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51. 3D+time acquisitions of 3D cell culture by means of lens-free tomographic microscopy

Author: Xavier Gidrol, Nathalie Picollet-D'hahan, Thomas Bordy, Bastien Laperrousaz, Cédric Allier, Sophie Morales, Anthony Berdeu, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université Grenoble Alpes, Inserm, CEA, BIG-Biologie du Cancer et de l’Infection, Grenoble, France, Clinatec - Centre de recherche biomédicale Edmond J.Safra (SCLIN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: Microscope, Materials science, Cell, Cell migration, Matrix (biology), law.invention, Extracellular matrix, 3D cell culture, medicine.anatomical_structure, law, Cell culture, [SDV.BC.IC]Life Sciences [q-bio]/Cellular Biology/Cell Behavior [q-bio.CB], Microscopy, medicine, [PHYS.PHYS.PHYS-INS-DET]Physics [physics]/Physics [physics]/Instrumentation and Detectors [physics.ins-det], Biological system
Abstract: Editors: Thomas G. Brown, Carol J. Cogswell, Tony Wilson; We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multi-angle acquisitions on 3D cell cultures embedded in extracellular matrix (ECM). We developed algorithms based on the Fourier diffraction theorem to perform fully 3D reconstructions of biological samples and we adapted the lens-free microscope to incubator conditions. Here we demonstrate for the first time, 3D+time lens-free acquisitions of 3D cell culture over 8 days directly into the incubator. The 3D reconstructed volume is as large as ~5 mm3 and provides a unique way to observe in the same 3D cell culture experiment multiple cell migration strategies. Namely, in a 3D cell culture of prostate epithelial cells embedded within a Matrigel® matrix, we are able to distinguish single cell ’leaders’, migration of cell clusters, migration of large aggregates of cells, and also close-gap and large-scale branching. In addition, we observe long-scale 3D deformations of the ECM that modify the geometry of the 3D cell culture. Interestingly, we also observed the opposite, i.e. we found that large aggregates of cells may deform the ECM by generating traction forces over very long distances. In sum we put forward a novel 3D lens-free microscopy tomographic technique to study the single and collective cell migrations, the cell-to-cell interactions and the cell-to-matrix interactions.
Published: 2018
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52. MicroRNA regulation of androgen signalling

Author: Damien A. Leach, Akifumi Shibakawa, Claire E. Fletcher, Charlotte L. Bevan, Alwyn Dart, Xavier Gidrol, Sean E. McGuire, Shawn E. Lupold, Bono Johann de, Ailsa Sita-Lumsden, Stéphanie Combe, and Eric Sulpice
Subjects: Signalling, medicine.drug_class, microRNA, medicine, Biology, Androgen, Cell biology
Published: 2018
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53. Lens-free Video Microscopy for the Dynamic and Quantitative Analysis of Adherent Cell Culture

Author: Lionel Hervé, Michel Bornens, Xavier Gidrol, Lamya Ghenim, Fabrice Navarro, Mathilde Menneteau, Olivier Cioni, Sophie Morales, Yves Usson, Thomas Bordy, Romaric Vincent, Sabine Bardin, Cédric Allier, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biomicrotechnologie et génomique fonctionnelle (BIOMICS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Mécanismes moléculaires du transport intracellulaire, Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Dynamique Cellulaire et Tissulaire- Interdisciplinarité, Modèles & Microscopies (TIMC-IMAG-DyCTiM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut Laue-Langevin (ILL), ILL, Laboratoire de biologie à grande échelle, Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Dynamique Cellulaire et Tissulaire- Interdisciplinarité, Modèles & Microscopies (TIMC-DyCTiM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC), and Dynamiques Cellulaire et Tissulaire - Interdisciplinarité, Modélisation & Microscopie (TIMC-IMAG-DyCTiM2)
Subjects: 0301 basic medicine, Microscope, Cell division, Computer science, General Chemical Engineering, [SDV]Life Sciences [q-bio], Holography, Cell Culture Techniques, Video microscopy, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, law.invention, 010309 optics, 03 medical and health sciences, law, 0103 physical sciences, Humans, ComputingMilieux_MISCELLANEOUS, Microscopy, Video, General Immunology and Microbiology, General Neuroscience, This Month in JoVE, Cell cycle, Lens (optics), 030104 developmental biology, Cell culture, Quantitative Microscopy, Biomedical engineering
Abstract: Here, we demonstrate that lens-free video microscopy enables us to simultaneously capture the kinetics of thousands of cells directly inside the incubator and that it is possible to monitor and quantify single cells along several cell cycles. We describe the full protocol used to monitor and quantify a HeLa cell culture for 2.7 days. First, cell culture acquisition is performed with a lens-free video microscope, and then the data is analyzed following a four-step process: multi-wavelength holographic reconstruction, cell-tracking, cell segmentation and cell division detection algorithms. As a result, we show that it is possible to gather a dataset featuring more than 10,000 cell cycle tracks and more than 2 x 10(6) cell morphological measurements.
Published: 2018
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54. 3D polyelectrolyte scaffolds to mimic exocrine glands: a step towards a prostate-on-chip platform

Author: Odile Filhol-Cochet, Frédérique Kermarrec, Maxime Legues, Xavier Gidrol, Guillaume Laffitte, Christophe A. Marquette, Jean-Pierre Alcaraz, Olivier Nicoud, Nathalie Picollet-D'hahan, Florence Rivera, Isabelle Vilgrain, Lavinia Liguori, Jacques Thélu, Patricia Obeid, Donald K. Martin, Ali Bouamrani, Sophie Gerbaud, Benoit Gilquin, Thibaud Moufle-Milot, Adrien Mombrun, Cristina Belda Marin, Axel Tollance, Biomicrotechnologie et génomique fonctionnelle (BIOMICS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut d'oncologie/développement Albert Bonniot de Grenoble (INSERM U823), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-EFS-Institut National de la Santé et de la Recherche Médicale (INSERM), Systèmes Nanobiotechnologiques et Biomimétiques (TIMC-IMAG-SyNaBi), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Génie Enzymatique, Membrane Biomimétique et Assemblages Supramoléculaires (GEMBAS), Institut de Chimie et Biochimie Moléculaires et Supramoléculaires (ICBMS), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-École Supérieure Chimie Physique Électronique de Lyon-Centre National de la Recherche Scientifique (CNRS), Invasion mechanisms in angiogenesis and cancer (IMAC), Biologie du Cancer et de l'Infection (BCI ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Gestes Medico-chirurgicaux Assistés par Ordinateur (TIMC-IMAG-GMCAO), Clinatec - Centre de recherche biomédicale Edmond J.Safra (SCLIN), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université de Lyon-Université de Lyon-École Supérieure de Chimie Physique Électronique de Lyon (CPE)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: Exocrine gland, 3D scaffolds, Biomedical Engineering, Nanotechnology, polyelectrolytes, 03 medical and health sciences, 0302 clinical medicine, Prostate, Genetics, medicine, Liquid biopsy, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, liquid biopsy, Polyelectrolyte, secretome, medicine.anatomical_structure, 030220 oncology & carcinogenesis, Molecular Medicine, organ-on-chip, TP248.13-248.65, Food Science, Biotechnology
Abstract: We report our approach to creating a microfluidic chip (namely UroLOC) that mimics the acinar/tubular structure and the luminal microenvironment of exocrine glands. The chip utilises a nanostructured membrane that is designed to provide a 3-dimensional supporting scaffold for the growth of exocrine acinus epithelial cells. The nanostructured membrane was produced using layer-by-layer assembly of polyelectrolytes, and formed into 3-dimensional hemispherical cavities and “finger-like” structures in order to mimic the natural architecture of acini found in exocrine glands. We utilised normal (PNT2) and cancerous (PC3, LNCaP) prostate epithelial cells to demonstrate the proof-of-concept of using MALDI (Matrix Assisted Laser Desorption Ionisation) profiling of secretions collected after 48 hours of cell growth, with no concentration or purification steps and without any a priori on the knowledge of targeted proteins. This MALDI profiling analysis of the crude supernatants from 3 different cell lines (PNT2, PC3 and LNCaP) demonstrated the capacity of the MALDI profiling approach to discriminate between the different secretome signatures. The UroLOC concept and secretome profiling that we describe opens new opportunities in terms of liquid-biopsy based diagnosis, particularly for the early stages of carcinogenesis.
Published: 2018
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55. Encapsulated organoids & organ-on-a-chip platform for cancer modeling

Author: Xavier Gidrol, A. Tollance, S. Porte, C. Belda-Marin, Vincent Haguet, Patricia Obeid, Nathalie Picollet-D'hahan, A. Romero-Millan, Bastien Laperrousaz, Donald K. Martin, Frédérique Kermarrec, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Physique de l'ENS Lyon (Phys-ENS), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Biogeochemistry Research Centre, School of Chemistry, Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL)
Subjects: 010302 applied physics, Scaffold, Computer science, [SDV]Life Sciences [q-bio], Microfluidics, Cancer, Nanotechnology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, medicine.disease, 01 natural sciences, Organ-on-a-chip, 3. Good health, 0103 physical sciences, Organoid, medicine, ComputingMilieux_MISCELLANEOUS
Abstract: This review highlights our major developments in the fields of organoids and organ-on-chip to address issues in fundamental and biomedical research by modeling development and cancer. We illustrate how contemporary miniaturized technologies (microfluidics, 3D scaffolding, 3D imaging) combined with RNAi-based organoids HTS (High-Throughput Screening), would help forming and analyzing consistent, efficient and reproducible organoids. Moreover, we illustrate the potential of engineered organ-on-chip devices for creating novel human organ and disease models, with particular focus on “prostate-on-a-chip”.
Published: 2017
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56. Microtubule stabilization drives 3D centrosome migration to initiate primary ciliogenesis

Author: Gaëlle Letort, Fabrice Senger, Xavier Gidrol, Laurent Guyon, Manuel Théry, Amandine Pitaval, James Sillibourne, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Physiologie cellulaire et végétale (LPCV), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Biologie du Cancer et de l'Infection (BCI ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut National de la Santé et de la Recherche Médicale (INSERM), Alloimmunité-Autoimmunité-Transplantation (A2T), Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), European Project: 310472,EC:FP7:ERC,ERC-2012-StG_20111109,SPICY(2012), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Universitaire d'Hématologie (IUH), Université Paris Diderot - Paris 7 (UPD7)-Université Paris Diderot - Paris 7 (UPD7), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: 0301 basic medicine, Axoneme, Time Factors, Cytoskeleton organization, Centrosome cycle, Retinal Pigment Epithelium, Microtubule dynamics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Transfection, Mechanotransduction, Cellular, Microtubules, Spindle pole body, Article, Cell Line, 03 medical and health sciences, 0302 clinical medicine, Primary ciliogenesis, Centrosome migration, Intraflagellar transport, Microtubule, Ciliogenesis, Humans, Actin network, Cilia, Cytoskeleton, Research Articles, 030304 developmental biology, Microtubule nucleation, Centrosome, 0303 health sciences, Protein Stability, Tumor Suppressor Proteins, Epithelial Cells, Cell Biology, Actin cytoskeleton, Human cells, Cell biology, Actin Cytoskeleton, 030104 developmental biology, Microtubule Proteins, RNA Interference, Centrosome positioning regulation, 030217 neurology & neurosurgery
Abstract: The classical view of centrosome decentering and migration to the cell periphery during ciliogenesis is that it is pulled toward its final destination. Here, Pitaval et al. argue that microtubule stabilization in the early stages of ciliogenesis generates pushing forces that propel the centrosome toward the apical pole., Primary cilia are sensory organelles located at the cell surface. Their assembly is primed by centrosome migration to the apical surface, yet surprisingly little is known about this initiating step. To gain insight into the mechanisms driving centrosome migration, we exploited the reproducibility of cell architecture on adhesive micropatterns to investigate the cytoskeletal remodeling supporting it. Microtubule network densification and bundling, with the transient formation of an array of cold-stable microtubules, and actin cytoskeleton asymmetrical contraction participate in concert to drive apical centrosome migration. The distal appendage protein Cep164 appears to be a key actor involved in the cytoskeleton remodeling and centrosome migration, whereas intraflagellar transport 88’s role seems to be restricted to axoneme elongation. Together, our data elucidate the hitherto unexplored mechanism of centrosome migration and show that it is driven by the increase and clustering of mechanical forces to push the centrosome toward the cell apical pole.
Published: 2017
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57. Deciphering Cell Intrinsic Properties: A Key Issue for Robust Organoid Production

Author: Xavier Gidrol, Nathalie Picollet-D'hahan, Donald K. Martin, Delphine Freida, Monika E. Dolega, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères] (LIPhy), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Systèmes Nanobiotechnologiques et Biomimétiques (TIMC-IMAG-SyNaBi), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: 0301 basic medicine, Cell type, Stromal cell, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV]Life Sciences [q-bio], Cell Culture Techniques, Bioengineering, Genomics, Nanotechnology, Computational biology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Regenerative Medicine, Regenerative medicine, 03 medical and health sciences, Organoid, Animals, Humans, Microfluidic Analytical Techniques, 3. Good health, Organoids, 030104 developmental biology, Drug development, Functional genomics, Function (biology), Biotechnology
Abstract: International audience; We highlight the disposition of various cell types to self-organize into complexorgan-like structures without necessarily the support of any stromal cells,provided they are placed into permissive 3D culture conditions. The goal ofgenerating organoids reproducibly and efficiently has been hampered by poorunderstanding of the exact nature of the intrinsic cell properties at the origin oforganoid generation, and of the signaling pathways governing their differen-tiation. Using microtechnologies like microfluidics to engineer organoidswould create opportunities for single-cell genomics and high-throughputfunctional genomics to exhaustively characterize cell intrinsic properties. Amore complete understanding of the development of organoids wouldenhance their relevance as models to study organ morphology, function,and disease and would open new avenues in drug development and regener-ative medicine.
Published: 2017
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58. 3D lens-free time-lapse microscopy for 3D cell culture

Author: Jean-Marc Dinten, Cédric Allier, Anthony Berdeu, Nathalie Picollet-D'hahan, Xavier Gidrol, Fabien Momey, Bastien Laperrousaz, Thomas Bordy, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Hubert Curien [Saint Etienne] (LHC), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique de l'ENS Lyon (Phys-ENS), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Laboratoire Hubert Curien (LHC), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Berdeu, Anthony
Subjects: Materials science, Super-resolution microscopy, business.industry, Scanning confocal electron microscopy, 02 engineering and technology, Inverse problem, 021001 nanoscience & nanotechnology, 01 natural sciences, Time-lapse microscopy, law.invention, 010309 optics, Lens (optics), 3D cell culture, Optics, law, 0103 physical sciences, Microscopy, Digital holographic microscopy, 0210 nano-technology, business, [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, [SPI.SIGNAL] Engineering Sciences [physics]/Signal and Image processing
Abstract: International audience; We propose a new imaging platform based on lens-free time-lapse microscopy for 3D cell culture and its dedicated algorithm lying on a fully 3D regularized inverse problem approach. First 3D + t results are presented.
Published: 2017
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59. Comparative study of fully three-dimensional reconstruction algorithms for lens-free microscopy

Author: Thomas Bordy, Anthony Berdeu, Jean-Marc Dinten, Cédric Allier, Fabien Momey, Bastien Laperrousaz, Nathalie Picollet-D'hahan, Xavier Gidrol, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire Hubert Curien [Saint Etienne] (LHC), Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS)-Institut d'Optique Graduate School (IOGS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire Hubert Curien (LHC), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Momey, Fabien
Subjects: Diffraction, Computer science, [SDV.IB.IMA]Life Sciences [q-bio]/Bioengineering/Imaging, Materials Science (miscellaneous), Phase contrast microscopy, 02 engineering and technology, Iterative reconstruction, 01 natural sciences, Industrial and Manufacturing Engineering, Time-lapse microscopy, law.invention, 010309 optics, symbols.namesake, Optics, law, 0103 physical sciences, Microscopy, Business and International Management, [SPI.SIGNAL] Engineering Sciences [physics]/Signal and Image processing, business.industry, Resolution (electron density), OCIS codes: (090.1970) Diffractive optics, (090.1995) Digital holography, (100.3010) Image reconstruction techniques, (170.0110) Imaging systems, (180.6900) Three-dimensional microscopy, Inverse problem, 021001 nanoscience & nanotechnology, [SDV.IB.IMA] Life Sciences [q-bio]/Bioengineering/Imaging, Fourier transform, symbols, 0210 nano-technology, Phase retrieval, business, Algorithm, [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, Digital holography
Abstract: International audience; We propose a three-dimensional (3D) imaging platform based on lens-free microscopy to perform multiangle acquisitions on 3D cell cultures embedded in extracellular matrices. Lens-free microscopy acquisitions present some inherent issues such as the lack of phase information on the sensor plane and a limited angular coverage. We developed and compared three different algorithms based on the Fourier diffraction theorem to obtain fully 3D reconstructions. These algorithms present an increasing complexity associated with a better reconstruction quality. Two of them are based on a regularized inverse problem approach. To compare the reconstruction methods in terms of artefact reduction, signal-to-noise ratio, and computation time, we tested them on two experimental datasets: an endothelial cell culture and a prostate cell culture grown in a 3D extracellular matrix with large reconstructed volumes up to ∼5 mm3∼5 mm3 with a resolution sufficient to resolve isolated single cells. The lens-free reconstructions compare well with standard microscopy.
Published: 2017
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60. The modulation of attachment, growth and morphology of cancerous prostate cells by polyelectrolyte nanofilms

Author: Xavier Gidrol, Ricky Bhajun, Philippe Cinquin, Nathalie Picollet-D'hahan, Eric Sulpice, Jean-Pierre Alcaraz, Frédérique Kermarrec, Jayesh Wagh, Donald K. Martin, Sophie Gerbaud, Patricia Obeid, Laurent Guyon, Monika E. Dolega, Laboratoire Biopuces (BIOPUCES), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Systèmes Nanobiotechnologiques et Biomimétiques (TIMC-IMAG-SyNaBi), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS), Structures et propriétés d'architectures moléculaire (SPRAM - UMR 5819), Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-IMAG-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-IMAG-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Institut Nanosciences et Cryogénie (INAC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: Male, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Materials science, Polymers, Carcinogenesis, Biophysics, Bioengineering, Epithelial cells, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Cell Line, Allylamine, Focal adhesion, Biomaterials, chemistry.chemical_compound, Animals, Cytotoxic T cell, [INFO.INFO-BT]Computer Science [cs]/Biotechnology, Cell adhesion, Cell proliferation, biology, Cell growth, Prostate, Prostatic Neoplasms, Adhesion, Models, Theoretical, Layer-by-layer, 021001 nanoscience & nanotechnology, Polyelectrolytes, Rats, 0104 chemical sciences, Fibronectin, chemistry, Cell culture, Mechanics of Materials, Immunology, biology.protein, Ceramics and Composites, 0210 nano-technology
Abstract: free download pdf : http://ac.els-cdn.com/S0142961213010715/1-s2.0-S0142961213010715-main.pdf?_tid=af9b5ff8-4bae-11e3-b1b0-00000aab0f27&acdnat=1384270119_77de34b330a3942315169996314d8ce3; International audience; The behaviour of cancerous epithelial prostatic cells (PC3) growing on polyelectrolytes (PE) coatings was compared to the behaviour of immortalized normal prostatic cells (PNT-2). The cell behaviour was evaluated and quantified in terms of initial cell attachment, growth, metabolic activity, morphometry, adhesion, apoptosis and stress related gene expression. Both the anionic PSS (poly(sodium 4-styrenesulphonate))-terminated surface and cationic PAH (poly(allylamine hydrochloride))-terminated surfaces were not cytotoxic. The initial attachment of cells was better on the PAH-terminated surface compared to fibronectin. However, the proliferation rate of PC3 cells was reduced on the PAH-terminated surface and slightly increased on the PSS coatings. Only PAH prevented the clustering phenotype of PC3 and reduced the number of focal adhesion points as compared to fibronectin or PSS coatings. In contrast, none of the PE surfaces significantly affected the biological responses of PNT-2 cells. PAH-terminating films provide a tool to preferentially modulate the growth of some cancerous phenotypes, in this case as a micro-environment that reduces the growth of metastatic PC3 cells.
Published: 2013
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61. Label-free analysis of prostate acini-like 3D structures by lensfree imaging

Author: Pierre Marcoux, Frédérique Kermarrec, Xavier Gidrol, Sophie Gerbaud, Nathalie Picollet-D'hahan, Monika E. Dolega, Jean-Marc Dinten, Cédric Allier, and Srikanth Vinjimore Kesavan
Subjects: Male, Cell Culture Techniques, Biomedical Engineering, Biophysics, Morphogenesis, Acinar Cells, Biology, medicine.disease_cause, Cell Line, Metastasis, 3D cell culture, Prostate, Cell Line, Tumor, Spheroids, Cellular, Image Processing, Computer-Assisted, Electrochemistry, medicine, Humans, Microscopy, Spheroid, Cell Polarity, Epithelial Cells, Equipment Design, General Medicine, medicine.disease, Cell biology, medicine.anatomical_structure, Carcinogenesis, Biotechnology, Transforming growth factor, Lumen (unit)
Abstract: We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.
Published: 2013
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62. High-Content Monitoring of Drug Effects in a 3D Spheroid Model

Author: Patricia Obeid, Xavier Gidrol, Vincent Haguet, Anastasiia V. Rulina, Maxim Y Balakirev, Frédérique Mittler, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Inflammasome NLRP3 – NLRP3 Inflammasome, Centre International de Recherche en Infectiologie (CIRI), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Université Jean Monnet - Saint-Étienne (UJM)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biomicrotechnologie et génomique fonctionnelle (BIOMICS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre International de Recherche en Infectiologie - UMR (CIRI), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: 0301 basic medicine, Drug, 3D model, Cancer Research, Cell cycle checkpoint, [SDV]Life Sciences [q-bio], media_common.quotation_subject, Cell, MLN4924, spheroids, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Computational biology, Biology, lcsh:RC254-282, drug discovery, 03 medical and health sciences, LNCaP, ubiquitin, medicine, Methods, ComputingMilieux_MISCELLANEOUS, media_common, Drug discovery, Spheroid, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, prostate cancer, Anticancer drug, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, Nedd8, 030104 developmental biology, medicine.anatomical_structure, Oncology, Cell culture, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, [SDV.IMM]Life Sciences [q-bio]/Immunology, HCS screening
Abstract: International audience; A recent decline in the discovery of novel medications challenges the widespread use of 2D monolayer cell assays in the drug discovery process. As a result, the need for more appropriate cellular models of human physiology and disease has renewed the interest in spheroid 3D culture as a pertinent model for drug screening. However, despite technological progress that has significantly simplified spheroid production and analysis, the seeming complexity of the 3D approach has delayed its adoption in many laboratories. The present report demonstrates that the use of a spheroid model may be straightforward and can provide information that is not directly available with a standard 2D approach. We describe a cost-efficient method that allows for the production of an array of uniform spheroids, their staining with vital dyes, real-time monitoring of drug effects, and an ATP-endpoint assay, all in the same 96-well U-bottom plate. To demonstrate the method performance, we analyzed the effect of the preclinical anticancer drug MLN4924 on spheroids formed by VCaP and LNCaP prostate cancer cells. The drug has different outcomes in these cell lines, varying from cell cycle arrest and protective dormancy to senescence and apoptosis. We demonstrate that by using high-content analysis of spheroid arrays, the effect of the drug can be described as a series of EC50 values that clearly dissect the cytostatic and cytotoxic drug actions. The method was further evaluated using four standard cancer chemotherapeutics with different mechanisms of action, and the effect of each drug is described as a unique multi-EC50 diagram. Once fully validated in a wider range of conditions, this method could be particularly valuable for phenotype-based drug discovery.
Published: 2017
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63. Label-Free Electric Monitoring of Human Cancer Cells as a Potential Diagnostic Tool

Author: Xavier Gidrol, Frédérique Kermarrec, Thibault Honegger, David Peyrade, Clarisse Vaillier, Laboratoire des technologies de la microélectronique (LTM ), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: Electrophoresis, Static Electricity, Nanotechnology, 02 engineering and technology, 01 natural sciences, Analytical Chemistry, Cell Movement, Cell Line, Tumor, Lab-On-A-Chip Devices, Neoplasms, Humans, Early Cancer Detection, Electrodes, Early Detection of Cancer, ComputingMilieux_MISCELLANEOUS, Label free, [PHYS]Physics [physics], Chemistry, 010401 analytical chemistry, Equipment Design, Human cell, Dielectrophoresis, 021001 nanoscience & nanotechnology, 0104 chemical sciences, On cells, Neoplasms diagnosis, 0210 nano-technology, Biological system, Electrorotation, Human cancer
Abstract: Dielectrophoresis is widely used for cell characterization, and the exerted force on cells depends on the difference of polarizability between the latter and the surrounding medium. This physical phenomenon is translated by the real part of the Clausius-Mossotti factor. It is mostly modeled from the imaginary part, measured by electrorotation. The method described here measures experimentally the real part of the Clausius-Mossotti factor. It relies on the cell velocity when submitted to pure dielectrophoresis, and it was conducted on several human cell lines, at different times. A variety of cell lines was evaluated, from different organs or representative of different stages of cancer, with promising findings for early cancer detection.
Published: 2016
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64. Distinct outcomes of CRL-Nedd8 pathway inhibition reveal cancer cell plasticity

Author: Anastasia V, Rulina, Frédérique, Mittler, Patricia, Obeid, Sophie, Gerbaud, Laurent, Guyon, Eric, Sulpice, Frédérique, Kermarrec, Nicole, Assard, Monika E, Dolega, Xavier, Gidrol, and Maxim Y, Balakirev
Subjects: Male, NEDD8 Protein, Transcription, Genetic, Cell Survival, Ubiquitin-Protein Ligases, Cell Plasticity, Prostatic Neoplasms, Cyclopentanes, Models, Biological, Pyrimidines, Transcriptional Regulator ERG, Receptors, Androgen, Cell Line, Tumor, Gene Knockdown Techniques, Spheroids, Cellular, Humans, Cell Lineage, Original Article, Ubiquitins, Cell Proliferation, Signal Transduction
Abstract: Inhibition of protein degradation by blocking Cullin-RING E3 ligases (CRLs) is a new approach in cancer therapy though of unknown risk because CRL inhibition may stabilize both oncoproteins and tumor suppressors. Probing CRLs in prostate cancer cells revealed a remarkable plasticity of cells with TMPRSS2-ERG translocation. CRL suppression by chemical inhibition or knockdown of RING component RBX1 led to reversible G0/G1 cell cycle arrest that prevented cell apoptosis. Conversely, complete blocking of CRLs at a higher inhibitor dose-induced cytotoxicity that was amplified by knockdown of CRL regulator Cand1. We analyzed cell signaling to understand how varying degrees of CRL inhibition translated to distinct cell fates. Both tumor suppressor and oncogenic cell signaling pathways and transcriptional activities were affected, with pro-metastatic Wnt/β-catenin as the most upregulated. Suppression of the NF-κB pathway contributed to anti-apoptotic effect, and androgen receptor (AR) and ERG played decisive, though opposite, roles: AR was involved in protective quiescence, whereas ERG promoted apoptosis. These data define AR–ERG interaction as a key plasticity and survival determinant in prostate cancer and suggest supplementary treatments that may overcome drug resistance mechanisms regulated by AR–ERG interaction.
Published: 2016

65. Lensfree diffractive tomography for the imaging of 3D cell cultures

Author: Xavier Gidrol, J.-M. Dinten, Fabien Momey, F. Kermarrec Marcel, Anthony Berdeu, Thomas Bordy, Nathalie Picollet-D'hahan, Cédric Allier, Laboratoire Hubert Curien / Eris, Laboratoire Hubert Curien [Saint Etienne] (LHC), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS)-Institut d'Optique Graduate School (IOGS)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS), Biomicrotechnologie et génomique fonctionnelle (BIOMICS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Berdeu, Anthony, Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS)-Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), and Laboratoire Hubert Curien (LHC)
Subjects: 0301 basic medicine, (1806900) Three-dimensional mi-croscopy References and links, Microscope, [SPI.OPTI] Engineering Sciences [physics]/Optics / Photonic, Computer science, Holography, OCIS codes: (0901970) Diffractive optics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 01 natural sciences, Article, law.invention, 010309 optics, 03 medical and health sciences, Optics, law, 0103 physical sciences, Microscopy, [SDV.BDD] Life Sciences [q-bio]/Development Biology, Organoid, (1700110) Imaging systems, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.BDD]Life Sciences [q-bio]/Development Biology, business.industry, Reconstruction algorithm, Atomic and Molecular Physics, and Optics, (1003010) Im-age reconstruction techniques, (0901995) Digital holography, 030104 developmental biology, [SPI.OPTI]Engineering Sciences [physics]/Optics / Photonic, Tomography, business, Phase retrieval, Digital holography, Biotechnology
Abstract: International audience; New microscopes are needed to help realize the full potential of 3D organoid culture studies bygathering large quantitative and systematic data over extended period of time while preserving the integrityof the living sample. On another hand, lensfree video microscopy is addressing these needs in the context of2D cell culture, providing label-free and non-phototoxic acquisition of large datasets. As scientistsroutinely adopt 3D culture techniques, the new challenging task is to extend lensfree microscopy techniquesto 3D structures.In order to image such large volume while preserving the ability to catch every single cell, we proposenew imaging platforms based on lensfree microscopy. We have built lensfree diffractive tomography setupsperforming multi-angle acquisitions of 3D organoid culture embedded in Matrigel® and developed adedicated 3D holographic reconstruction algorithm based on the Fourier diffraction theorem.With a first prototype we have been able to reconstruct a 3D volume as large as 21.5 mm^3 of a 3Dorganoid culture of prostatic RWPE1 cells in Matrigel® showing the ability of these cells to assemble in 3Dintricate cellular network at the mesoscopic scale. Comparisons with 2D images showed that it ispossible to detect single cells isolated from the main cellular structure.From results of the first prototype and numerical simulations, a second prototype wasdesigned to improve the reconstructions quality, especially on the vertical axis. First results on dust andbubbles lying at the bottom of a Petri® dish are promising.The adaptation of lensfree microscopy techniques to 3D organoid cultures imaging and thepresentation of the associated algorithms are the scopes of the present communication.
Published: 2016
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66. Characterization of the transcriptional signature of C/EBPbeta isoforms (LAP/LIP) in Hep3B cells: Implication of LIP in pro-survival functions

Author: Xavier Gidrol, Jean-Louis Danan, Eric Sulpice, Martine Hiron, Jean-Philippe Salier, Simon Tendil, Gaëlle Saint-Auret, Maryvonne Daveau, and Celine A. Blache
Subjects: Gene isoform, Carcinoma, Hepatocellular, Paclitaxel, Transcription, Genetic, Cell Survival, Biology, Models, Biological, Transcriptome, Transcription (biology), Cell Line, Tumor, Humans, Protein Isoforms, Gene, Transcription factor, Cell Proliferation, Oligonucleotide Array Sequence Analysis, Cell Death, Hepatology, CCAAT-Enhancer-Binding Protein-beta, Gene Expression Profiling, Liver Neoplasms, digestive, oral, and skin physiology, Staurosporine, Cell biology, Gene expression profiling, Cell culture, Immunology, Genetic Engineering, Chromatin immunoprecipitation
Abstract: Background & Aims C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist. In this study, we used the opposing transcriptional effects driven by LAP and LIP to determine the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line. We subsequently investigated the role of each of the LAP and LIP isoforms in drug-induced Hep3B cell death. Methods We engineered Hep3B cells with regulated LAP or LIP expression using the Tet-off expression system. The genes that showed inverse regulation by LAP and LIP were identified by cDNA array analysis. The cohort of direct-C/EBPbeta-targets was distinguished from indirect-targets by ChIP-on-chip analysis. Results We characterized 676 genes by this approach. Among these genes, 39 are novel direct targets of C/EBPbeta. Eleven of these new direct targets are involved in cell survival, suggesting critical roles for LAP/LIP isoforms in this cellular process. Therefore, we examined the effects of LAP and LIP over-expression on cell survival. We show that LIP promotes survival in staurosporine- or taxol-induced Hep3B cell death. Conclusions Our study provides new molecular and cellular insights into the role of C/EBPbeta in cells of hepatic origin.
Published: 2011
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67. Large scale RNAi screen reveals that the inhibitor of DNA binding 2 (ID2) protein is repressed by p53 family member p63 and functions in human keratinocyte differentiation

Author: Paul-Henri Romeo, Olivier Alibert, M A Viganò, David Castel, Ning Wu, Xavier Gidrol, Marie-Anne Debily, Kristiina Iljin, and Roberto Mantovani
Subjects: Keratinocytes, Cellular differentiation, Mitosis, Cell Cycle Proteins, Biology, Biochemistry, SDG 3 - Good Health and Well-being, E2F1, Gene silencing, Humans, Gene Regulation, RNA, Small Interfering, Promoter Regions, Genetic, Molecular Biology, Gene, Transcription factor, Inhibitor of Differentiation Protein 2, Regulation of gene expression, Tumor Suppressor Proteins, Nuclear Proteins, Promoter, Cell Differentiation, Cell Biology, Cell cycle, Phosphoproteins, Molecular biology, DNA-Binding Proteins, HEK293 Cells, Gene Expression Regulation, Tumor Suppressor Protein p53, Transcription Factors
Abstract: The inhibitor of DNA binding 2, dominant negative helix-loop-helix protein, ID2, acts as an oncogene and elevated levels of ID2 have been reported in several malignancies. Whereas some inducers of the ID2 gene have been characterized, little is known regarding the proteins capable to repress its expression. We developed siRNA microarrays to perform a large scale loss-of-function screen in human adult keratinocytes engineered to express GFP under the control of the upstream region of ID2 gene. We screened the effect of siRNA-dependent inhibition of 220 cancer-associated genes on the expression of the ID2::GFP reporter construct. Three genes NBN, RAD21, and p63 lead to a repression of ID2 promoter activity. Strikingly NBN and RAD21 are playing on major role in cell cycle progression and mitosis arrest. These results underline the pregnant need to silence ID2 expression at transcript level to promote cell cycle exit. Central to this inhibitory mechanism we find p63, a key transcription factor in epithelial development and differentiation, which binds specific cis-acting sequence within the ID2 gene promoter both in vitro and in vivo. P63 would not suppress ID2 expression, but would rather prevent excessive expression of that protein to enable the onset of keratinocyte differentiation.
Published: 2011
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68. 2D and 3D cell microarrays in pharmacology

Author: Xavier Gidrol, Nathalie Picollet-D'hahan, Lamya Ghenim, Vincent Haguet, Brigitte Fouque, and Béatrice Schaack
Subjects: Automation, Laboratory, Pharmacology, Miniaturization, Microarray, Drug discovery, Microarray analysis techniques, Cell, Cell Culture Techniques, Nanotechnology, Biology, Microarray Analysis, High-Throughput Screening Assays, medicine.anatomical_structure, Cell culture, Drug Discovery, medicine, Animals, Humans, Biological Assay, DNA microarray
Abstract: To analyze the phenotypic consequences of perturbing mammalian cells with drugs, there is an increasing need for systematic cell-based assays in an HTS format. Cell microarrays provide an attractive solution as they offer more than a simple miniaturization and mechanization of conventional microtiter plates. While standard monolayer two-dimensional culture conditions are poor mimics of the cellular environment in situ, microfabricated systems enable three-dimensional organotypic cell cultures and have the potential to provide biological insight not achievable before. This article compares different cell microarray formats and evaluates their potential use in the drug discovery process.
Published: 2009
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69. Inférence des réseaux de régulation transcriptionnelle

Author: Ning Wu, Xavier Gidrol, Marie-Anne Debily, and Vincent Frouin
Subjects: Physics, Cell network, General Medicine, Molecular biology, General Biochemistry, Genetics and Molecular Biology
Abstract: La cellule est un systeme infiniment complexe et meme si la biologie moleculaire a permis des avancees remarquables, les biologistes cherchent aujourd’hui a aborder cette complexite a l’echelle du systeme, esperant ainsi mieux decrypter les mysteres cellulaires. L’inference des reseaux de regulation transcriptionnelle, de leur topologie, de leur evolution dynamique, de leur role dans les grandes fonctions biologiques est au coeur de cette approche. Nous proposons une demarche systemique, fondee sur l’integration de donnees a large echelle telles que les profils d’expression, l’analyse par puce des produits d’immunoprecipitation de chromatine, et l’ARN interference a haut debit pour inferer ces reseaux et caracteriser les phenotypes associes. A plus long terme, la comprehension des proprietes des reseaux pourrait permettre la decouverte de nouvelles cibles therapeutiques et la mise a jour de nouvelles strategies medicamenteuses.
Published: 2008
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70. A 3D Toolbox to Enhance Physiological Relevance of Human Tissue Models

Author: Christophe A. Marquette, Xavier Gidrol, Nathalie Picollet-D'hahan, Séverine Le Gac, Monika E. Dolega, Donald K. Martin, and Lavinia Liguori
Subjects: 0301 basic medicine, Tissue Engineering, Tissue Scaffolds, Computer science, Cell Culture Techniques, METIS-321608, Bioengineering, Nanotechnology, 3d model, Computational biology, Microfluidic Analytical Techniques, Models, Biological, Toolbox, Extracellular Matrix, Molecular analysis, Organoids, 03 medical and health sciences, 030104 developmental biology, Tissue engineering, Tissue scaffolds, Tissue Array Analysis, Humans, Relevance (information retrieval), Cells, Cultured, Biotechnology
Abstract: We discuss the current challenges and future prospects of flow-based organoid models and 3D self-assembling scaffolds. The existing paradigm of 3D culture suffers from a lack of control over organoid size and shape; can be an obstacle for cell harvesting and extended cellular and molecular analysis; and does not provide access to the function of exocrine glands. Moreover, existing organ-on-chip models are mostly composed of 2D extracellular matrix (ECM)-coated elastomeric membranes that do not mimic real organ architectures. A new comprehensive 3D toolbox for cell biology has emerged to address some of these issues. Advances in microfabrication and cell-culturing approaches enable the engineering of sophisticated models that mimic organ 3D architectures and physiological conditions, while supporting flow-based drug screening and secretomics-based diagnosis.
Published: 2016

71. 3D LENSFREE MICROSCOPY FOR 3D CELL CULTURE

Author: Anthony Berdeu, Fabien Momey, Jean-Marc Dinten, Nathalie Picollet-d'Hahan, Xavier Gidrol, cedric allier, Berdeu, Anthony, Laboratoire Hubert Curien [Saint Etienne] (LHC), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Laboratoire d'Exploration Fonctionnelle des Génomes (LEFG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS), and Laboratoire Hubert Curien (LHC)
Subjects: [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, [SPI.SIGNAL] Engineering Sciences [physics]/Signal and Image processing
Abstract: National audience; New microscopes are needed to help realize the full potential of 3D organoid culture studies by gathering large quantitative and systematic data over extended period of time while preserving the integrity of the living sample. In order to reconstruct large volume while keeping the ability to catch every single cell, we propose new imaging platforms based on lensfree microscopy, a technic which is addressing these needs in the context of 2D cell culture, providing label-free and non-phototoxic acquisition of large datasets. We have built lensfree diffractive tomography setups performing multi-angle acquisitions of 3D organoid culture embedded in Matrigel ® and developed dedicated 3D holographic reconstruction algorithms based on the Fourier diffraction theorem. Nonetheless, holographic setups do not record the phase of the incident wavefront and the biological samples in Petri dish strongly limit the angular coverage. These limitations introduces numerous artefacts in the sample reconstruction. We developed several methods to overcome them, such as multi wavelength imaging or iterative phase retrieval. The most promising technic currently developed is based on a regularized inverse problem approach directly performed on the 3D volume to reconstruct. 3D reconstructions were realized on several complex samples such as 3D networks or spheroids embedded in capsules with large reconstructed volumes up to ~25 mm^3 while still being able to identify single cells. To our knowledge, this is the first time that such an inverse problem approach is implemented in the context of lensfree diffractive tomography enabling to reconstruct large volume of unstained biological samples.
Published: 2016

72. Refilins are short-lived Actin-bundling proteins that regulate lamellipodium protrusion dynamics

Author: Joël Lachuer, Christian Delphin, Xavier Gidrol, Pascal Stuelsatz, Jacques Baudier, Nicole Assard, Benoit Gilquin, Bertacchi Griffi, Nathalie, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), INSERM U836, équipe 1, Physiopathologie du cytosquelette, Grenoble Institut des Neurosciences (GIN), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Santé et de la Recherche Médicale (INSERM), Métabolisme et pathologies du cerveau : imagerie et modélisation (GMPIB), Université Paris-Sud - Paris 11 (UP11), Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)
Subjects: RefilinA, 0301 basic medicine, QH301-705.5, Science, [SDV]Life Sciences [q-bio], Arp2/3 complex, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, General Biochemistry, Genetics and Molecular Biology, Cell membrane, Protein filament, 03 medical and health sciences, Filamin binding, Actin remodeling of neurons, 0302 clinical medicine, FilaminA, medicine, Actin-binding protein, Biology (General), FAM101B, FAM101A, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Actin, ComputingMilieux_MISCELLANEOUS, RefilinB, biology, Cell biology, [SDV] Life Sciences [q-bio], 030104 developmental biology, medicine.anatomical_structure, biology.protein, Lamellipodium, General Agricultural and Biological Sciences, 030217 neurology & neurosurgery, Research Article, Polydendrocyte
Abstract: Refilins (RefilinA and RefilinB) are members of a novel family of Filamin binding proteins that function as molecular switches to conformationally alter the Actin filament network into bundles. We show here that Refilins are extremely labile proteins. An N-terminal PEST/DSG(X)2-4S motif mediates ubiquitin-independent rapid degradation. A second degradation signal is localized within the C-terminus. Only RefilinB is protected from rapid degradation by an auto-inhibitory domain that masks the PEST/DSG(X)2-4S motif. Dual regulation of RefilinA and RefilinB stability was confirmed in rat brain NG2 precursor cells (polydendrocyte). Using loss- and gain-of-function approaches we show that in these cells, and in U373MG cells, Refilins contribute to the dynamics of lamellipodium protrusion by catalysing Actin bundle formation within the lamella Actin network. These studies extend the Actin bundling function of the Refilin-Filamin complex to dynamic regulation of cell membrane remodelling., Summary: The newly identified Refilin protein family are unique short-lived proteins that complex with Filamin to regulate plasma membrane dynamics though the promotion of Actin filament bundling.
Published: 2016
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73. Time-lapse contact microscopy of cell cultures based on non-coherent illumination

Author: François Perraut, Nathalie Picollet-D'hahan, Francois Chatelain, Marion Gabriel, Vincent Haguet, Cyrille Pornin, Marc R. Block, Stéphane Gétin, Dorothée Balle, Stéphanie Bigault, Xavier Gidrol, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Département d'Optronique (DOPT), Laboratoire d'Electronique et des Technologies de l'Information (CEA-LETI), Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Département Microtechnologies pour la Biologie et la Santé (DTBS), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Dynamique des systèmes d'adhérence et différenciation (DySAD), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
Subjects: Materials science, [SDV]Life Sciences [q-bio], Population, Cell Culture Techniques, Holography, Video microscopy, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Time-Lapse Imaging, Article, law.invention, Optics, Cell Movement, law, Microscopy, Cell Adhesion, Humans, Image sensor, education, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, Cell Proliferation, education.field_of_study, Microscopy, Video, Multidisciplinary, business.industry, Ray, Pinhole (optics), Photonics, business
Abstract: Video microscopy offers outstanding capabilities to investigate the dynamics of biological and pathological mechanisms in optimal culture conditions. Contact imaging is one of the simplest imaging architectures to digitally record images of cells due to the absence of any objective between the sample and the image sensor. However, in the framework of in-line holography, other optical components, e.g., an optical filter or a pinhole, are placed underneath the light source in order to illuminate the cells with a coherent or quasi-coherent incident light. In this study, we demonstrate that contact imaging with an incident light of both limited temporal and spatial coherences can be achieved with sufficiently high quality for most applications in cell biology, including monitoring of cell sedimentation, rolling, adhesion, spreading, proliferation, motility, death and detachment. Patterns of cells were recorded at various distances between 0 and 1000 μm from the pixel array of the image sensors. Cells in suspension, just deposited or at mitosis focalise light into photonic nanojets which can be visualised by contact imaging. Light refraction by cells significantly varies during the adhesion process, the cell cycle and among the cell population in connection with every modification in the tridimensional morphology of a cell.
Published: 2015
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74. Phi-score: A cell-to-cell phenotypic scoring method for sensitive and selective hit discovery in cell-based assays

Author: Ricky Bhajun, Stéphanie Combe, Jean-Philippe Vert, Eric Sulpice, J. Pablo Radicella, Philippe Rouillier, Christian Lajaunie, Mélissa Mary, Xavier Gidrol, Anna Campalans, Frédéric Fer, Guillaume Pinna, Patricia Obeid, Laurent Guyon, Laboratoire de Biologie à Grande Échelle ( BGE - UMR S1038 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Grenoble Alpes [Saint Martin d'Hères]-Institut National de la Santé et de la Recherche Médicale ( INSERM ), Cancer et génôme: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, MINES ParisTech - École nationale supérieure des mines de Paris-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -INSTITUT CURIE, Centre de Bioinformatique ( CBIO ), MINES ParisTech - École nationale supérieure des mines de Paris-PSL Research University ( PSL ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Cellules Souches et Radiations ( SCSR - U 967 ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris Diderot - Paris 7 ( UPD7 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), This work was funded by grants from the Agence Nationale de la Recherche (Investissements d’avenir), the pôle de compétitivité MEDICEN and CEA. The screen was funded by a grant from the Association pour la Recherche sur le Cancer (n°PJA 20131200165 to JPR)., Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Cancer et génome: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, Mines Paris - PSL (École nationale supérieure des mines de Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Bioinformatique (CBIO), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Stabilité génétique, Cellules Souches et Radiations (SCSR (U_967)), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Paris (UP), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), MINES ParisTech - École nationale supérieure des mines de Paris-Institut Curie-Institut National de la Santé et de la Recherche Médicale (INSERM), MINES ParisTech - École nationale supérieure des mines de Paris-PSL Research University (PSL), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Cellules Souches et Radiations (SCSR (U967 / UMR-E_008)), Université Paris-Sud - Paris 11 (UP11)-Université Paris Diderot - Paris 7 (UPD7)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA), Centre de Géosciences (GEOSCIENCES), PARi (PARI), Département Plateforme (PF I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de Radiobiologie Cellulaire et Moléculaire (IRCM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay, Cellules Souches et Radiations (SCSR - U 967), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire d'Informatique, de Traitement de l'Information et des Systèmes (LITIS), Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Université Le Havre Normandie (ULH), Normandie Université (NU), PSL Research University (PSL)-MINES ParisTech - École nationale supérieure des mines de Paris, and Institut Curie
Subjects: [SDV]Life Sciences [q-bio], Phenotypic screening, Cell, Excision-Repair, Robust statistics, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Population Context, 01 natural sciences, Article, 03 medical and health sciences, RNA interference, medicine, Statistical-Methods, Hit selection, Primary cell, Pathways, Patterns, [ SDV.BIBS ] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Genetics, 0303 health sciences, Multidisciplinary, Scale RNAi Screens, Transfection, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], 0104 chemical sciences, 010404 medicinal & biomolecular chemistry, medicine.anatomical_structure
Abstract: Phenotypic screening monitors phenotypic changes induced by perturbations, including those generated by drugs or RNA interference. Currently-used methods for scoring screen hits have proven to be problematic, particularly when applied to physiologically relevant conditions such as low cell numbers or inefficient transfection. Here, we describe the Φ-score, which is a novel scoring method for the identification of phenotypic modifiers or hits in cell-based screens. Φ-score performance was assessed with simulations, a validation experiment and its application to gene identification in a large-scale RNAi screen. Using robust statistics and a variance model, we demonstrated that the Φ-score showed better sensitivity, selectivity and reproducibility compared to classical approaches. The improved performance of the Φ-score paves the way for cell-based screening of primary cells, which are often difficult to obtain from patients in sufficient numbers. We also describe a dedicated merging procedure to pool scores from small interfering RNAs targeting the same gene so as to provide improved visualization and hit selection.
Published: 2015
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75. A kinome-targeted RNAi-based screen links FGF signaling to H2AX phosphorylation in response to radiation

Author: Vincent Frouin, Ning Wu, Amandine Pitaval, Paul-Henri Romeo, Xavier Gidrol, Claudie Lemercier, Alexandre Papine, Sami Benzina, Françoise Soussaline, Celine Lustremant, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Neuroimagerie Assistée par Ordinateur (LNAO), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), IMSTAR, IMSTAR S.A., Institut Cochin (UMR_S567 / UMR 8104), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)
Subjects: Keratinocytes, DNA Repair, [SDV]Life Sciences [q-bio], Cell Cycle Proteins, DNA-Activated Protein Kinase, environment and public health, DSB, Phosphorylation cascade, Histones, 0302 clinical medicine, DNA Breaks, Double-Stranded, Kinome, γH2AX, Phosphorylation, RNA, Small Interfering, ComputingMilieux_MISCELLANEOUS, 0303 health sciences, Radiation, biology, Kinase, Nuclear Proteins, Chromatin, DNA-Binding Proteins, Histone, 030220 oncology & carcinogenesis, Molecular Medicine, RNA Interference, Signal transduction, biological phenomena, cell phenomena, and immunity, Research Article, Signal Transduction, DNA repair, Cell Line, 03 medical and health sciences, Cellular and Molecular Neuroscience, Humans, Mitogen-Activated Protein Kinase 8, Receptor, Fibroblast Growth Factor, Type 4, Molecular Biology, 030304 developmental biology, Pharmacology, JNK1, Cell Biology, Fibroblast Growth Factors, enzymes and coenzymes (carbohydrates), ATM, FGFR4, biology.protein, Cancer research
Abstract: A general radioprotective effect by fibroblast growth factor (FGF) has been extensively described since the early 1990s; however, the molecular mechanisms involved remain largely unknown. Radiation-induced DNA double-strand breaks (DSBs) lead to a complex set of responses in eukaryotic cells. One of the earliest consequences is phosphorylation of histone H2AX to form nuclear foci of the phosphorylated form of H2AX (γH2AX) in the chromatin adjacent to sites of DSBs and to initiate the recruitment of DNA-repair molecules. Upon a DSB event, a rapid signaling network is activated to coordinate DNA repair with the induction of cell-cycle checkpoints. To date, three kinases (ATM, ATR, and DNA-PK) have been shown to phosphorylate histone H2AX in response to irradiation. Here, we report a kinome-targeted small interfering RNA (siRNA) screen to characterize human kinases involved in H2AX phosphorylation. By analyzing γH2AX foci at a single-nucleus level, we identified 46 kinases involved either directly or indirectly in H2AX phosphorylation in response to irradiation in human keratinocytes. Furthermore, we demonstrate that in response to irradiation, the FGFR4 signaling cascade promotes JNK1 activation and direct H2AX phosphorylation leading, in turn, to more efficient DNA repair. This can explain, at least partially, the radioprotective effect of FGF. Electronic supplementary material The online version of this article (doi:10.1007/s00018-015-1901-7) contains supplementary material, which is available to authorized users.
Published: 2015
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76. Lensfree video microscopy: high throughput monitoring and cell tracking of 2D cell cultures

Author: Olivier Cioni, Thomas Bordy, Cédric Allier, Fabien Momey, Xavier Gidrol, L. Hervé, J.-M. Dinten, Fabrice Navarro, Mathilde Menneteau, Eric Sulpice, Sophie Morel, S. Vinjimore Kesavan, D. Freida, and Bernard Chalmond
Subjects: Standard cell, Microscope, Cell division, business.industry, Cell, Video microscopy, Biology, Frame rate, law.invention, medicine.anatomical_structure, law, Cell culture, medicine, Computer vision, Artificial intelligence, Cell adhesion, business, Biomedical engineering
Abstract: In order to extend the analysis of the datasets produced by lensfree video microscopy we have implemented a cell tracking algorithm to combine and correlate cell motility to the previously devised metrics to quantify e.g. cell adhesion and spreading, cell division, and cell death. In this paper we present the assessment of these new methodology on experiments involving three different cell lines, namely 3T3 fibroblast cells, primary HUVEC cells and macrophage THP1 cells. We demonstrate that the good spatial resolution and the fast frame rate obtained with of our lensfree video microscope allows standard cell tracking algorithm to be computed. The results is the possibility to analyze thousands of cells successfully tracked over tens of hours. The results is the possibility to compare different cell cultures in terms of e.g. cell motility and cell confinement ration. Ultimately we managed to measure the doubling time at single cell level over a large number of N=235 cells tracked over two days.
Published: 2015
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77. Author response: Wss1 metalloprotease partners with Cdc48/Doa1 in processing genotoxic SUMO conjugates

Author: David F. Lindsey, Nicole Assard, Eric Sulpice, Adrien Favier, Xavier Gidrol, James E. Mullally, Anastasia V Rulina, Keith D. Wilkinson, and Maxim Y Balakirev
Subjects: Metalloproteinase, Biochemistry, Chemistry, Conjugate
Published: 2015
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78. Involvement of membrane proteins and ion channels on the self-rotation of human cells in a non-rotating AC electric field

Author: Clarisse Vaillier, Frédérique Kermarrec, David Peyrade, Xavier Gidrol, Thibault Honegger, Laboratoire des technologies de la microélectronique (LTM), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Clot, Marielle, Université Joseph Fourier - Grenoble 1 (UJF)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Laboratoire des technologies de la microélectronique [2001-2015] (LTM [2001-2015])
Subjects: Rotation, Cell Survival, [SDV]Life Sciences [q-bio], Clinical Biochemistry, Cell Culture Techniques, Nanotechnology, Biochemistry, Ion Channels, Analytical Chemistry, [PHYS] Physics [physics], Electricity, Humans, Ion channel, ComputingMilieux_MISCELLANEOUS, [PHYS]Physics [physics], Chemistry, HEK 293 cells, Membrane Proteins, Dielectrophoresis, Transmembrane protein, Membrane, HEK293 Cells, Membrane protein, Cytoplasm, Gene Knockdown Techniques, Cancer cell, Biophysics, RNA Interference
Abstract: International audience; Dielectrophoresis is a force that has been exploited in microsystems for label‐free characterization and separation of cells, when their electrical signature is known. However, the polarization effect of cells at the transmembrane protein level is not well established. In this work, we have use the self‐rotation effect of cells in a non‐rotating field, known as the “Quincke effect,” in order to measure the maximum rotation frequency (frotmax) of different cell populations when modifying the composition of their membrane. We investigated the influence of active ionic transportation of membrane protein concentration on frotmax of HEK cells. Our results show that ionic transportation is responsible for the reduction of conductivity within the cytoplasm, which results in higher frotmax. However, the influence of the concentration of proteins in the membrane, achieved by silencing gene expression in cancer cells, changes significantly frotmax, which is not explained by the changes of ionic conductivity within the cell.
Published: 2015
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79. Cell microarrays in drug discovery

Author: Marie-Anne Debily, Xavier Gidrol, Amandine Pitaval, and David Castel
Subjects: High-throughput screening, Cell, Protein Array Analysis, Gene Expression, Nanotechnology, Large range, Computational biology, Pharmaceutical technology, Drug Discovery, Cell Adhesion, medicine, Animals, Humans, Microtechnology, RNA, Small Interfering, Cells, Cultured, Oligonucleotide Array Sequence Analysis, Pharmacology, Extracellular Matrix Proteins, Drug discovery, business.industry, Reproducibility of Results, medicine.anatomical_structure, Tissue Array Analysis, Drug Design, RNA Interference, DNA microarray, business
Abstract: There is an increasing need for systematic cell-based assays in a high-throughput screening (HTS) format to analyze the phenotypic consequences of perturbing mammalian cells with drugs, genes, interfering RNA. Taking advantage of the recent progress in microtechnology, new cell microarrays are being developed and applied to a large range of issues in metazoan cells. This article compares different approaches and evaluates their potential use in the drug discovery process. Although still an emerging technology, cell microarrays hold great promise to optimize the efficiency:cost ratio in cell-based HTS.
Published: 2006
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80. Role of the iron mobilization and oxidative stress regulons in the genomic response of yeast to hydroxyurea

Author: Carl Mann, Régis Courbeyrette, Cyrille Petat, Xavier Gidrol, Anne Chevalier, and Caroline Dubacq
Subjects: YAP1, Gene Expression Profiling, Iron, Mutant, DNA replication, Saccharomyces cerevisiae, General Medicine, Protein Serine-Threonine Kinases, Biology, Regulon, Molecular biology, Yeast, Oxidative Stress, Ribonucleotide reductase, Drug Resistance, Fungal, Ribonucleotide Reductases, Genetics, Hydroxyurea, Enzyme Inhibitors, Genome, Fungal, DNA microarray, Transcription Factor Gene, Molecular Biology, Oligonucleotide Array Sequence Analysis
Abstract: Hydroxyurea (HU) is a specific inhibitor of ribonucleotide reductase and thus impairs dNTP synthesis and DNA replication. The long-term transcriptional response of yeast cells to hydroxyurea was investigated using DNA microarrays containing all yeast coding sequences. We show that the redox-responsive Yap regulon and the iron-mobilization Aft regulon are activated in yeast cells treated with HU. Yap1 accumulates in the nucleus in response to HU, but HU activation of the Yap regulon was only partially dependent on Yap1 and yap1Delta mutants were not hypersensitive to HU. In contrast, deletion of the AFT1 and AFT2 transcription factor genes blocked the HU activation of a subset of the Aft regulon and the aft1Delta aft2Delta double mutant was hypersensitive to HU in an iron-suppressible manner. These results highlight the importance of the redox and iron mobilization regulons in the cellular response to HU.
Published: 2005
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81. Global transcriptional characterization of SP and MP cells from the myogenic C2C12 cell line: effect of FGF6

Author: Olivier Danos, Sylvie Bortoli, Philippe Rameau, Geneviève Piétu, Diana Tronik-Le Roux, Luis Garcia, Christelle Rochon, Xavier Gidrol, Rachid Benchaouir, Charles Decraene, Marie-Agnès Dillies, David Israeli, and Amandine Pitaval
Subjects: Transcription, Genetic, Physiology, Fibroblast Growth Factor 6, Cellular differentiation, Population, Down-Regulation, Cell Separation, Biology, Myoblasts, Mice, Side population, Proto-Oncogene Proteins, Genetics, Animals, education, Cells, Cultured, Principal Component Analysis, education.field_of_study, Staining and Labeling, Reverse Transcriptase Polymerase Chain Reaction, Gene Expression Profiling, Reproducibility of Results, Cell Differentiation, DNA, Flow Cytometry, Microarray Analysis, Phenotype, Molecular biology, Up-Regulation, Gene expression profiling, Cell culture, Benzimidazoles, Stem cell, DNA Probes, C2C12
Abstract: With the use of Hoechst staining techniques, we have previously shown that the C2C12 myogenic cell line contains a side population (SP) that is largely increased in the presence of fibroblast growth factor 6 (FGF6). Here, we compared transcriptional profiles from SP and main population (MP) cells from either C2C12 or FGF6-expressing C2C12. Expression profiles of SPs show that these cells are less differentiated than MPs and display some similarities to stem cells. Moreover, principal component analysis made it possible to distinguish specific contributions of either FGF6 or differentiation effects on gene expression profiles. This demonstrated that FGF6-expanded SPs were similar to parental C2C12-derived SPs. Conversely, FGF6-treated MPs differed from parental MPs and were more related to SP cells. These results show that FGF6 pushed committed myogenic cells toward a more immature phenotype resulting in the accumulation of cells with a SP phenotype. We propose that FGF6 conditioning could provide a way to expand the pool of immature cells by myoblast dedifferentiation.
Published: 2005
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82. Puces à cellules et génomique fonctionnelle

Author: Xavier Gidrol, Amandine Pitaval, Sandrine Baghdoyan, Yoann Roupioz, and David Castel
Subjects: Complementary DNA, Human genome, General Medicine, Computational biology, DNA microarray, Biology, Gene, Genome, Functional genomics, General Biochemistry, Genetics and Molecular Biology, Loss function, Function (biology)
Abstract: 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.
Published: 2005
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83. DNA microarray allows molecular profiling of rheumatoid arthritis and identification of pathophysiological targets

Author: Xavier Gidrol, Amandine Pitaval, Gilles Chiocchia, Carlo Lucchesi, Sylvie Mistou, Maxime Breban, S Marion, P Anract, G Falgarone, Valérie Devauchelle, M Hamadouche, Franck Letourneur, O Alibert, M Dougados, X Ayral, Nicolas Cagnard, and Catherine Fournier
Subjects: Adult, Male, Microarray, Cathepsin L, Immunology, Gene Expression, Arthritis, Computational biology, Biology, Bioinformatics, Polymerase Chain Reaction, law.invention, Arthritis, Rheumatoid, DEAD-box RNA Helicases, GTP-Binding Proteins, law, Osteoarthritis, Synovial Fluid, Gene expression, Genetics, medicine, Humans, RNA, Messenger, Gene, Cells, Cultured, Genetics (clinical), Polymerase chain reaction, Aged, DNA Primers, Glycoproteins, Oligonucleotide Array Sequence Analysis, Aged, 80 and over, Reverse Transcriptase Polymerase Chain Reaction, Lactoylglutathione Lyase, Membrane Proteins, Nucleic Acid Hybridization, Middle Aged, medicine.disease, Cathepsins, Cysteine Endopeptidases, Clusterin, Real-time polymerase chain reaction, Gene chip analysis, Female, DNA microarray, RNA Helicases, Molecular Chaperones
Abstract: This study was undertaken to evaluate the possibility to obtain a molecular signature of rheumatoid arthritis (RA) comparatively osteoarthritis (OA), and to lay the bases to develop new diagnostic tools and identify new targets. Microarray technology was used for such an analysis. The gene expression profiles of synovial tissues from patients with confirmed RA, and patients with OA were established and compared. A set of 63 genes was selected, based, more specifically, on their overexpression or underexpression in RA samples compared to OA. Results for six of these genes have been verified by quantitative PCR using both samples identical to those used in the microarray experiments and entirely separate samples. Expression profile of the 48 known genes allowed the correct classification of additional RA and OA patients. Furthermore, the distinct expression of three of the selected genes was also studied by quantitative RT-PCR in cultured synovial cells. Detailed analysis of the expression profile of the selected genes provided evidence for dysregulated biological pathways, pointed out to chromosomal location and revealed novel genes potentially involved in RA. It is proposed that such an approach allows valuable diagnosis/prognostics tools in RA to be established and potential targets for combating the disease to be identified.
Published: 2004
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84. Expression profiling of genes and proteins in HaCaT keratinocytes: Proliferating versus differentiated state

Author: Cyrille Petat, Pierre Vaigot, Amandine Pitaval, Pascal Soularue, Gilles Lemaitre, Jérôme Garin, Michèle T. Martin, Xavier Gidrol, Stephan Bouet, Jérôme Lamartine, and Gilles Waksman
Subjects: Epidermis (botany), Cellular differentiation, Cell, Cell Biology, Cell cycle, Biology, Proteomics, Biochemistry, Molecular biology, Cell biology, Gene expression profiling, HaCaT, medicine.anatomical_structure, Gene expression, medicine, Molecular Biology
Abstract: The knowledge of the mechanism of keratinocyte differentiation in culture is still uncompleted. The emergence of new technologies, such as cDNA microarrays or 2D electrophoresis followed by mass spectrometry analysis, has allowed the identification of genes and proteins expressed in biological processes in keratinocytes. Here, we report a genome wide analysis of proliferating versus differentiated human HaCaT keratinocytes. We found that genes and proteins which take part in the cell cycle control, carbohydrate metabolism, cell auto-immunity, adhesion and cytokine signal transduction pathways were regulated in differentiated HaCaT keratinocytes. In addition, we identified seven proteins and 33 transcripts that had not been previously described as differentially expressed in proliferating versus differentiated HaCaT cells. Furthermore, some of these transcripts or proteins were similarly regulated in human primary keratinocytes and in human epidermis. The present study opens new areas of investigation in the comprehension of keratinocyte differentiation. (C) 2004 Wiley-Liss, Inc.
Published: 2004
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85. Tomographie diffractive sans lentille appliquée à la culture cellulaire 3D

Author: Anthony Berdeu, Fabien Momey, Nathalie Picollet-d'Hahan, Frédérique Marcel, Xavier Gidrol, Thomas Bordy, Jean-Marc Dinten, cedric allier, Berdeu, Anthony, Laboratoire Hubert Curien [Saint Etienne] (LHC), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet - Saint-Étienne (UJM)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire Conception pour Imageurs et Biopuces (LCIB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Institut d'Optique Graduate School (IOGS)-Université Jean Monnet [Saint-Étienne] (UJM)-Centre National de la Recherche Scientifique (CNRS), and Laboratoire Hubert Curien (LHC)
Subjects: [PHYS.PHYS.PHYS-OPTICS] Physics [physics]/Physics [physics]/Optics [physics.optics], [PHYS.PHYS.PHYS-OPTICS]Physics [physics]/Physics [physics]/Optics [physics.optics]
Abstract: National audience; The study of in vitro cell populations is a major concern for biologists in the realms of drug screening and oncology for example. With standard microscopy techniques, crucial information at the mesoscopic scale is still difficult to reach due to reduced field of view and complexity to monitor cell behaviors in real time over large periods of time. Lensfree video microscopy has already overcome these difficulties in the context of 2D cell biology. Based on the in-line holography principle, it puts cells directly between a coherent light source and a sensor allowing large field of view, e.g. 30 mm 2. With the growth of 3D cell cultures, the new challenging task is to extend this imaging technique to acquisitions and 3D reconstructions of cellular culture. For this purpose, we have developed an experimental optical bench dedicated to lensfree diffractive tomography and studied several 3D reconstruction algorithms on acquisitions of 3D prostatic cell cultures.
Published: 2015

86. Point-of-care diagnostics for ricin exposure

Author: Chantal Mourton-Gilles, Dorothée Jary, Jean Berthier, Cathy Philippe, Mohamed Lemine Youba Diakite, Jérôme Rollin, Didier Sauvaire, Guillaume Delapierre, Xavier Gidrol, Génétique, immunothérapie, chimie et cancer (GICC), UMR 7292 CNRS [2012-2017] (GICC UMR 7292 CNRS), Université de Tours (UT)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Agence Nationale de Sécurité du Médicament et des produits de santé, ANSM, Agence nationale de sécurité du médicament et des produits de santé [Saint-Denis] (ANSM), Laboratoire d'Exploration Fonctionnelle des Génomes (LEFG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris Diderot - Paris 7 (UPD7), Département Microtechnologies pour la Biologie et la Santé (DTBS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Université de Tours-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Electronique et des Technologies de l'Information (CEA-LETI), Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Direction de Recherche Technologique (CEA) (DRT (CEA)), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Theranostic Nanomedicine, Point-of-care testing, Point-of-Care Systems, [SDV]Life Sciences [q-bio], Biomedical Engineering, Host response, Poison control, Bioengineering, Biological Warfare Agents, Ricin, Computer security, computer.software_genre, Biochemistry, chemistry.chemical_compound, Cartridge, Mice, Lab-On-A-Chip Devices, Animals, Humans, ComputingMilieux_MISCELLANEOUS, Whole blood, Mice, Inbred BALB C, Chromatography, General Chemistry, 3. Good health, Ricin toxin, chemistry, computer
Abstract: A long-sought milestone in the defense against bioterrorism is the development of rapid, simple, and near-patient assays for diagnostic and theranostic purposes. Here, we present a powerful test based on a host response to a biological weapon agent, namely the ricin toxin. A signature for exposure to ricin was extracted and characterized in mice and then integrated into a plastic microfluidic cartridge. This enabled early diagnosis of exposure to ricin in mice using a drop of whole blood in less than 1 h and 30 min. The cartridge stores the reagents and implements all of the steps of the analysis, including mRNA extraction from a drop of blood, followed by tens of parallel RT-qPCR reactions. The simple and low-cost microfluidic cartridge developed here may find other applications in point-of-care diagnostics.
Published: 2015
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87. Culture Conditions can Counterbalance the Thermosensitive Properties of Poly (N-Isopropylacrylamide) during Cell Harvesting

Author: Agache, Vinjimore Kesavan S, P. Pouteau, Nathalie Picollet-D'hahan, Monika E. Dolega, Gerbaud S, Xavier Gidrol, F. Abeille, Beatrice Icard, Cédric Allier, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: Microscope, Materials science, [SDV]Life Sciences [q-bio], Cell, Nanotechnology, 02 engineering and technology, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 010402 general chemistry, 021001 nanoscience & nanotechnology, Polymer brush, 01 natural sciences, 0104 chemical sciences, law.invention, chemistry.chemical_compound, medicine.anatomical_structure, chemistry, law, Cell culture, Poly(N-isopropylacrylamide), Biophysics, medicine, 0210 nano-technology, Fibroblastic cell, ComputingMilieux_MISCELLANEOUS
Abstract: Poly (N-isopropylacrylamide) (PNIPAM) has been widely referenced as an alternative solution for proteolyticenzyme- based cell harvesting. However, the reversibility (i.e., consecutive attachment-detachment cycles) of PNIPAM for cell harvesting has not been studied. In this work, we synthesized PNIPAM-grafted glass surfaces to study the efficiency of fibroblastic cell detachment after iterative use. For robust quantification of thermally triggered cell detachment, an innovative wide-field lens free video microscope was introduced. The cell detachment efficiency was shown to decrease significantly after each cell culture experiment due to the loss of polymer brush thermosensitivity. The influence of the temperature variation rate on cell detachment from the re-used PNIPAM-grafted surfaces was further characterized. The cell detachment behavior on these substrates was shown to be quite similar to that on bare glass substrates. Furthermore, it was demonstrated that non-negligible cell detachment occurs and is modulated according to the temperature variation rate. This study aims to inform scientists using PNIPAM-grafted substrates for cell detachment about possible issues, to help scientists to more carefully monitor culture experiments, and to aid scientists in the interpretation of results obtained from cell detachment experiments performed on PNIPAM-grafted surfaces.
Published: 2015
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88. A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

Author: Vincent Haguet, Stéphanie Combe, Ricky Bhajun, Christian Lajaunie, Xavier Gidrol, Amandine Pitaval, Eric Sulpice, Patricia Obeid, Laurent Guyon, Itebeddine Ghorbel, Biomicrotechnologie et génomique fonctionnelle (BIOMICS), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre de Bioinformatique (CBIO), MINES ParisTech - École nationale supérieure des mines de Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Cancer et génome: Bioinformatique, biostatistiques et épidémiologie d'un système complexe, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut Curie [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR et CEA, Mines Paris - PSL (École nationale supérieure des mines de Paris), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Centre de Géosciences (GEOSCIENCES), MINES ParisTech - École nationale supérieure des mines de Paris-PSL Research University (PSL), and MINES ParisTech, Bibliothèque
Subjects: Kinase, Mesenchymal Transition, [SDV]Life Sciences [q-bio], Cells, Gene regulatory network, Gene-Expression, Breast Neoplasms, Biology, Discovery, Article, Cell Movement, Myosin, microRNA, Gene expression, Humans, Gene Regulatory Networks, Disease, ComputingMilieux_MISCELLANEOUS, Cellular Senescence, Cell Proliferation, Monomeric GTP-Binding Proteins, Genetics, Regulation of gene expression, [SDV.BIBS] Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Multidisciplinary, Gene Expression Profiling, Computational Biology, Molecular Sequence Annotation, Animal Development, Actin cytoskeleton, [SDV.BIBS]Life Sciences [q-bio]/Quantitative Methods [q-bio.QM], Cell biology, Gene Expression Regulation, Neoplastic, Gene expression profiling, Actin Cytoskeleton, MicroRNAs, Gene Ontology, Female, Databases, Nucleic Acid, Prediction, Signal Transduction
Abstract: International audience; MiRNAs 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 OPEN
Published: 2015
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89. Erratum: A large-scale RNAi screen identifies LCMR1 as a critical regulator of Tspan8-mediated melanoma invasion

Author: Odile Berthier-Vergnes, Laetitia Barbollat-Boutrand, Xavier Gidrol, T Voeltzel, Emmanuelle Berger, Eric Sulpice, Jérôme Lamartine, Manale El Kharbili, Ingrid Masse, Françoise Degoul, A de la Fouchardière, Ricky Bhajun, G Agaësse, Centre de génétique et de physiologie moléculaire et cellulaire (CGPhiMC), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Biologie à Grande Echelle, Institut National de la Santé et de la Recherche Médicale (INSERM), Imagerie Moléculaire et Therapie Vectorisee, Université d'Auvergne - Clermont-Ferrand I (UdA), Département de Biopathologie, Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Institut National de la Recherche Agronomique (INRA), Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), French Society for Dermatological Research (SRD), La Ligue Contre le Cancer (Comite Ardeche), association 'Vaincre le Melanome', Roche Company, Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National des Sciences Appliquées de Lyon (INSA Lyon), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université d'Auvergne (Clermont Ferrand 1) (UdA), Université de Lyon-Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Centre de Recherche en Cancérologie de Lyon (CRCL), Université de Lyon-Université de Lyon-Centre Léon Bérard [Lyon]-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre Jean Perrin [Clermont-Ferrand] (UNICANCER/CJP), UNICANCER, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Planétologie et Géodynamique [UMR 6112] (LPG), Université d'Angers (UA)-Université de Nantes - UFR des Sciences et des Techniques (UN UFR ST), Université de Nantes (UN)-Université de Nantes (UN)-Institut national des sciences de l'Univers (INSU - CNRS)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: 0301 basic medicine, Male, mélanome, Cancer Research, Skin Neoplasms, Tetraspanins, [SDV]Life Sciences [q-bio], cancer de la peau, medicine.disease_cause, métastase, Molecular oncology, Mice, 0302 clinical medicine, RNA interference, Vemurafenib, Melanoma, ComputingMilieux_MISCELLANEOUS, cellular proliferation, Mediator Complex, Tumor Protein, Translationally-Controlled 1, Cell cycle, 3. Good health, 030220 oncology & carcinogenesis, invasion cellulaire, Heterografts, RNA Interference, medicine.drug, Signal Transduction, endocrine system, Mice, Nude, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Transfection, 03 medical and health sciences, Cell Line, Tumor, medicine, Genetics, PTEN, Animals, Humans, Neoplasm Invasiveness, Molecular Biology, prolifération cellulaire, medicine.disease, 030104 developmental biology, Immunology, Cancer research, biology.protein, Skin cancer, Carcinogenesis
Abstract: Melanoma is the deadliest form of skin cancer owing to its proclivity to metastasise, and recently developed therapies have not yielded the expected results, because almost all patients relapse. Therefore, understanding the molecular mechanisms that underlie early invasion by melanoma cells is crucial to improving patient survival. We have previously shown that, whereas the Tetraspanin 8 protein (Tspan8) is undetectable in normal skin and benign lesions, its expression arises with the progression of melanoma and is sufficient to increase cell invasiveness. Therefore, to identify Tspan8 transcriptional regulators that could explain the onset of Tspan8 expression, thereby conferring an invasive phenotype, we performed an innovative RNA interference-based screen, which, for the first time, identified several Tspan8 repressors and activators, such as GSK3 beta, PTEN, IQGAP1, TPT1 and LCMR1. LCMR1 is a recently identified protein that is overexpressed in numerous carcinomas; its expression and role, however, had not previously been studied in melanoma. The present study identified Tspan8 as the first LCMR1 target that could explain its function in carcinogenesis. LCMR1 modulation was sufficient to positively regulate endogenous Tspan8 expression, with concomitant in vitro phenotypic changes such as loss of melanoma cell-matrix adherence and increase in invasion, and Tspan8 expression promoted tumourigenicity in vivo. Moreover, LCMR1 and Tspan8 overexpression were shown to correlate in melanoma lesions, and both proteins could be downregulated in vitro by vemurafenib. In conclusion, this study highlights the importance of Tspan8 and its regulators in the control of early melanoma invasion and suggests that they may be promising new therapeutic targets downstream of the RAF-MEK-ERK signalling pathway.
Published: 2017
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90. VIDÉO-MICROSCOPIE SANS LENTILLE POUR LA BIOLOGIE CELLULAIRE 2D ET 3D

Author: Fabien Momey, cedric allier, Olivier Cioni, Jean-Guillaume Coutard, Lionel Hervé, Vinjimore Kesavan Srikanth, Jean-Marc Dinten, Nathalie Picollet-D 'Hahan, Monika Dolega, Xavier Gidrol, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Eyraud, Philippe, Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])
Subjects: [SPI]Engineering Sciences [physics], [SPI] Engineering Sciences [physics], microscopie sans lentille, Holographie numérique, culture cellulaire 2D, culture cellulaire 3D
Abstract: International audience; L'étude de l'évolution et de l'organisation de populations de cellules cultivées in vitro intéresse les biologistes depuis plusieurs dizaines d'années. À ces fins, d'importants progrès ont été réalisés dans les méthodes d'imagerie à l'échelle microscopique. Cependant, certaines informations demeurent inaccessibles, notamment à l'échelle mésoscopique, en raison du champ de vue réduit, ainsi que la complexité et le coût pour réaliser des acquisitions hors incubateur en temps réel sur de longues périodes. En réponse à ces limitations, nous avons développé la vidéo-microscopie sans lentille, en plaçant directement les cellules vivantes sur un capteur numérique en regard d'une illumination cohérente selon le principe de l'holographie en ligne. Cette technique permet l'observation d'une culture cellulaire sur un large champ de vue (24 mm² soit plusieurs dizaines de milliers de cellules), et ce à l'intérieur même de l'incubateur, autorisant de surcroît des acquisitions dynamiques couvrant des périodes allant de quelques jours à plusieurs semaines. À partir des images holographiques brutes acquises, nous pouvons remonter aux images refocalisées par reconstruction numérique jusqu'à une résolution de 2µm. Le traitement de ces images donne accès à des niveaux d'information quantifiables allant de la cellule unique à l'organisation inter-individus de la population. Avec des premières études sur des cultures standard de cellules sur substrat 2D, nous sommes aujourd'hui en mesure, avec notre dispositif et la force de l'imagerie holographique, d'explorer et d'étudier la vie cellulaire en 3D, nous rapprochant un peu plus de la réalité physiologique des phénomènes biologiques.
Published: 2014

91. High-throughput monitoring of major cell functions by means of lensfree video microscopy

Author: Fabien Momey, Xavier Gidrol, S. Vinjimore Kesavan, Nelly Dubrulle, Eric Sulpice, Jean-Marc Dinten, Brigitte David-Watine, Olivier Cioni, Bernard Chalmond, D. Freida, Spencer L. Shorte, Cédric Allier, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Imagerie Dynamique (Plate-Forme) (PFID), Institut Pasteur [Paris] (IP), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Centre de Mathématiques et de Leurs Applications (CMLA), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Laboratoire d'Electronique et des Technologies de l'Information ( CEA-LETI ), Université Grenoble Alpes ( UGA ) -Direction de Recherche Technologique (CEA) ( DRT (CEA) ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Imagerie Dynamique (Plate-Forme) ( PFID ), Université de Cergy Pontoise ( UCP ), Université Paris-Seine, Centre de Mathématiques et de Leurs Applications ( CMLA ), École normale supérieure - Cachan ( ENS Cachan ) -Centre National de la Recherche Scientifique ( CNRS ), and Bertacchi Griffi, Nathalie
Subjects: Programmed cell death, Cell division, [SDV]Life Sciences [q-bio], Primary Cell Culture, Cell, Population, Video Recording, Cell Count, Video microscopy, Biology, Article, Cell Line, Tumor, Cell Adhesion, medicine, Humans, RNA, Small Interfering, education, [ SDV.IB.IMA ] Life Sciences [q-bio]/Bioengineering/Imaging, education.field_of_study, Microscopy, Video, Osteoblasts, Multidisciplinary, Cell Death, Mesenchymal stem cell, [ SDV.BC.BC ] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Mesenchymal Stem Cells, Transfection, Fibroblasts, Cell biology, [SDV] Life Sciences [q-bio], medicine.anatomical_structure, Cell culture, Cell Division
Abstract: International audience; Quantification of basic cell functions is a preliminary step to understand complex cellular mechanisms, for e.g., to test compatibility of biomaterials, to assess the effectiveness of drugs and siRNAs, and to control cell behavior. However, commonly used quantification methods are label-dependent, and end-point assays. As an alternative, using our lensfree video microscopy platform to perform high-throughput real-time monitoring of cell culture, we introduce specifically devised metrics that are capable of non-invasive quantification of cell functions such as cell-substrate adhesion, cell spreading, cell division, cell division orientation and cell death. Unlike existing methods, our platform and associated metrics embrace entire population of thousands of cells whilst monitoring the fate of every single cell within the population. This results in a high content description of cell functions that typically contains 25,000 - 900,000 measurements per experiment depending on cell density and period of observation. As proof of concept, we monitored cell-substrate adhesion and spreading kinetics of human Mesenchymal Stem Cells (hMSCs) and primary human fibroblasts, we determined the cell division orientation of hMSCs, and we observed the effect of transfection of siCellDeath (siRNA known to induce cell death) on hMSCs and human Osteo Sarcoma (U2OS) Cells.
Published: 2014
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92. An EWOD-based microfluidic chip for single-cell isolation, mRNA purification and subsequent multiplex qPCR

Author: Dorothée Jary, Yves Fouillet, G. Castellan, A. Bellemin-Comte, Xavier Gidrol, C. Delattre, A. Rival, Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)
Subjects: [SDV]Life Sciences [q-bio], Microfluidics, Biomedical Engineering, Bioengineering, Nanotechnology, 02 engineering and technology, Computational biology, Cell Separation, Cellular level, Biology, 01 natural sciences, Biochemistry, Cell Line, Single-cell analysis, Humans, Multiplex, Cell isolation, RNA, Messenger, ComputingMilieux_MISCELLANEOUS, Ability to work, 010401 analytical chemistry, General Chemistry, Equipment Design, Microfluidic Analytical Techniques, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Microfluidic chip, Electrowetting, 0210 nano-technology, Multiplex Polymerase Chain Reaction
Abstract: Single cell analysis circumvents the need to average data from large populations by observing each cell individually, thus enabling the analysis of cell-to-cell variability. The ability to work on this scale presents many new opportunities for the life sciences and biomedical applications. Microfluidics has become a tool of choice for such studies and electrowetting on dielectric (EWOD) technology is well adapted for samples with reduced size and biological studies at the single cell level. In the present manuscript, for the first time, we present an integrated and automated system based on EWOD that can process the complete workflow on a single device, from the isolation of a single cell to mRNA purification and gene expression analysis.
Published: 2014
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93. Induction of cytochrome P450 1A1 gene expression, oxidative stress, and genotoxicity by carbaryl and thiabendazole in transfected human HepG2 and lymphoblastoid cells22Abbreviations: AhR, aryl hydrocarbon receptor; CAT, chloramphenicol acetyl transferase; CYP1A1, cytochrome P450 1A1; FBS, fetal bovine serum; GADD, growth arrest and DNA damage; GRP78, 78-kD glucose-regulated protein; GST, glutathione S-transferase; HMTII, human metallothionein II; HSP, heat shock protein; 3-MC, 3-methylcholanthrene; NF-κBRE, nuclear factor of κ enhancer responsive element; RARE, retinoid acid responsive element; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDF, 2,3,7,8-tetrachlorodibenzofuran; UDS, unscheduled DNA synthesis; and XRE, xenobiotic responsive element

Author: Marie P. Piechocki, Xavier Gidrol, C. Delescluse, Roger Rahmani, N. Ledirac, Ronald N. Hines, and Rouya Li
Subjects: Pharmacology, Reporter gene, DNA repair, DNA damage, Promoter, Biology, medicine.disease_cause, Biochemistry, Molecular biology, Gene expression, Transcriptional regulation, Aryl hydrocarbon receptor binding, medicine, Genotoxicity
Abstract: Carbaryl and thiabendazole, two widely used pesticides, have been shown to induce cytochrome P450 1A1 (CYP1A1) expression, but neither compound is capable of displacing [3H] 2,3,7,8-tetrachlorodibenzo-P-dioxin from its aryl hydrocarbon receptor binding site. In the present study, we investigated the transcriptional regulation of CYP1A1 as well as other genes in various human hepatoma HepG2 cell lines stably transfected with the chloramphenicol acetyl transferase (CAT) reporter gene and cloned under the control of each of 14 promoters or response elements from relevant stress genes. Carbaryl and thiabendazole were found to activate CYP1A1 at the level of transcription, as demonstrated by the dose-dependent increase in reporter CAT and CYP1A1 mRNAs. Moreover, this effect appeared to be mediated via the xenobiotic responsive element (XRE), because both pesticides specifically activated various fusion constructs containing XRE sequences (CYP1A, glutathione S-transferase, and XRE). Carbaryl and to a lesser extent thiabendazole also activated other stress genes such as c-fos and NF-kappaBRE, HSP70 and GRP78, and GADD153 at a transcriptional level. These data suggest that these molecules induce early alert genes, including those known to be sensitive to oxidative stress. This led us to examine the genotoxic effect of carbaryl and thiabendazole by an in vitro DNA repair solid-phase assay. Both compounds provoked a strong DNA-damaging activity in the human lymphoblastoid cell line that constitutively expresses human CYP1A1 cDNA, but not in the parental line, indicating that CYP1A1 is chiefly implicated in carbaryl and thiabendazole genotoxicity. This effect was confirmed on HepG2 cells. These observations support the notion that intracellular signals leading to CYP1A1 induction, oxidative stress, and genotoxicity are intimately related.
Published: 2001
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94. Statistical modeling and analyses of a base-specific Salmonella mutagenicity assay

Author: Susan J Simmons, Xavier Gidrol, Barry H. Margolin, Pauline Gee, Errol Zeiger, and Walter W. Piegorsch
Subjects: Salmonella typhimurium, Salmonella, Genotype, Health, Toxicology and Mutagenesis, Biology, medicine.disease_cause, Ames test, Toxicology, Benzene Derivatives, Genetics, medicine, Food science, Analysis of Variance, Models, Statistical, Mutagenicity Tests, Test procedures, Standard plate, Statistical model, Replicate, biology.organism_classification, Enterobacteriaceae, Serology, Nitrofurantoin, Mutation, Analysis of variance, Mutagens
Abstract: Statistical features of a base-specific Salmonella mutagenicity assay are considered in detail, following up on a previous report comparing responses of base-specific Salmonella (Ames II) strains with those of traditional tester strains. In addition to using different Salmonella strains, the new procedure also differs in that it is performed as a microwell fluctuation test, as opposed to the standard plate or preincubation test. This report describes the statistical modeling of data obtained from the use of these new strains in the microwell test procedure. We emphasize how to assess any significant interactions between replicate cultures and exposure doses, and how to identify a significant increase in the mutagenic response to a series of concentrations of a test substance.
Published: 2000
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95. Comparison of responses of base-specific Salmonella tester strains with the traditional strains for identifying mutagens: the results of a validation study

Author: R.B Burris, C.H Sommers, P Gee, Xavier Gidrol, A.S Melick, M.E Nelson, M.D Todd, Errol Zeiger, and R.C Klemm
Subjects: Salmonella typhimurium, Validation study, Salmonella, biology, Strain (chemistry), Mutagenicity Tests, Chemistry, Health, Toxicology and Mutagenesis, Mutant, Reproducibility of Results, food and beverages, Mutagen, biology.organism_classification, medicine.disease_cause, Enterobacteriaceae, Ames test, Toxicology, Genetics, medicine, Food science, Bacteria
Abstract: The ability of a TA7000 series of Salmonella his- mutant tester strains to detect mutagens as classified by the traditional tester strains (TA100, TA98, TA1535, TA1537, TA97, TA102 and TA104) was evaluated using 30 coded chemicals, 5 of which were duplicates with different code numbers. The TA7000 series of tester strains were TA7001, TA7002, TA7003, TA7004, TA7005 and TA7006, each of which reverts by a specific base substitution. In addition, each chemical was tested in a mixture of the base-specific strains (the Mix), plus the traditional strains, TA98 and TA1537. A liquid version of the Salmonella mutagenicity assay was performed in microtiter plates to allow partial automation for increased throughput. The results were compared to those in the National Toxicology Program (NTP) database, which were obtained from the traditional strains in the preincubation assay. In the two strains common to both protocols, TA98 and TA1537, the agreement was 80% and 85%, respectively. When compared to the NTP results for TA100, the Mix gave a 72% concordance, while the addition of the frameshift tester strain, TA98, increased the agreement to 76%. The overall agreement on positive or negative classifications of mutagenicity was 88% for the 25 chemicals tested. There were three notable exceptions to the overall agreement. Benzaldehyde was detected as a mutagen in TA7005 in contrast to its classification as a non-mutagen in the NTP database. This does not necessarily contradict the NTP results because the base-specific strains may respond to different mutagens. Two weak mutagens in the NTP database, 1-chloro-2-propanol and isobutyl nitrite, were not detected as mutagens in the base-specific new strains in the liquid protocol. While there are a number of major differences in the two assays, it was concluded that the results from each procedure are comparable.
Published: 1998
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96. Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8

Author: Michael Durchschlag, Lorenzo Galluzzi, Frank Madeo, Mickaël Michaud, L Zitvogel, Laura Senovilla, E Sulpice, Erika Vacchelli, Frédéric Schlemmer, Yuting Ma, Antonella Sistigu, Christoph Ruckenstuhl, Isabelle Martins, Abdul Qader Sukkurwala, Guido Kroemer, Oliver Kepp, Wang Y, Xavier Gidrol, Schlumberger, Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Princeton University, Institut Gustave Roussy (IGR), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Service d'Oncologie Médicale [CHRU Besançon], Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon)-Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Immunologie des tumeurs et immunothérapie (UMR 1015), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Gustave Roussy (IGR)-Université Paris-Sud - Paris 11 (UP11), Apoptose, cancer et immunité (U848), Université Paris-Sud - Paris 11 (UP11)-Institut Gustave Roussy (IGR)-Institut National de la Santé et de la Recherche Médicale (INSERM), Plateforme de métabolomique, Direction de la recherche [Gustave Roussy], Institut Gustave Roussy (IGR)-Institut Gustave Roussy (IGR), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)
Subjects: musculoskeletal diseases, Chemokine, Saccharomyces cerevisiae Proteins, [SDV]Life Sciences [q-bio], Chemokine CXCL2, Antineoplastic Agents, Apoptosis, Saccharomyces cerevisiae, Receptors, Interleukin-8B, Receptors, Interleukin-8A, Mice, Immune system, Cell Line, Tumor, Neoplasms, Animals, Humans, CXC chemokine receptors, Autocrine signalling, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Original Paper, Mice, Inbred BALB C, biology, Interleukin-8, Dendritic Cells, Cell Biology, HCT116 Cells, Cell biology, Mice, Inbred C57BL, CXCL2, Cancer cell, biology.protein, Mitoxantrone, Signal transduction, Calreticulin, Transcriptome, HeLa Cells, Signal Transduction
Abstract: The exposure of calreticulin (CRT) on the surface of stressed and dying cancer cells facilitates their uptake by dendritic cells and the subsequent presentation of tumor-associated antigens to T lymphocytes, hence stimulating an anticancer immune response. The chemotherapeutic agent mitoxantrone (MTX) can stimulate the peripheral relocation of CRT in both human and yeast cells, suggesting that the CRT exposure pathway is phylogenetically conserved. Here, we show that pheromones can act as physiological inducers of CRT exposure in yeast cells, thereby facilitating the formation of mating conjugates, and that a large-spectrum inhibitor of G protein-coupled receptors (which resemble the yeast pheromone receptor) prevents CRT exposure in human cancer cells exposed to MTX. An RNA interference screen as well as transcriptome analyses revealed that chemokines, in particular human CXCL8 (best known as interleukin-8) and its mouse ortholog Cxcl2, are involved in the immunogenic translocation of CRT to the outer leaflet of the plasma membrane. MTX stimulated the production of CXCL8 by human cancer cells in vitro and that of Cxcl2 by murine tumors in vivo. The knockdown of CXCL8/Cxcl2 receptors (CXCR1/Cxcr1 and Cxcr2) reduced MTX-induced CRT exposure in both human and murine cancer cells, as well as the capacity of the latter-on exposure to MTX-to elicit an anticancer immune response in vivo. Conversely, the addition of exogenous Cxcl2 increased the immunogenicity of dying cells in a CRT-dependent manner. Altogether, these results identify autocrine and paracrine chemokine signaling circuitries that modulate CRT exposure and the immunogenicity of cell death.
Published: 2014
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97. Human bone marrow mesenchymal stem cells regulate biased DNA segregation in response to cell adhesion asymmetry

Author: Séverine Lecourt, Xavier Gidrol, Laurent Guyon, Valérie Vanneaux, Gaëlle Letort, Jérôme Larghero, Audrey Cras, Delphine Freida, Manuel Thery, Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Unité de Thérapie Cellulaire, Hopital St Louis, Laboratoire de physiologie cellulaire végétale (LPCV), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Recherche Agronomique (INRA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), European Research Council [ERC-310472], Agence National pour la Recherche [ANR-05PRIB01103, ANR-10-IBHU-0002], Martin-Laffon, Jacqueline, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Larghero, Jerome, Thery, Manuel, Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA)
Subjects: Cell type, Cell division, Cellular differentiation, micropatterns, Bone Marrow Cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Chromatids, Biology, General Biochemistry, Genetics and Molecular Biology, Chromosome segregation, 03 medical and health sciences, 0302 clinical medicine, Chromosome Segregation, Humans, Sister chromatids, human, Cell adhesion, lcsh:QH301-705.5, Mitosis, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Metaphase, 030304 developmental biology, Genetics, mitosis, 0303 health sciences, mesenchymal stem cells, spatial distribution, Mesenchymal stem cell, Cell Differentiation, cell adhesion, DNA, Fibroblasts, Fibronectins, Cell biology, lcsh:Biology (General), Cell Division, 030217 neurology & neurosurgery
Abstract: International audience; Biased DNA segregation is a mitotic event in which the chromatids carrying the original template DNA strands and those carrying the template copies are not segregated randomly into the two daughter cells. Biased segregation has been observed in several cell types, but not in human mesenchymal stem cells (hMSCs), and the factors affecting this bias have yet to be identified. Here, we have investigated cell adhesion geometries as a potential parameter by plating hMSCs from healthy donors on fibronectin-coated micropatterns. On symmetric micropatterns, the segregation of sister chromatids to the daughter cells appeared random. In contrast, on asymmetric micropatterns, the segregation was biased. This sensitivity to asymmetric extracellular cues was reproducible in cells from all donors but was not observed in human skin-derived fibroblasts or in a fibroblastic cell line used as controls. We conclude that the asymmetry of cell adhesion is a major factor in the regulation of biased DNA segregation in hMSCs.
Published: 2013
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98. Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress

Author: Xavier Gidrol, Yip Soo Fern, Anil Kush, and Paolo A. Sabelli
Subjects: Annexins, Molecular Sequence Data, Mutant, Arabidopsis, Biology, Genes, Plant, medicine.disease_cause, Gene product, Bacterial Proteins, Gene Expression Regulation, Plant, Annexin, Complementary DNA, Escherichia coli, medicine, Amino Acid Sequence, RNA, Messenger, Cloning, Molecular, Plant Proteins, Multidisciplinary, cDNA library, Escherichia coli Proteins, Genetic Complementation Test, Gene Expression Regulation, Bacterial, Hydrogen Peroxide, biology.organism_classification, Molecular biology, DNA-Binding Proteins, Repressor Proteins, Oxidative Stress, Regulon, Peroxidases, Genes, Bacterial, bacteria, Sequence Alignment, Research Article, Transcription Factors
Abstract: Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.
Published: 1996
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99. The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

Author: Véronique Collin-Faure, Sylvie Elsen, Xavier Gidrol, Claudie Lemercier, Lemercier, Claudie, Pathogénie bactérienne et réponses cellulaires, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Institut de Biosciences et de Biotechnologies de Grenoble (ex-IRTSV) (BIG), Université Joseph Fourier - Grenoble 1 (UJF)-Institut National de la Recherche Agronomique (INRA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Commissariat à l'Energie Atomique et aux Energies Renouvelables (CEA), the Centre National de la Recherche Scientifique (CNRS) and the Université Joseph Fourier (UJF Grenoble)., Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut National de la Santé et de la Recherche Médicale (INSERM)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Recherche Agronomique (INRA)-Université Grenoble Alpes (UGA), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Grenoble Alpes (UGA)
Subjects: MESH: Signal Transduction, DNA Repair, MESH: ADP Ribose Transferases, [SDV]Life Sciences [q-bio], MESH: DNA Breaks, Double-Stranded, Ataxia Telangiectasia Mutated Proteins, medicine.disease_cause, Histones, chemistry.chemical_compound, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Chromosome instability, DNA Breaks, Double-Stranded, H2AX, Phosphorylation, MESH: Ataxia Telangiectasia Mutated Proteins, MESH: Bacterial Proteins, ADP Ribose Transferases, MESH: DNA Repair, MESH: Histones, 0303 health sciences, biology, Kinase, Intracellular Signaling Peptides and Proteins, DNA double strand breaks, 3. Good health, [SDV] Life Sciences [q-bio], Histone, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Pseudomonas aeruginosa, MESH: Pseudomonas aeruginosa, [SDV.MHEP.MI] Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Molecular Medicine, Signal transduction, Tumor Suppressor p53-Binding Protein 1, Infection, Signal Transduction, MESH: Cell Line, Tumor, DNA repair, Bacterial Toxins, HL-60 Cells, [SDV.CAN]Life Sciences [q-bio]/Cancer, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Microbiology, MESH: Chromosomal Instability, 03 medical and health sciences, Cellular and Molecular Neuroscience, Bacterial Proteins, [SDV.CAN] Life Sciences [q-bio]/Cancer, MESH: HL-60 Cells, Cell Line, Tumor, Chromosomal Instability, MESH: Intracellular Signaling Peptides and Proteins, medicine, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, [SDV.MP] Life Sciences [q-bio]/Microbiology and Parasitology, 030304 developmental biology, Pharmacology, MESH: Humans, MESH: Phosphorylation, 030306 microbiology, Mutagenesis, JNK Mitogen-Activated Protein Kinases, Cell Biology, MESH: JNK Mitogen-Activated Protein Kinases, chemistry, MESH: Bacterial Toxins, ATM, biology.protein, DNA
Abstract: International audience; Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.
Published: 2013
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100. Impedance Sensing of Biological Processes in Mammalian Cells

Author: Xavier Gidrol, Matsuhiko Nishizawa, Hirokazu Kaji, and Lamya Ghenim
Subjects: Materials science, Motility, Impedance sensing, Biosensor, Mitosis, Cell biology
Published: 2012
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