 46% women) with high-gradient (mean pressure gradient >= 40 mm Hg and/or aortic peak jet velocity >= 4 m/s) SAS, left ventricular ejection fraction >= 50%, and no or mild symptoms were studied. The impact of AT/ET 0.35 on all-cause mortality was retrospectively studied. During a median follow-up of 39 (25th-75th percentile, 23-62) months, 70 patients died. AT/ET >0.35 was associated with a considerable increased mortality risk after adjustment for established prognostic factors in SAS under medical and/or surgical management (adjusted hazard ratio [HR], 2.54; 95% CI, 1.47-4.37; P0.35 improved the predictive performance of models including established risk factors in SAS with better global model fit, reclassification, and discrimination. After propensity matching, increased mortality risk persisted when AT/ET >0.35 (adjusted HR, 2.10; 95% CI, 1.12-3.90; P0.35 is a strong predictor of outcome in patients with SAS and no or only mild symptoms and identifies a subgroup of patients at higher risk of death who may derive benefit from earlier aortic valve replacement

Assessment of left ventricular size and function by 3-dimensional transthoracic echocardiography: Impact of the echocardiography platform and analysis software

International audienceBackground: Whether echocardiography platform and analysis software impact left ventricular (LV) volumes, ejection fraction (EF), and stroke volume (SV) by transthoracic tridimensional echocardiography (3DE) has not yet been assessed. Hence, our aim was to compare 3DE LV end-diastolic and end-systolic volumes (EDV and ESV), LVEF, and SV obtained with echocardiography platform from 2 different manufacturers. Methods: 3DE was performed in 84 patients (65% of screened consecutive patients), with equipment from 2 different manufacturers, with subsequent off-line postprocessing to obtain parameters of LV function and size (Philips QLAB 3DQ and General Electric EchoPAC 4D autoLVQ). Twenty-five patients with clinical indication for cardiac magnetic resonance imaging served as a validation subgroup. Results: LVEDV and LVESV from 2 vendors were highly correlated (r=0.93), but compared with 4D autoLVQ, the use of Qlab 3DQ resulted in lower LVEDV and LVESV (bias: 11 mL, limits of agreement: -25 to + 47 and bias: 6 mL, limits of agreement: -22 to + 34, respectively). The agreement between LVEF values of each software was poor (intraclass correlation coefficient 0.62) despite no or minimal bias. SVs were also lower with Qlab 3DQ advanced compared with 4D autoLVQ, and both were poorly correlated (r=0.66). Consistently, the underestimation of LVEDV, LVESV, and SV by 3DE compared with cardiac magnetic resonance imaging was more pronounced with Philips QLAB 3DQ advanced than with 4D autoLVQ. Conclusions: The echocardiography platform and analysis software significantly affect the values of LV parameters obtained by 3DE. Intervendor standardization and improvements in 3DE modalities are needed to broaden the use of LV parameters obtained by 3DE in clinical practice. (C) 2018 Elsevier Inc. All rights reserved

Enhanced tunability and temperature-dependent dielectric characteristics at microwaves of K0.5Na0.5NbO3 thin films epitaxially grown on (100)MgO substrates

International audienceK0.5Na0.5NbO3 thin films were deposited by pulsed laser deposition on (100)MgO substrates for microwave device applications. A fine epitaxial growth of pure perovskite phase was evidenced by X-ray diffraction. Dielectric characterizations were performed from 1 to 40 GHz using coplanar microwave devices printed on the 500 nm-thick K0.5Na0.5NbO3 thin films. Dielectric permittivity εr = 355 and loss tangent tanδ = 0.35 at 10 GHz were retrieved without biasing. A comparison of the results with those retrieved from the resonant cavity method (to characterize as-deposited films) showed no deleterious influence neither from the device patterning nor the thin film-device interface. A frequency tunability up to 22% was measured under a moderate external DC bias electric field Ebias = 94 kV/cm. Temperature measurements from 20° to 240°C exhibited a permittivity increase up to εr = 975 coupled to a loss decrease tanδ = 0.25 at 10 GHz. According to such measurements, an orthorhombic-tetragonal phase transition was evidenced close to 220 °C with an increase of the frequency tunability up to 34%. Comparison of the properties of such films with those grown on R-plane sapphire substrates demonstrated the benefit brought by the epitaxial growth of K0.5Na0.5NbO3 films on (100) MgO