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17 results on '"Mariano Gonzalez-Pisfil"'

1. Cytosolic S100A8/A9 promotes Ca2+ supply at LFA-1 adhesion clusters during neutrophil recruitment

Author

Matteo Napoli, Roland Immler, Ina Rohwedder, Valerio Lupperger, Johannes Pfabe, Mariano Gonzalez Pisfil, Anna Yevtushenko, Thomas Vogl, Johannes Roth, Melanie Salvermoser, Steffen Dietzel, Marjan Slak Rupnik, Carsten Marr, Barbara Walzog, Markus Sperandio, and Monika Pruenster

Subjects
neutrophil recruitment, intracellular S100A8/A9, calcium signaling, LFA-1 integrin clustering, acute inflammation, intracellular signaling, Medicine, Science, Biology (General), QH301-705.5
Abstract

S100A8/A9 is an endogenous alarmin secreted by myeloid cells during many acute and chronic inflammatory disorders. Despite increasing evidence of the proinflammatory effects of extracellular S100A8/A9, little is known about its intracellular function. Here, we show that cytosolic S100A8/A9 is indispensable for neutrophil post-arrest modifications during outside-in signaling under flow conditions in vitro and neutrophil recruitment in vivo, independent of its extracellular functions. Mechanistically, genetic deletion of S100A9 in mice caused dysregulated Ca2+ signatures in activated neutrophils resulting in reduced Ca2+ availability at the formed LFA-1/F-actin clusters with defective β2 integrin outside-in signaling during post-arrest modifications. Consequently, we observed impaired cytoskeletal rearrangement, cell polarization, and spreading, as well as cell protrusion formation in S100a9-/- compared to wildtype (WT) neutrophils, making S100a9-/- cells more susceptible to detach under flow, thereby preventing efficient neutrophil recruitment and extravasation into inflamed tissue.

Published
2024
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2. Extracellular vesicle-mediated trafficking of molecular cues during human brain development

Author

Andrea Forero, Fabrizia Pipicelli, Sylvain Moser, Natalia Baumann, Christian Grätz, Mariano Gonzalez Pisfil, Michael W. Pfaffl, Benno Pütz, Pavel Kielkowski, Filippo M. Cernilogar, Giuseppina Maccarrone, Rossella Di Giaimo, and Silvia Cappello

Subjects
CP: Neuroscience, CP: Developmental biology, Biology (General), QH301-705.5
Abstract

Summary: Cellular crosstalk is an essential process influenced by numerous factors, including secreted vesicles that transfer nucleic acids, lipids, and proteins between cells. Extracellular vesicles (EVs) have been the center of many studies focusing on neurodegenerative disorders, but whether EVs display cell-type-specific features for cellular crosstalk during neurodevelopment is unknown. Here, using human-induced pluripotent stem cell-derived cerebral organoids, neural progenitors, neurons, and astrocytes, we identify heterogeneity in EV protein content and dynamics in a cell-type-specific and time-dependent manner. Our results support the trafficking of key molecules via EVs in neurodevelopment, such as the transcription factor YAP1, and their localization to differing cell compartments depending on the EV recipient cell type. This study sheds new light on the biology of EVs during human brain development.

Published
2024
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3. Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

Author

Mariano Gonzalez Pisfil, Iliya Nadelson, Brigitte Bergner, Sonja Rottmeier, Andreas W. Thomae, and Steffen Dietzel

Subjects
Medicine, Science
Abstract

Abstract Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600–630) and two in the far red channel (633/641–680), supplemented by a single further redshifted fluorochrome (670/701–750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

Published
2022
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4. Disrupting Roquin-1 interaction with Regnase-1 induces autoimmunity and enhances antitumor responses

Author

Stephanie L. Edelmann, Laura S. de Jonge, Lisa Kifinger, Wolfgang Wurst, Florian Giesert, Christine Hohn, Naoto Kawakami, Sebastian Theurich, Mingui Fu, Dierk Niessing, Nina Kronbeck, Martin E. Kirmaier, Vigo Heissmeyer, Timsse Raj, Thomas Monecke, Elena S. Davydova, Elaine H. Wong, Stefan Feske, Gesine Behrens, and Mariano Gonzalez Pisfil

Subjects
Cytotoxicity, Immunologic, Male, Skin Neoplasms, T-Lymphocytes, Melanoma, Experimental, Autoimmunity, medicine.disease_cause, Zc3h12a protein, mouse, Immunotherapy, Adoptive, immunology [T-Lymphocytes], Tumor Microenvironment, Immunology and Allergy, genetics [Ribonucleases], metabolism [Repressor Proteins], genetics [Ubiquitin-Protein Ligases], Mutation, therapy [Skin Neoplasms], transplantation [T-Lymphocytes], metabolism [Skin Neoplasms], Cell biology, medicine.anatomical_structure, therapy [Melanoma, Experimental], Phenotype, Female, Protein Binding, T cell, Ubiquitin-Protein Ligases, Immunology, Mice, Transgenic, Biology, immunology [Melanoma, Experimental], Article, Proinflammatory cytokine, genetics [Skin Neoplasms], Immune system, metabolism [Ubiquitin-Protein Ligases], Ribonucleases, medicine, Animals, Humans, ddc:610, metabolism [T-Lymphocytes], Autoantibody, genetics [Melanoma, Experimental], Germinal center, Immunity, Humoral, Mice, Inbred C57BL, Repressor Proteins, genetics [Repressor Proteins], HEK293 Cells, immunology [Skin Neoplasms], Rc3h1 protein, mouse, metabolism [Melanoma, Experimental], metabolism [Ribonucleases], roquin-2 protein, mouse, CD8, HeLa Cells
Abstract

Roquin and Regnase-1 proteins bind and post-transcriptionally regulate proinflammatory target messenger RNAs to maintain immune homeostasis. Either the sanroque mutation in Roquin-1 or loss of Regnase-1 cause systemic lupus erythematosus-like phenotypes. Analyzing mice with T cells that lack expression of Roquin-1, its paralog Roquin-2 and Regnase-1 proteins, we detect overlapping or unique phenotypes by comparing individual and combined inactivation. These comprised spontaneous activation, metabolic reprogramming and persistence of T cells leading to autoimmunity. Here, we define an interaction surface in Roquin-1 for binding to Regnase-1 that included the sanroque residue. Mutations in Roquin-1 impairing this interaction and cooperative regulation of targets induced T follicular helper cells, germinal center B cells and autoantibody formation. These mutations also improved the functionality of tumor-specific T cells by promoting their accumulation in the tumor and reducing expression of exhaustion markers. Our data reveal the physical interaction of Roquin-1 with Regnase-1 as a hub to control self-reactivity and effector functions in immune cell therapies. Mutations in the RNA-binding proteins Roquin-1 or Regnase-1 cause systemic autoimmunity. Heissmeyer and colleagues show that Roquin-1 and Regnase-1 physically interact and thereby regulate CD4+ and CD8+ T cell metabolism and functionality.

Published
2021
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5. Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms.

Author

François Sipieter, Benjamin Cappe, Mariano Gonzalez Pisfil, Corentin Spriet, Jean-François Bodart, Katia Cailliau-Maggio, Peter Vandenabeele, Laurent Héliot, and Franck B Riquet

Subjects
Medicine, Science
Abstract

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues.

Published
2015
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6. Triple-Color STED Nanoscopy: Sampling Absorption Spectra Differences for Efficient Linear Species Unmixing

Author

Mariano Gonzalez Pisfil, Marcelle König, Benedikt Krämer, Rainer Erdmann, Felix Koberling, Sumeet Rohilla, and Matthias Patting

Subjects
Physics, Digital image correlation, Microscopy, Confocal, 010304 chemical physics, STED microscopy, Nanosecond, 010402 general chemistry, 01 natural sciences, Fluorescence, 0104 chemical sciences, Surfaces, Coatings and Films, Wavelength, Microscopy, Fluorescence, 0103 physical sciences, Materials Chemistry, Fluorescence microscope, Stimulated emission, Physical and Theoretical Chemistry, Biological system, Absorption (electromagnetic radiation), Algorithms
Abstract

Stimulated emission depletion (STED) in confocal fluorescence microscopy enables a visualization of biological structures within cells far below the optical diffraction limit. To meet the demand in the field for simultaneous investigations of multiple species within a cell, a couple of different STED techniques have been proposed, each with their own challenges. By systemically exploiting spectral differences in the absorption of fluorescent labels, we present a novel, beneficial approach to multispecies STED nanoscopy. By using three excitation wavelengths in nanosecond pulsed interleaved excitation (PIE) mode, we probe quasi simultaneously multiple species with fluorescent labels having absorption maxima as close as 13 nm. The acquired image is decomposed into its single species contributions by application of a linear unmixing algorithm based on present reference patterns. For multispecies images containing single species regions, we introduce the image correlation map (ICM). Here, the single species regions easily can be identified in order to generate the necessary single species reference patterns. This avoids the otherwise cumbersome and artifact prone preparation and recording of additional reference samples. The power of the proposed imaging scheme persists in species separation quality at high speed shown for up to three species with established reference samples and dyes commonly used for cellular STED imaging.

Published
2021

7. Scanning FCS and super-resolution microscopy on 2D lipid membranes (Conference Presentation)

Author

Rhys Dowler, Uwe Ortmann, Marcelle König, Rainer Erdmann, Felix Koberling, Sumeet Rohilla, Benedikt Krämer, and Mariano Gonzalez Pisfil

Subjects
Membrane, Materials science, Super-resolution microscopy, Energy transfer, Multi species, Lipid bilayer, Photobleaching, Fluorescence, Biomedical engineering
Abstract

Scanning FCS (sFCS) is a great tool for studying slowly diffusing species as is often the case in cell membranes. In sFCS, the excitation volume is scanned rapidly through the sample allowing for simultaneous measurement at multiple locations. The shorter residence times also lead to lower photon doses experienced by each detected molecule, reducing the risk of photobleaching. Here, we show results from sFCS measurements on supported lipid bilayers (SLBs) where fluorescence lifetime information is used to achieve an axial nanometric localization based on Metal Induced Energy Transfer (MIET).

Published
2020
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9. HEXIM1 Diffusion in the Nucleus Is Regulated by Its Interactions with Both 7SK and P-TEFb

Author

Corentin Le Nézet, Gabriel Bidaux, Thorsten Wohland, Mélanie Henry, Laurent Héliot, Alessandro Furlan, Bernard Vandenbunder, O. Bensaude, Dorian Champelovier, Mariano Gonzalez-Pisfil, Marc Lefranc, Aymeric Leray, Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Laboratoire Interdisciplinaire Carnot de Bourgogne [Dijon] (LICB), Université de Technologie de Belfort-Montbeliard (UTBM)-Université de Bourgogne (UB)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Hospices Civils de Lyon (HCL), Université de Bourgogne (UB)-Université de Technologie de Belfort-Montbeliard (UTBM)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biophotonique Cellulaire Fonctionelle, Institut de Recherche Interdisciplinaire, 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), Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Laboratoire Interdisciplinaire Carnot de Bourgogne [Dijon] (ICB), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Institut National des Sciences Appliquées (INSA)-Université de Lyon-Institut National des Sciences Appliquées (INSA)-Hospices Civils de Lyon (HCL)-Institut National de la Santé et de la Recherche Médicale (INSERM), bidaux, gabriel, Laboratoire Interdisciplinaire Carnot de Bourgogne (ICB), Université de Technologie de Belfort-Montbeliard (UTBM)-Université de Bourgogne (UB)-Université Bourgogne Franche-Comté [COMUE] (UBFC)-Centre National de la Recherche Scientifique (CNRS), 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), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Département de Biologie - ENS Paris, and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Anomalous diffusion, [SDV]Life Sciences [q-bio], Population, Biophysics, Plasma protein binding, Diffusion, 03 medical and health sciences, 0302 clinical medicine, Cell Line, Tumor, 7SK RNA, medicine, Humans, Computer Simulation, Positive Transcriptional Elongation Factor B, Nuclear protein, P-TEFb, education, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Cell Nucleus, 0303 health sciences, Molecular diffusion, education.field_of_study, Chemistry, RNA-Binding Proteins, Articles, [SDV] Life Sciences [q-bio], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Spectrometry, Fluorescence, medicine.anatomical_structure, RNA, Long Noncoding, Nucleus, 030217 neurology & neurosurgery, Protein Binding, Transcription Factors
Abstract

International audience; How nuclear proteins diffuse and find their targets remains a key question in the transcription field. Dynamic proteins in the nucleus are classically subdiffusive and undergo anomalous diffusion, yet the underlying physical mechanisms are still debated. In this study, we explore the contribution of interactions to the generation of anomalous diffusion by the means of fluorescence spectroscopy and simulation. Using interaction-deficient mutants, our study indicates that HEXIM1 interactions with both 7SK RNA and positive transcription elongation factor b are critical for HEXIM1 subdiffusion and thus provides evidence of the effects of protein-RNA interaction on molecular diffusion. Numerical simulations allowed us to establish that the proportions of distinct oligomeric HEXIM1 subpopulations define the apparent anomaly parameter of the whole population. Slight changes in the proportions of these oligomers can lead to significant shifts in the diffusive features and recapitulate the modifications observed in cells with the various interaction-deficient mutants. By combining simulations and experiments, our work opens new prospects in which the anomaly a coefficient in diffusion becomes a helpful tool to infer alterations in molecular interactions.

Published
2019
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10. [P-TEFb and Brd4: actors of the transcription pause release as therapeutical targets]

Author

Alessandro, Furlan, Florence, Agbazahou, Mélanie, Henry, Mariano, Gonzalez-Pisfil, Corentin, Le Nézet, Dorian, Champelovier, Marie, Fournier, Bernard, Vandenbunder, Gabriel, Bidaux, and Laurent, Héliot

Subjects
Transcription, Genetic, Humans, Nuclear Proteins, Cell Cycle Proteins, Positive Transcriptional Elongation Factor B, Molecular Targeted Therapy, RNA Polymerase II, Transcription Factors
Abstract

Most cell physiology events are dictated by the integration of perceived signals and the elaboration by cells of adapted answers via the execution of proper transcriptional programs. In order to ensure an optimal control of these answers, many regulation mechanisms have been selected throughout the evolution, thus allowing to fine-tune transcript expression. The transcriptional pause and its release by P-TEFb (Positive Transcription Elongation Factor) have been evidenced two decades ago. Since then, the importance of such mechanisms has been highlighted by the association between alterations of this machinery and the appearance of diseases. P-TEFb and Brd4 have thus recently emerged as potential therapeutical targets for cancers and AIDS notably. In this review, we present a brief case history and an up-to-date synthesis of models for transcriptional pause release. We later discuss on the pathophysiological processes associated with this mechanism and clinical trials targeting Brd4 and P-TEFb.

Published
2018

11. P-TEFb et Brd4: Acteurs de la levée de pause transcriptionnelle, possibles cibles thérapeutiques

Author

Dorian Champelovier, Mariano Gonzalez-Pisfil, Mélanie Henry, Florence Agbazahou, Alessandro Furlan, Gabriel Bidaux, Laurent Héliot, Corentin Le Nézet, Bernard Vandenbunder, Marie Fournier, Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Cell physiology, BRD4, Transcription elongation, [SDV]Life Sciences [q-bio], General Medicine, Biology, General Biochemistry, Genetics and Molecular Biology, 3. Good health, 03 medical and health sciences, 030104 developmental biology, Transcription (biology), Cell Cycle Protein, P-TEFb, Neuroscience, Gene transcript
Abstract

Most cell physiology events are dictated by the integration of perceived signals and the elaboration by cells of adapted answers via the execution of proper transcriptional programs. In order to ensure an optimal control of these answers, many regulation mechanisms have been selected throughout the evolution, thus allowing to fine-tune transcript expression. The transcriptional pause and its release by P-TEFb (Positive Transcription Elongation Factor) have been evidenced two decades ago. Since then, the importance of such mechanisms has been highlighted by the association between alterations of this machinery and the appearance of diseases. P-TEFb and Brd4 have thus recently emerged as potential therapeutical targets for cancers and AIDS notably. In this review, we present a brief case history and an up-to-date synthesis of models for transcriptional pause release. We later discuss on the pathophysiological processes associated with this mechanism and clinical trials targeting Brd4 and P-TEFb.

Published
2018
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12. Scanning FCS and multi-species STED microscopy for diffusion studies in membranes (Conference Presentation)

Author

Marcelle Koenig, Mariano Gonzalez Pisfil, Felix Koberling, Benedikt Kraemer, Rhys Dowler, Paja Reisch, and Matthias Patting

Subjects
Membrane, Materials science, Microscope, law, STED microscopy, Fluorescence correlation spectroscopy, Stimulated emission, Diffusion (business), Biological system, Photobleaching, Photon counting, law.invention
Abstract

Fluorescence Correlation Spectroscopy (FCS) is an important technique for understanding molecular dynamics and motion on timescales ranging from nanoseconds to seconds. The high concentrations found in some biological systems reduce the significance of FCS, as too many fluorophores are present within a standard confocal volume. Combining FCS with Stimulated Emission Depletion (STED) is one technique to overcome this problem by reducing the observation volume in the sample, reducing the number of molecules within this volume. This technique also allows the observation volume to be tuned to access additional information, such as the parameters which characterize hindered diffusion. In addition, by utilizing galvoscanners, scanning FCS can increase the number of transits recorded and reduce the residence time of each molecule, increasing the statistics and reducing the effects of photobleaching respectively. This technique is also useful for increasing the number of transits observed for slowly diffusing species at low concentrations, such as observed in membranes. Measuring multiple species simultaneously saves time and allows interactions between molecules to be investigated, which may not be clear from multiple experiments investigating single species. By exploiting the lifetime information available from a microscope equipped with Time Correlated Single Photon Counting (TCSPC) hardware, pattern matching can be used to separate similar fluorophores allowing up to three superresolved species to be resolved using a single STED laser. This pattern matching analysis can be combined with STED-FCS and scanning FCS to investigate complex diffusion in membranes.

Published
2018
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13. FRET Image Correlation Spectroscopy Reveals RNAPII-Independent P-TEFb Recruitment on Chromatin

Author

Mariano Gonzalez Pisfil, Mélanie Henry, Gabriel Bidaux, O. Bensaude, Corentin Le Nézet, Alessandro Furlan, Laurent Héliot, Bernard Vandenbunder, Cardiovasculaire, métabolisme, diabétologie et nutrition (CarMeN), 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), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut de biologie de l'ENS Paris (IBENS), Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Biophotonique Cellulaire Fonctionelle, Institut de Recherche Interdisciplinaire, FURLAN, Alessandro, Institut de biologie de l'ENS Paris (UMR 8197/1024) (IBENS), École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Département de Biologie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Département de Biologie - ENS Paris

Subjects
0301 basic medicine, Fluorescence-lifetime imaging microscopy, Transcription, Genetic, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Biophysics, RNA polymerase II, Bone Neoplasms, Histones, 03 medical and health sciences, 0302 clinical medicine, Transcription (biology), Histone H2A, Fluorescence Resonance Energy Transfer, Image Processing, Computer-Assisted, Tumor Cells, Cultured, Humans, Positive Transcriptional Elongation Factor B, Phosphorylation, P-TEFb, ComputingMilieux_MISCELLANEOUS, Osteosarcoma, Nucleic Acids and Genome Biophysics, biology, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Chemistry, Chromatin, 030104 developmental biology, Förster resonance energy transfer, biology.protein, RNA Polymerase II, 030217 neurology & neurosurgery, Protein Binding
Abstract

Biochemical studies have revealed that the RNA Polymerase II (RNAPII) pause release is triggered by phosphorylation of the transcription machinery by the positive transcription elongation factor b (P-TEFb). However, there are no direct report that P-TEFb and RNA polymerase II interact in single living cells and the biophysical mechanisms mediating this association are still unclear. Forster resonance energy transfer (FRET) detects molecular interactions at the subcellular level. Time domain fluorescence lifetime imaging provides an accurate quantification of FRET efficiency, E FRET , because it is fluorochrome concentration-independent and insensitive to fluorescence bleed-through. However, the way FRET signal is usually analyzed does not provide information about the areas where protein-protein interactions take place. In this work, we developed a method, dubbed FRET image correlation spectroscopy (FICS), which relied on FRET fluorescence lifetime imaging image acquisition and image correlation spectroscopy of E FRET clusters to quantify the spatial distribution of interaction clusters in the nucleus. The combination of high content FRET microscopy with batch image analysis allowed a robust statistical analysis. By applying FICS, we characterized the area and density of interaction clusters between P-TEFb and RNAPII or histone H2A in single living cells. The FICS method applied to cells expressing genetically engineered mutated proteins confirmed that the histidine-rich domain of P-TEFb is required for its interaction with RNAPII. Furthermore, it demonstrated that P-TEFb was also located in close vicinity to histone H2A, independently of its interactions with RNAPII. These results support the hypothesis that P-TEFb dynamics on chromatin regulate its recruitment on RNAPII.

Published
2018
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14. Improved photodynamic effect through encapsulation of two photosensitizers in lipid nanocapsules

Author

Nadia Skandrani, Solomiya Paryzhak, Gabriel Bidaux, Tijani Gharbi, Sabine Szunerits, Mariano Gonzalez Pisfil, Laurent Héliot, Alexandre Barras, V’yacheslav Lehen’kyi, Rabah Boukherroub, Rostyslav Bilyy, Aurélien Haustrate, Hatem Boulahdour, Tetiana Dumych, Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), NanoBioInterfaces - IEMN (NBI - IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Nanomédecine, imagerie, thérapeutique - UFC (EA 4662) (NIT / NANOMEDECINE), Université de Franche-Comté (UFC), Université Bourgogne Franche-Comté [COMUE] (UBFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Danylo Halytskiy Lviv National Medical University, Laboratoire de Physiologie Cellulaire : Canaux ioniques, inflammation et cancer - U 1003 (PHYCELL), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille, Centre Hospitalier Régional Universitaire de Besançon (CHRU Besançon), Centre National de la Recherche Scientifique (CNRS), Universite de Lille, Hauts-de-France region, CPER for Society, Institut National du Cancer (INCa), EUH2020-MSCE-RISE-2015 690836 PANG, European Project: 690836,H2020,H2020-MSCA-RISE-2015,PANG(2016), Nanomédecine, imagerie, thérapeutique - UFC (UR 4662) (NIT / NANOMEDECINE), BARRAS, ALEXANDRE, Pathogen and Graphene - PANG - - H20202016-01-01 - 2019-12-31 - 690836 - VALID, NanoBioInterfaces - IEMN [NBI - IEMN], Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 [IEMN], Laboratoire de Physiologie Cellulaire : Canaux ioniques, inflammation et cancer - U 1003 [PHYCELL], and Université Lille Nord de France (COMUE)

Subjects
Biodistribution, medicine.medical_treatment, [CHIM.THER] Chemical Sciences/Medicinal Chemistry, Biomedical Engineering, Photodynamic therapy, 02 engineering and technology, [CHIM.THER]Chemical Sciences/Medicinal Chemistry, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, 01 natural sciences, HeLa, chemistry.chemical_compound, medicine, Photosensitizer, General Materials Science, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, chemistry.chemical_classification, Reactive oxygen species, Protoporphyrin IX, biology, 010405 organic chemistry, General Chemistry, General Medicine, [SDV.SP]Life Sciences [q-bio]/Pharmaceutical sciences, 021001 nanoscience & nanotechnology, biology.organism_classification, 3. Good health, 0104 chemical sciences, Hypericin, [SDV.SP] Life Sciences [q-bio]/Pharmaceutical sciences, chemistry, Biophysics, 0210 nano-technology, Phototoxicity
Abstract

International audience; Photodynamic therapy (PDT) has developed into a new clinical and non-invasive treatment for cancer over the past 30 years. By the combination of three non-toxic partners, i.e. a photosensitizer (PS), molecular oxygen (O2) and light, cytotoxic reactive oxygen species (ROS) are locally produced leading to irreversible vascular and cellular damage. In the present study, we report for the first time that the combination of two photosensitizers (2 PSs: Protoporphyrin IX, PpIX and Hypericin, Hy) loaded in the same lipid nanocapsules (LNCs) leads to enhanced photodynamic therapy efficiency when compared with previously reported systems. The 2 PS-loaded LNCs are shown to increase the in vitro phototoxicity at the nanomolar range (IC50 = 274 and 278 nM on HeLa and MDA-MB-231 cell lines, respectively), whereas the corresponding single PS-loaded LNCs at the same concentration exhibit a phototoxicity two times lower. Intracellular localization in HeLa cells indicates a subcellular asymmetry of PpIX and Hy, in the plasma, ER membranes and round internal structures. The biodistribution of LNCs was studied upon different routes of injection into Swiss nude mice; based on the obtained data, LNCs were injected intratumorally and used to slow the growth of xenograft tumors in mice. The results obtained in this study suggest that the combination of two or more PSs may be a promising strategy to improve the efficacy of conventional photodynamic therapy as well as to reduce dark toxicity.

Published
2018
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15. Compounds Triggering ER Stress Exert Anti-Melanoma Effects and Overcome BRAF Inhibitor Resistance

Author

Emilie Jaune, Hella Amdouni, Anne-Sophie Dabert-Gay, Jean-Sébastien Annicotte, Magali Plaisant, Solange Moréra, Delphine Debayle, Corine Bertolotto, Patricia Abbe, Thierry Passeron, Abdelali Lehraiki, Laurent Héliot, Robert Ballotti, Philippe Gual, Baharia Mograbi, Cyril Ronco, Mariano Gonzalez-Pisfil, Thomas Botton, Damien Alcor, Caroline Robert, Michael Cerezo, Stéphane Rocchi, Nabil Rabhi, Véronique Hofman, Armelle Vigouroux, Antoine Millet, Rachid Benhida, Maruf M.U. Ali, Paul Hofman, Florian Rouaud, Université Nice Sophia Antipolis - Faculté de Médecine (UNS UFR Médecine), Université Nice Sophia Antipolis (... - 2019) (UNS), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA), Centre méditerranéen de médecine moléculaire (C3M), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institutions et Dynamiques Historiques de l'Économie et de la Société (IDHES), École normale supérieure - Cachan (ENS Cachan)-Université Panthéon-Sorbonne (UP1)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Centre National de la Recherche Scientifique (CNRS)-Université d'Évry-Val-d'Essonne (UEVE), Institut de Chimie de Nice (ICN), Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS), Service de Dermatologie [Nice], Hôpital Archet 2 [Nice] (CHU), Institut de Recherche sur le Cancer et le Vieillissement (IRCAN), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Nice Sophia Antipolis (... - 2019) (UNS), Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre d'Etudes Lasers Intenses et Applications (CELIA), Université de Bordeaux (UB)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Génomique Intégrative et Modélisation des Maladies Métaboliques (EGID), Université de Lille-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Centre National de la Recherche Scientifique (CNRS)-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 (PhLAM), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Français de Recherche pour l'Exploitation de la Mer - Brest (IFREMER), Institut Français de Recherche pour l'Exploitation de la Mer (IFREMER), Microbiologie et enzymologie structurale (MESB3S), Département Biochimie, Biophysique et Biologie Structurale (B3S), Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique (CNRS), University of Stavanger, Laboratoire Chrono-environnement - UFC (UMR 6249) (LCE), Centre National de la Recherche Scientifique (CNRS)-Université de Franche-Comté (UFC)-Université Bourgogne Franche-Comté [COMUE] (UBFC), Centre méditérannéen de médecine moléculaire ( C3M ), Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Université Nice Sophia Antipolis - Faculté de Médecine ( UNS UFR Médecine ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ), Institut de Chimie de Nice ( ICN ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Centre National de la Recherche Scientifique ( CNRS ), Hôpital Archet 2 [Nice] ( CHU ), Institut de Recherche sur le Cancer et le Vieillissement ( IRCAN ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Pathologie Clinique et Expérimentale, Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Hôpital pasteur [Colmar]-CHU Nice, Institut de pharmacologie moléculaire et cellulaire ( IPMC ), Génomique Intégrative et Modélisation des Maladies Métaboliques ( EGID ), Institut Pasteur de Lille, Réseau International des Instituts Pasteur ( RIIP ) -Réseau International des Instituts Pasteur ( RIIP ) -Université de Lille-Centre Hospitalier Régional Universitaire [Lille] ( CHRU Lille ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de Physique des Lasers, Atomes et Molécules - UMR 8523 ( PhLAM ), Université de Lille-Centre National de la Recherche Scientifique ( CNRS ), Département de médecine oncologique [Gustave Roussy], Institut Gustave Roussy ( IGR ), Microbiologie et enzymologie structurale ( MESB3S ), Département Biochimie, Biophysique et Biologie Structurale ( B3S ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Sud - Paris 11 ( UP11 ), Department of Life Sciences, Imperial College London, Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Université Paris 1 Panthéon-Sorbonne (UP1)-Université Paris 8 Vincennes-Saint-Denis (UP8)-Université Paris Nanterre (UPN)-Université d'Évry-Val-d'Essonne (UEVE)-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Metabolic functional (epi)genomics and molecular mechanisms involved in type 2 diabetes and related diseases - UMR 8199 - UMR 1283 (EGENODIA (GI3M)), Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-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), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Hôpital pasteur [Colmar]-Centre Hospitalier Universitaire de Nice (CHU Nice), Institut Gustave Roussy (IGR), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA)-Université Nice Sophia Antipolis (... - 2019) (UNS), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Metabolic functional (epi)genomics and molecular mechanisms involved in type 2 diabetes and related diseases - UMR 8199 - UMR 1283 (GI3M), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur de Lille, Réseau International des Instituts Pasteur (RIIP)-Réseau International des Instituts Pasteur (RIIP), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de Bordeaux (UB), Institut Français de Recherche pour l'Exploitation de la Mer - Brest (IFREMER Centre de Bretagne), Institutions et Dynamiques Historiques de l'Économie et de la Société ( IDHES ), École normale supérieure - Cachan ( ENS Cachan ) -Université Panthéon-Sorbonne ( UP1 ) -Université Paris 8 Vincennes-Saint-Denis ( UP8 ) -Université Paris Nanterre ( UPN ) -Université d'Évry-Val-d'Essonne ( UEVE ) -Centre National de la Recherche Scientifique ( CNRS ), Centre d'Etudes Lasers Intenses et Applications ( CELIA ), Université de Bordeaux ( UB ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ), Institut Français de Recherche pour l'Exploitation de la Mer - Brest ( IFREMER ), Institut Français de Recherche pour l'Exploitation de la Mer ( IFREMER ), Centre National de la Recherche Scientifique ( CNRS ), Laboratoire Chrono-environnement ( LCE ), Université Bourgogne Franche-Comté ( UBFC ) -Centre National de la Recherche Scientifique ( CNRS ) -Université de Franche-Comté ( UFC ), and Houel, Ludivine

Subjects
0301 basic medicine, Proto-Oncogene Proteins B-raf, Cancer Research, Programmed cell death, [SDV]Life Sciences [q-bio], Antineoplastic Agents, Pharmacology, Biology, Transcriptome, 03 medical and health sciences, Mice, 0302 clinical medicine, Cell Line, Tumor, medicine, Animals, Humans, Endoplasmic Reticulum Chaperone BiP, Melanoma, Protein Kinase Inhibitors, neoplasms, Heat-Shock Proteins, 030304 developmental biology, Cell Proliferation, Regulation of gene expression, 0303 health sciences, Sulfonamides, [ SDV ] Life Sciences [q-bio], Autophagy, Cell Biology, medicine.disease, Endoplasmic Reticulum Stress, Xenograft Model Antitumor Assays, [SDV] Life Sciences [q-bio], Gene Expression Regulation, Neoplastic, 030104 developmental biology, Oncology, Cell culture, Apoptosis, Drug Resistance, Neoplasm, 030220 oncology & carcinogenesis, Unfolded protein response, Cancer research
Abstract

International audience; Hepatitis B virus (HBV) is a small DNA virus that replicates by reverse transcription of a terminally redundant RNA, the pregenome. Specific packaging of this transcript into viral capsids is mediated by interaction of the reverse transcriptase, P protein, with the 5'-proximal encapsidation signal epsilon, epsilon-function is correlated with the formation of a hairpin structure containing a bulge and a loop, each consisting of 6 nt. To analyse the importance of primary sequence in these regions, we have combined selection of encapsidation competent individuals from pools of randomized epsilon-sequences in transfected cells with in vitro amplification, thus bypassing the current experimental limitations of the HBV system. While no alterations of the authentic loop sequence were detectable, many different sequences were tolerated in the 3'-part of the bulge. However, at the two 5'-proximal bulge positions the wt sequence was strongly selected for, indicating that for RNA packaging close contacts between protein and the 5'- but not the 3'-part of the bulge are important. Such a bipartite organisation provides a structural basis for the recently demonstrated special role of the 3'-part of the bulge as template for the first nucleotides of (-)-strand DNA in HBV reverse transcription.

Published
2016
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16. Plasmonic photothermal destruction of uropathogenic E. coli with reduced graphene oxide and core/shell nanocomposites of gold nanorods/reduced graphene oxide

Author

Julie Boukaert, Amaia Pesquera, Aritz Yanguas Serrano, Laurent Héliot, Kostiantyn Turcheniuk, Iban Larroulet, Rabah Boukherroub, Sabine Szunerits, Mariano Gonzalez Pisfil, Jolanda Spadavecchia, Charles-Henri Hage, Amaia Zurutuza, Institut de Recherche Interdisciplinaire [Villeneuve d'Ascq] (IRI), Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Droit et Santé-Université de Lille, Sciences et Technologies, Laboratoire de Réactivité de Surface (LRS), Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS), Université de Lille, Sciences et Technologies-Université de Lille, Droit et Santé-Centre National de la Recherche Scientifique (CNRS), Institut d’Électronique, de Microélectronique et de Nanotechnologie - UMR 8520 (IEMN), Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), NanoBioInterfaces - IEMN (NBI - IEMN), and Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)-Centrale Lille-Institut supérieur de l'électronique et du numérique (ISEN)-Université de Valenciennes et du Hainaut-Cambrésis (UVHC)-Université de Lille-Centre National de la Recherche Scientifique (CNRS)-Université Polytechnique Hauts-de-France (UPHF)

Subjects
Materials science, Graphene, Biomedical Engineering, Oxide, technology, industry, and agriculture, Nanoparticle, Nanotechnology, General Chemistry, General Medicine, Photothermal therapy, law.invention, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, chemistry.chemical_compound, chemistry, law, Core shell nanocomposites, General Materials Science, Nanorod, Uropathogenic E. coli, Plasmon
Abstract

The development of non-antibiotic based treatments against bacterial infections by Gram-negative uropathogenic E. coli is a complex task. New strategies to treat such infections are thus urgently needed. This report illustrates the development of pegylated reduced graphene oxide nanoparticles (rGO-PEG) and gold nanorods (Au NRs) coated with rGO-PEG (rGO-PEG-Au NRs) for the selective killing of uropathogenic E. coli UTI89. We took advantage of the excellent light absorption properties of rGO-PEG and Au NR particles in the near-infrared (NIR) region to photothermally kill Gram-negative pathogens up to 99% in 10 min by illumination of solutions containing the bacteria. The rGO-PEG-Au NRs demonstrated better photothermal efficiency towards E. coli than rGO-PEG. Targeted killing of E. coli UTI89 could be achieved with rGO-PEG-Au NRs functionalized with multimeric heptyl α-D-mannoside probes. This currently offers a unique biocompatible method for the ablation of pathogens with the opening of probably a new possibility for clinical treatments of patients with urinary infections.

Published
2015
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17. Implementation of transportation distance for analyzing FLIM and FRET experiments

Author

Laurent Héliot, Philippe Heinrich, Aymeric Leray, Jonas Kahn, and Mariano Gonzalez Pisfil

Subjects
Fluorescence-lifetime imaging microscopy, Time Factors, General Mathematics, Cells, Immunology, Living cell, General Biochemistry, Genetics and Molecular Biology, Cell Line, Nuclear magnetic resonance, Robustness (computer science), Fluorescence Resonance Energy Transfer, Humans, Computer Simulation, Cluster analysis, General Environmental Science, Fluorescent Dyes, Pharmacology, Physics, Models, Statistical, Pixel, General Neuroscience, Mathematical Concepts, Transportation distance, Förster resonance energy transfer, HEK293 Cells, Computational Theory and Mathematics, Microscopy, Fluorescence, Comparison study, General Agricultural and Biological Sciences, Biological system, Monte Carlo Method
Abstract

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Forster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.

Published
2014

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