11 results on '"Clarissa, Liesche"'
Search Results
2. Single-Fluorescent Protein Reporters Allow Parallel Quantification of Natural Killer Cell-Mediated Granzyme and Caspase Activities in Single Target Cells
- Author
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Clarissa Liesche, Patricia Sauer, Isabel Prager, Doris Urlaub, Maren Claus, Roland Eils, Joël Beaudouin, and Carsten Watzl
- Subjects
natural killer cells ,cytotoxic lymphocytes ,single-fluorescent protein reporters ,granzyme and caspase activity ,apoptosis and cell death ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Natural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes via perforin pores or by activation of caspases via death receptors. In order to understand how NK cells combine different cell death mechanisms, it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here, we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 min. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K, and H. The approach presented here is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.
- Published
- 2018
- Full Text
- View/download PDF
3. Supramolecular assembly of the Escherichia coli LdcI upon acid stress
- Author
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Grégory Effantin, Jean-Philippe Kleman, Megghane Baulard, Maria Bacia-Verloop, Dominique Bourgeois, Karine Huard, Virgile Adam, Angélique Fraudeau, Jan Felix, Clarissa Liesche, Irina Gutsche, Matthew Jessop, Ambroise Desfosses, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-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)-Université Grenoble Alpes (UGA), Unit for Structural Biology, VIB-UGent Center for Inflammation Research [Gand, Belgique] (IRC), VIB [Belgium]-VIB [Belgium], The cryoelectron microscopy platform, The M4D Cell Imaging Platform, The nanobody generation platform of the AFMB laboratory, ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: grant no. 647784, European Project: ALTF441-2017, and European Project: 789385,RespViRALI
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DYNAMICS ,DOMAINS ,Cryo-electron microscopy ,lysine decarboxylase ,ORGANIZATION ,medicine.disease_cause ,Supramolecular assembly ,03 medical and health sciences ,LdcI ,REVEALS ,Extracellular ,medicine ,ENZYMATIC-ACTIVITIES ,SMLM ,Escherichia coli ,BACTERIAL-CELL BIOLOGY ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Lysine decarboxylase ,[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM] ,030306 microbiology ,Chemistry ,Biology and Life Sciences ,LOCALIZATION ,Negative stain ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology ,Cytosol ,Biophysics ,cryo-EM ,VISUALIZATION ,COMPLEXES ,Function (biology) - Abstract
International audience; Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.
- Published
- 2021
4. Supramolecular assembly of the
- Author
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Matthew, Jessop, Clarissa, Liesche, Jan, Felix, Ambroise, Desfosses, Megghane, Baulard, Virgile, Adam, Angélique, Fraudeau, Karine, Huard, Grégory, Effantin, Jean-Philippe, Kleman, Maria, Bacia-Verloop, Dominique, Bourgeois, and Irina, Gutsche
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Adenosine Triphosphatases ,Models, Molecular ,Carboxy-Lyases ,Escherichia coli Proteins ,Cryoelectron Microscopy ,lysine decarboxylase ,Hydrogen-Ion Concentration ,Biological Sciences ,Crystallography, X-Ray ,Biochemistry ,LdcI ,Microscopy, Fluorescence ,Stress, Physiological ,Escherichia coli ,cryo-EM ,Amino Acid Sequence ,Protein Multimerization ,SMLM ,Protein Binding - Abstract
Significance Bacteria possess a sophisticated arsenal of defense mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, Escherichia coli and many other enterobacteria activate a proton-consuming resistance system based on the acid stress-inducible lysine decarboxylase LdcI. Here we develop generally applicable tools to uncover the spatial localization of LdcI inside the cell by superresolution fluorescence microscopy and investigate the in vitro supramolecular organization of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations., Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.
- Published
- 2020
5. Supramolecular assembly of the E. coli LdcI upon acid stress
- Author
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Jean-Philippe Kleman, Matthew Jessop, Grégory Effantin, Maria Bacia-Verloop, Irina Gutsche, Karine Huard, Dominique Bourgeois, Ambroise Desfosses, Virgile Adam, Clarissa Liesche, Angélique Fraudeau, Jan Felix, and Megghane Baulard
- Subjects
0303 health sciences ,Fluorescence-lifetime imaging microscopy ,Lysine decarboxylase ,030306 microbiology ,Chemistry ,Negative stain ,In vitro ,Supramolecular assembly ,03 medical and health sciences ,Cytosol ,Extracellular ,Biophysics ,Function (biology) ,030304 developmental biology - Abstract
Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid-stress response regulation of E. coli LdcI by combining biochemical and biophysical characterisation with negative stain and cryo-electron microscopy, and wide-field and super-resolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localisation of nanobody-labelled endogenous wild-type LdcI in acid-stressed E. coli cells, and show that it organises into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerisation as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-electron microscopy, and reveal the molecular determinants of LdcI polymerisation, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organisation in the acid stress response.Significance statementBacteria possess a sophisticated arsenal of defence mechanisms that allow them to survive in adverse conditions. Adaptation to acid stress and hypoxia is crucial for the enterobacterial transmission in the gastrointestinal tract of their human host. When subjected to low pH, E. coli and many other enterobacteria activate a proton-consuming resistance system based on the acid-stress inducible lysine decarboxylase LdcI. Here we develop generally-applicable tools to uncover the spatial localisation of LdcI inside the cell by super-resolution fluorescence microscopy, and investigate the in vitro supramolecular organisation of this enzyme by cryo-EM. We build on these results to propose a mechanistic model for LdcI function and offer tools for further in vivo investigations.
- Published
- 2020
- Full Text
- View/download PDF
6. Automated Analysis of Single-Molecule Photobleaching Data by Statistical Modeling of Spot Populations
- Author
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Kristin S. Grußmayer, Roland Eils, Michael Ludwig, Stefan Wörz, Karl Rohr, Joël Beaudouin, Dirk-Peter Herten, and Clarissa Liesche
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Models, Molecular ,Fluorophore ,Population ,Biophysics ,Noise (electronics) ,Automation ,chemistry.chemical_compound ,Optics ,Microscopy ,Image Processing, Computer-Assisted ,Molecule ,fas Receptor ,Protein Structure, Quaternary ,education ,Fluorescent Dyes ,education.field_of_study ,Models, Statistical ,Photobleaching ,Base Sequence ,business.industry ,Statistical model ,DNA ,Fluorescent labelling ,chemistry ,Nucleic Acid Conformation ,Protein Multimerization ,Proteins and Nucleic Acids ,business ,Biological system - Abstract
The number of fluorophores within a molecule complex can be revealed by single-molecule photobleaching imaging. A widely applied strategy to analyze intensity traces over time is the quantification of photobleaching step counts. However, several factors can limit and bias the detection of photobleaching steps, including noise, high numbers of fluorophores, and the possibility that several photobleaching events occur almost simultaneously. In this study, we propose a new approach, to our knowledge, to determine the fluorophore number that correlates the intensity decay of a population of molecule complexes with the decay of the number of visible complexes. We validated our approach using single and fourfold Atto-labeled DNA strands. As an example we estimated the subunit stoichiometry of soluble CD95L using GFP fusion proteins. To assess the precision of our method we performed in silico experiments showing that the estimates are not biased for experimentally observed intensity fluctuations and that the relative precision remains constant with increasing number of fluorophores. In case of fractional fluorescent labeling, our simulations predicted that the fluorophore number estimate corresponds to the product of the true fluorophore number with the labeling fraction. Our method, denoted by spot number and intensity correlation (SONIC), is fully automated, robust to noise, and does not require the counting of photobleaching events.
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- 2015
- Full Text
- View/download PDF
7. NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing
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Björn Önfelt, Isabel Prager, Niklas Sandström, Clarissa Liesche, Quentin Verron, Hanna van Ooijen, Doris Urlaub, Roland Eils, Frank Fasbender, Maren Claus, Joël Beaudouin, and Carsten Watzl
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Cytotoxicity, Immunologic ,Programmed cell death ,Immunology ,Cell ,Granzymes ,Article ,medicine ,Immunology and Allergy ,Cytotoxic T cell ,Humans ,fas Receptor ,Cytotoxicity ,Caspase ,Research Articles ,Caspase 8 ,biology ,Chemistry ,Perforin ,Receptors, Death Domain ,Fas receptor ,Cell biology ,Granzyme B ,Killer Cells, Natural ,Kinetics ,medicine.anatomical_structure ,biology.protein ,HeLa Cells - Abstract
Natural killer cells can kill infected and transformed cells via two different cell death mechanisms. Prager et al. show that NK cells quickly kill their first targets by releasing cytotoxic granules and only use the slower death receptor–mediated cytotoxicity for their final kill., NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor–mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor–mediated cytotoxicity are differentially regulated during NK cell serial killing., Graphical Abstract
- Published
- 2018
8. Single-fluorescent protein reporters allow parallel quantification of NK cell-mediated granzyme and caspase activities in single target cells
- Author
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Maren Claus, Carsten Watzl, Joël Beaudouin, Roland Eils, Clarissa Liesche, and Patricia Sauer
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Programmed cell death ,biology ,Chemistry ,Cell ,Cell biology ,Granzyme B ,medicine.anatomical_structure ,Perforin ,Granzyme ,Granzyme A ,medicine ,biology.protein ,Cytotoxic T cell ,Caspase - Abstract
1.AbstractNatural killer (NK) cells eliminate infected and tumorigenic cells through delivery of granzymes via perforin pores or by activation of caspases via death receptors. In order to understand how NK cells combine different cell death mechanisms it is important to quantify target cell responses on a single cell level. However, currently existing reporters do not allow the measurement of several protease activities inside the same cell. Here we present a strategy for the comparison of two different proteases at a time inside individual target cells upon engagement by NK cells. We developed single-fluorescent protein reporters containing the RIEAD or the VGPD cleavage site for the measurement of granzyme B activity. We show that these two granzyme B reporters can be applied in combination with caspase-8 or caspase-3 reporters. While we did not find that caspase-8 was activated by granzyme B, our method revealed that caspase-3 activity follows granzyme B activity with a delay of about 6 minutes. Finally, we illustrate the comparison of several different reporters for granzyme A, M, K and H. The here presented approach is a valuable means for the investigation of the temporal evolution of cell death mediated by cytotoxic lymphocytes.
- Published
- 2018
- Full Text
- View/download PDF
9. CD95 receptor activation by ligand-induced trimerization is independent of its partial pre-ligand assembly
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Sabine Aschenbrenner, Mike Heilemann, Johanna Berndt, Clarissa Liesche, Roland Eils, Franziska Fricke, and Joël Beaudouin
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biology ,Chemistry ,Biophysics ,biology.protein ,Receptor clustering ,FADD ,biological phenomena, cell phenomena, and immunity ,Fas receptor ,Ligand (biochemistry) ,Receptor ,Transmembrane protein ,Fas ligand ,Death domain - Abstract
CD95 (Fas, APO-1, TNFRSF6) is a widely expressed single-pass transmembrane protein that is implicated in cell death, inflammatory response, proliferation and cell migration. CD95 ligand (CD95L, FasL, TNFSF6), is a potent apoptotic inducer in the membrane form but not when cleaved into soluble CD95L (sCD95L). Here, we aimed at understanding the relation between ligand-receptor multimerization and receptor activation by correlating the kinetics of ligand binding, receptor oligomerization, FADD (FAS-Associated via Death Domain) recruitment and caspase-8 activation inside living cells. Using single molecule localization microscopy and Förster resonance energy transfer imaging we show that the majority of CD95 receptors on the plasma membrane are monomeric at rest. This was confirmed functionally as the wild-type receptor is not blocked by a receptor mutant that cannot bind ligand. Moreover, using time-resolved fluorescence imaging approaches we demonstrated that receptor multimerization follows instantaneously ligand binding, whereas FADD recruitment is delayed. This process can explain the typical delay time seen with caspase-8 activity reporters. Finally, the low activity of sCD95L, which was caused by inefficient FADD recruitment, was not explained by the low avidity for the receptor but by a receptor clustering mechanism that was different from the one induced by the strong apoptosis inducer IZ-sCD95L. Our results reveal that receptor activation is modulated by the capacity of its ligand to trimerize it.HighlightsAt a density of less than 10 receptors per µm2CD95 exists as monomer (58%) and dimer (42%)Pre-formed dimers do not contribute to ligand-induced CD95 apoptotic signalingThe PLAD of CD95 attenuates overexpression-induced, ligand-independent cell deathsoluble CD95L can rapidly multimerize CD95 after binding but it is still a poor inducer of apoptosis through inefficient FADD recruitmentFADD recruitment kinetics but not ligand binding kinetics correlates with caspase-8 onset of activity
- Published
- 2018
10. Caspase-8 cleaves its substrates from the plasma membrane upon CD95-induced apoptosis
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S. Aschenbrenner, Maximilian Hörner, Roland Eils, Clarissa Liesche, and Joël Beaudouin
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Programmed cell death ,Receptor complex ,Calnexin ,Active Transport, Cell Nucleus ,Apoptosis ,Caspase 3 ,Caspase 6 ,Caspase 8 ,Substrate Specificity ,Mitochondrial Proteins ,Humans ,Amino Acid Sequence ,fas Receptor ,Cycloheximide ,Molecular Biology ,Caspase ,Fluorescent Dyes ,Original Paper ,biology ,Tumor Necrosis Factor-alpha ,Keratin-8 ,Cell Membrane ,Cell Biology ,Protein Structure, Tertiary ,Cell biology ,Enzyme Activation ,biology.protein ,Signal transduction ,BH3 Interacting Domain Death Agonist Protein ,HeLa Cells ,Signal Transduction - Abstract
Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells.
- Published
- 2013
11. Death receptor-based enrichment of Cas9-expressing cells
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Roland Eils, Stefanie Grosse, Joël Beaudouin, Clarissa Liesche, L. Venkatraman, S. Aschenbrenner, and Dirk Grimm
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0301 basic medicine ,T7E1 ,Population ,Apoptosis ,Biology ,Genome ,570 Life sciences ,03 medical and health sciences ,symbols.namesake ,0302 clinical medicine ,Genome editing ,INDEL Mutation ,Cell Line, Tumor ,CRISPR ,Humans ,Guide RNA ,Indel ,Cloning, Molecular ,education ,Gene ,CRISPR/Cas9 ,Editing efficiency ,Sanger sequencing ,Genetics ,education.field_of_study ,Cas9 ,Receptors, Death Domain ,Cell biology ,030104 developmental biology ,Phenotype ,symbols ,Puromycin ,CRISPR-Cas Systems ,030217 neurology & neurosurgery ,Biotechnology ,Research Article ,HeLa Cells - Abstract
Background The CRISPR/Cas9 genome editing system has greatly facilitated and expanded our capacity to engineer mammalian genomes, including targeted gene knock-outs. However, the phenotyping of the knock-out effect requires a high DNA editing efficiency. Results Here, we report a user-friendly strategy based on the extrinsic apoptosis pathway that allows enrichment of a polyclonal gene-edited cell population, by selecting Cas9-transfected cells that co-express dominant-negative mutants of death receptors. The extrinsic apoptosis pathway can be triggered in many mammalian cell types, and ligands are easy to produce, do not require purification and kill much faster than the state-of-the-art selection drug puromycin. Stringent assessment of our advanced selection strategy via Sanger sequencing, T7 endonuclease I (T7E1) assay and direct phenotyping confirmed a strong and rapid enrichment of Cas9-expressing cell populations, in some cases reaching up to 100 % within one hour. Notably, the efficiency of target DNA cleavage in these enriched cells reached high levels that exceeded the reliable range of the T7E1 assay, a conclusion that can be generalized for editing efficiencies above 30 %. Moreover, our data emphasize that the insertion and deletion pattern induced by a specific gRNA is reproducible across different cell lines. Conclusions The workflow and the findings reported here should streamline a wide array of future low- or high-throughput gene knock-out screens, and should largely improve data interpretation from CRISPR experiments. Electronic supplementary material The online version of this article (doi:10.1186/s12896-016-0250-4) contains supplementary material, which is available to authorized users.
- Published
- 2016
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