UMR9187 / U1196 – Chemistry, Modelling and Imaging for Biology (CMIB)

Unit publications

Year of publication 2019

Nathalie Grandin, Bruno Pereira, Camille Cohen, Pauline Billard, Caroline Dehais, Catherine Carpentier, Ahmed Idbaih, Franck Bielle, François Ducray, Dominique Figarella-Branger, Jean-Yves Delattre, Marc Sanson, Patrick Lomonte, Delphine Poncet, Pierre Verrelle, Michel Charbonneau, (2019 Nov 11)

The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas.

Acta neuropathologica communications : 7 : 175 : DOI : 10.1186/s40478-019-0833-0 Learn more
Summary

All cancer cells need to maintain functional telomeres to sustain continuous cell division and proliferation. In human diffuse gliomas, functional telomeres are maintained due either to reactivation of telomerase expression, the main pathway in most cancer types, or to activation of a mechanism called the alternative lengthening of telomeres (ALT). The presence of IDH1/2 mutations (IDH-mutant) together with loss of ATRX expression (ATRX-lost) are frequently associated with ALT in diffuse gliomas. However, detection of ALT, and a fortiori its quantification, are rarely, if ever, measured in neuropathology laboratories. We measured the level of ALT activity using the previously described quantitative “C-circle” assay and analyzed it in a well characterized cohort of 104 IDH-mutant and ATRX-lost adult diffuse gliomas. We report that in IDH-mutant ATRX-lost anaplastic astrocytomas, the intensity of ALT was inversely correlated with age (p < 0.001), the younger the patient, the higher the intensity of ALT. Strikingly, glioblastomas having progressed from anaplastic astrocytomas did not exhibit this correlation. ALT activity level in the tumor did not depend on telomere length in healthy tissue cells from the same patient. In summary, we have uncovered the existence, in anaplastic astrocytomas but not in glioblastomas with the same IDH and ATRX mutations, of a correlation between patient age and the level of activity of ALT, a telomerase-independent pathway of telomere maintenance.

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Priyanka Toshniwal, Michelle Nguyen, Aurore Guédin, Helena Viola, Diwei Ho, Yongeun Kim, Uditi Bhatt, Charles Bond, Livia Hool, Laurence H Hurley, Jean-Louis Mergny, Mark Fear, Fiona Wood, K Swaminathan Iyer, Nicole M Smith (2019 Nov 3)

TGF-β-induced fibrotic stress increases G-quadruplex formation in human fibroblasts.

FEBS letters : Epub ahead of print : DOI : 10.1002/1873-3468.13658 Learn more
Summary

Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-β- induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.

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Chen J., Zhang Y., Cheng M., Mergny J.L., Lin Q., Zhou J., Ju H. (2019 Nov 1)

Highly active G-quadruplex/hemin DNAzyme for sensitive colorimetric determination of lead(II)

Microchimica Acta : 186 : 786 : DOI : 10.1007/s00604-019-3950-3 Learn more
Summary

A UV-vis, CD, and differential pulse voltammetric study was performed on the deactivation of the activity of parallel G-quadruplex/hemin DNAzymes (G4 DNAzymes) by Pb(II). The G4 DNAzyme carries a d[TC] sequence at its 3extasciiacutex end and is stabilized by potassium(I). On addition of Pb(II), the K(I) ions in the parallel G4 are replaced by Pb(II) to keep the parallel topology. Intruded Pb(II) decrease the affinity between the topology and hemin, this leads to a decrease of DNAzyme activity for catalyzing the oxidation of 2,2extasciiacutex-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by hydrogen peroxide to form a green dye with an absorption maximum at 420 nm. The assay does not use any amplification, and has a linear response in the 0.01 to 10 muM Pb(II) concentration range and a 7.1 nM limit of detection. The method was successfully applied to the analysis of spiked water samples.

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Tao X., Chen H., Trepout S., Cen J., Ling J., Li M.H. (2019 Oct 1)

Polymersomes with Aggregation-Induced Emission Based on Amphiphilic Block Copolypeptoids

Chem. Commun. : Accepted Manuscript : - : DOI : 10.1039/C9CC07501A Learn more
Summary

Biocompatible polymersomes are prepared from amphiphilic block copolypeptoids with aggregation-induced emission, where the hydrophobic block P(TPE-NAG) is a tetraphenylethylene (TPE)-modified poly(N-allylglycine) and the hydrophilic block is polysarcosine. These nanoparticles are non-cytotoxic and show strong fluorescence emission in aqueous solution.

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Franck Court, Elisa Le Boiteux, Anne Fogli, Mélanie Müller-Barthélémy, Catherine Vaurs-Barrière, Emmanuel Chautard, Bruno Pereira, Julian Biau, Jean-Louis Kemeny, Toufic Khalil, Lucie Karayan-Tapon, Pierre Verrelle, Philippe Arnaud (2019 Sep 20)

Transcriptional alterations in glioma result primarily from DNA methylation-independent mechanisms.

Genome research : 22 : 1605-1621 : DOI : 10.1101/gr.249219.119 Learn more
Summary

In cancer cells, aberrant DNA methylation is commonly associated with transcriptional alterations, including silencing of tumor suppressor genes. However, multiple epigenetic mechanisms, including polycomb repressive marks, contribute to gene deregulation in cancer. To dissect the relative contribution of DNA methylation-dependent and -independent mechanisms to transcriptional alterations at CpG island/promoter-associated genes in cancer, we studied 70 samples of adult glioma, a widespread type of brain tumor, classified according to their isocitrate dehydrogenase (IDH1) mutation status. We found that most transcriptional alterations in tumor samples were DNA methylation-independent. Instead, altered histone H3 trimethylation at lysine 27 (H3K27me3) was the predominant molecular defect at deregulated genes. Our results also suggest that the presence of a bivalent chromatin signature at CpG island promoters in stem cells predisposes not only to hypermethylation, as widely documented, but more generally to all types of transcriptional alterations in transformed cells. In addition, the gene expression strength in healthy brain cells influences the choice between DNA methylation- and H3K27me3-associated silencing in glioma. Highly expressed genes were more likely to be repressed by H3K27me3 than by DNA methylation. Our findings support a model in which altered H3K27me3 dynamics, more specifically defects in the interplay between polycomb protein complexes and the brain-specific transcriptional machinery, is the main cause of transcriptional alteration in glioma cells. Our study provides the first comprehensive description of epigenetic changes in glioma and their relative contribution to transcriptional changes. It may be useful for the design of drugs targeting cancer-related epigenetic defects.

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Angrand G., Quillévéré A., Loaëc N., Daskalogianni C., Granzhan A., Teulade-Fichou M.P., Fahraeus R., Prado Martins R., Blondel M. (2019 Sep 1)

Sneaking Out for Happy Hour: Yeast-Based Approaches to Explore and Modulate Immune Response and Immune Evasion

Genes : 10 : 667-689 : DOI : 10.3390/genes10090667 Learn more
Summary

Many pathogens (virus, bacteria, fungi, or parasites) have developed a wide variety of mechanisms to evade their host immune system. The budding yeast Saccharomyces cerevisiae has successfully been used to decipher some of these immune evasion strategies. This includes the cis-acting mechanism that limits the expression of the oncogenic Epstein–Barr virus (EBV)-encoded EBNA1 and thus of antigenic peptides derived from this essential but highly antigenic viral protein. Studies based on budding yeast have also revealed the molecular bases of epigenetic switching or recombination underlying the silencing of all except one members of extended families of genes that encode closely related and highly antigenic surface proteins. This mechanism is exploited by several parasites (that include pathogens such as Plasmodium, Trypanosoma, Candida, or Pneumocystis) to alternate their surface antigens, thereby evading the immune system. Yeast can itself be a pathogen, and pathogenic fungi such as Candida albicans, which is phylogenetically very close to S. cerevisiae, have developed stealthiness strategies that include changes in their cell wall composition, or epitope-masking, to control production or exposure of highly antigenic but essential polysaccharides in their cell wall. Finally, due to the high antigenicity of its cell wall, yeast has been opportunistically exploited to create adjuvants and vectors for vaccination.

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Abegão L.M.G., Fonseca R.D., Santos F.A., Rodrigues J.J., Kamada K., Mendonça C.R., Piguel S., De Boni L. (2019 Aug 23)

First molecular electronic hyperpolarizability of series of π-conjugated oxazole dyes in solution: an experimental and theoretical study

RSC Adv. : 9 : 26476-26482 : DOI : 10.1039/C9RA05246A Learn more
Summary

In this work, we report the experimental and theoretical first molecular electronic hyperpolarizability (βHRS) of eleven π-conjugated oxazoles compounds in toluene medium. The Hyper-Rayleigh Scattering (HRS) technique allowed the determination of the experimental dynamic βHRS values, by exciting the compounds with a picosecond pulse trains from a Q-switched and mode-locked Nd:YAG laser tuned at 1064 nm. Theoretical predictions based on time-dependent density functional theory level using the Gaussian 09 program package were performed with three different functionals (B3LYP, CAM-B3LYP, and M06-2X), to calculate both static and dynamic theoretical βHRS values. Good accordance was found between the experimental and theoretical values, in particular for the CAM-B3LYP and M06-2X functionals.

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Michaela Krafcikova, Simon Dzatko, Coralie Caron, Anton Granzhan, Radovan Fiala, Tomas Loja, Marie-Paule Teulade-Fichou, Tomas Fessl, Robert Hänsel-Hertsch, Jean-Louis Mergny, Silvie Foldynova-Trantirkova, Lukas Trantirek (2019 Aug 9)

Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

Journal of the Americal Chemical Society : 141 : 13281-13285 : DOI : 10.1021/jacs.9b03031 Learn more
Summary

Monitoring DNA–Ligand Interactions in Living Human Cells Using NMR Spectroscopy

Studies on DNA–ligand interactions in the cellular environment are problematic due to the lack of suitable biophysical tools. To address this need, we developed an in-cell NMR-based approach for monitoring DNA–ligand interactions inside the nuclei of living human cells. Our method relies on the acquisition of NMR data from cells electroporated with preformed DNA–ligand complexes. The impact of the intracellular environment on the integrity of the complexes is assessed based on in-cell NMR signals from unbound and ligand-bound forms of a given DNA target. This technique was tested on complexes of two model DNA fragments and four ligands, namely, a representative DNA minor-groove binder (netropsin) and ligands binding DNA base-pairing defects (naphthalenophanes). In the latter case, we demonstrate that two of the three in vitro-validated ligands retain their ability to form stable interactions with their model target DNA in cellulo, whereas the third one loses this ability due to off-target interactions with genomic DNA and cellular metabolites. Collectively, our data suggest that direct evaluation of the behavior of drug-like molecules in the intracellular environment provides important insights into the development of DNA-binding ligands with desirable biological activity and minimal side effects resulting from off-target binding.

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Xiao Xie, Michela Zuffo, Marie-Paule Teulade-Fichou, Anton Granzhan (2019 Aug 6)

Identification of optimal fluorescent probes for G-quadruplex nucleic acids through systematic exploration of mono- and distyryl dye libraries

Beilstein Journal of Organic Chemistry : 15 : 1872–1889 : DOI : 10.3762/bjoc.15.183 Learn more
Summary

Identification of optimal fluorescent probes for G-quadruplex nucleic acids

A library of 52 distyryl and 9 mono-styryl cationic dyes was synthesized and investigated with respect to their optical properties, propensity to aggregation in aqueous medium, and capacity to serve as fluorescence “light-up” probes for G-quadruplex (G4) DNA and RNA structures. Among the 61 compounds, 57 dyes showed preferential enhancement of fluorescence intensity in the presence of one or another G4-DNA or RNA structure, while no dye displayed preferential response to double-stranded DNA or single-stranded RNA analytes employed at equivalent nucleotide concentration. Thus, preferential fluorimetric response towards G4 structures appears to be a common feature of mono- and distyryl dyes, including long-known mono-styryl dyes used as mitochondrial probes or protein stains. However, the magnitude of the G4-induced “light-up” effect varies drastically, as a function of both the molecular structure of the dyes and the nature or topology of G4 analytes. Although our results do not allow to formulate comprehensive structure–properties relationships, we identified several structural motifs, such as indole- or pyrrole-substituted distyryl dyes, as well as simple mono-stryryl dyes such as DASPMI [2-(4-(dimethylamino)styryl)-1-methylpyridinium iodide] or its 4-isomer, as optimal fluorescent light-up probes characterized by high fluorimetric response (I/I0 of up to 550-fold), excellent selectivity with respect to double-stranded DNA or single-stranded RNA controls, high quantum yield in the presence of G4 analytes (up to 0.32), large Stokes shift (up to 150 nm) and, in certain cases, structural selectivity with respect to one or another G4 folding topology. These dyes can be considered as promising G4-responsive sensors for in vitro or imaging applications. As a possible application, we implemented a simple two-dye fluorimetric assay allowing rapid topological classification of G4-DNA structures.

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Birzu C., Hillairet A., Giry M., Grandin N., Verrelle P., Mokhtari K., Marie Y., Hoang-Xuan K., Delattre J., Idbaih A., Charbonneau M., Sanson M., Alentorn A. (2019 Aug 1)

OS9.7 Telomere length, TERTp mutation and ALT status in adult diffuse gliomas

Neuro-Oncology : 21 : iii19-iii20 : DOI : 10.1093/neuonc/noz126.065 Learn more
Summary

BACKGROUND

The current classification of adult diffuse gliomas integrates two alternative telomere maintenance mechanisms: reactivation of telomerase activity by TERT promoter (TERTp) mutations or ATRX mutations associated with alternative length telomere (ALT). We investigated here the relation between these two mechanisms, telomere length, and outcome in a large series of diffuse gliomas.

MATERIAL AND METHODS

We performed C-circle assay (CCA) to determine ALT status, determined telomere length in tumor (RTLt) and leukocyte (RTLl) in a cohort of 354 adult diffuse gliomas, and sequenced ATRX gene. We calculated an age-adjusted telomere score considering tumor and leukocyte (blood) telomere length and corrected by age. This score was used in univariate and multivariate survival analyses to evaluate the potential impact of telomere length on the prognosis of gliomas. We used the TCGA LGG-GBM dataset to validate our findings in an independent cohort.

RESULTS

RTLl and RTLt were associated with ATRX mutation and ALT phenotype, and negatively associated with age and TERTp mutations. ATRX mutations (found in 52% (64/123) of samples) were mostly transitions (C>T or T>C), and were associated with ALT phenotype. None of 1p/19q co-deleted oligodendrogliomas harbored an ALT phenotype. No patients with TERTp mutations had ALT phenotype except for a very small subgroup of patients (3/87, 3.4%) suggesting that multiple ways of telomere maintenance, may co-exist in a single tumor, probably expressed in different clones. Telomere age-adjusted score was independently associated with better outcome (HR= 0.73 [95% CI 0.56–0.97], p-value 0.03 adjusted for age, TERTp mutation, IDH mutation, 1p/19q co-deletion and WHO grade). These results were validated using the LGG-GBM TCGA dataset.

CONCLUSION

We unravel the relation between RTLl and RTLt, TERTp mutation and ALT phenotype and describe a novel telomere age-adjusted score independently associated with better prognosis in adult diffuse gliomas.

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Yangwei Deng, Hui Chen, Xinfeng Tao, Fangyi Cao, Sylvain Trépout, Jun Ling, Min-Hui Li (2019 Jul 31)

Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids.

Biomacromolecules : 20 : 3435-3444 : DOI : 10.1021/acs.biomac.9b00713 Learn more
Summary

Stimuli-responsive polymersomes formed by amphiphilic block copolymers have attracted substantial attention as smart and robust containers for drug delivery and nano/microreactors. Biosourced amphiphilic diblock copolypeptoids were developed that can self-assemble into oxidation-responsive unilamellar vesicles. These vesicles can burst under the action of reactive oxygen species which can be the hydrogen peroxide or the singlet oxygen produced by light-activation of a photosensitizer with spatiotemporal control. Polysarcosine (PSar, also called poly(-methyl glycine)) was selected as the hydrophilic block because of its resistance to protein adsorption and low toxicity, similar to poly(ethylene glycol) (PEG). We designed and synthesized poly(-3-(methylthio)propyl glycine) as the hydrophobic block. Its polyglycine backbone is the same as that of PSar, and especially, its hydrophobic N-substituents, thioether side chains, can be oxidized to hydrophilic sulfoxides. These oxidation-responsive polymersomes entirely based on N-substituted poly(amino acid)s were biocompatible as confirmed by cell viability tests and may find applications in drug delivery, biosensing, biodetection, and nano/microreactors.

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Paulina Prorok, Marie Artufel, Antoine Aze, Philippe Coulombe, Isabelle Peiffer, Laurent Lacroix, Aurore Guédin, Jean-Louis Mergny, Julia Damaschke, Aloys Schepers, Benoit Ballester, Marcel Méchali (2019 Jul 24)

Involvement of G-quadruplex regions in mammalian replication origin activity.

Nature communications : 3274 : DOI : 10.1038/s41467-019-11104-0 Learn more
Summary

Genome-wide studies of DNA replication origins revealed that origins preferentially associate with an Origin G-rich Repeated Element (OGRE), potentially forming G-quadruplexes (G4). Here, we functionally address their requirements for DNA replication initiation in a series of independent approaches. Deletion of the OGRE/G4 sequence strongly decreased the corresponding origin activity. Conversely, the insertion of an OGRE/G4 element created a new replication origin. This element also promoted replication of episomal EBV vectors lacking the viral origin, but not if the OGRE/G4 sequence was deleted. A potent G4 ligand, PhenDC3, stabilized G4s but did not alter the global origin activity. However, a set of new, G4-associated origins was created, whereas suppressed origins were largely G4-free. In vitro Xenopus laevis replication systems showed that OGRE/G4 sequences are involved in the activation of DNA replication, but not in the pre-replication complex formation. Altogether, these results converge to the functional importance of OGRE/G4 elements in DNA replication initiation.

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S Houari, E Picard, T Wurtz, E Vennat, N Roubier, T D Wu, J L Guerquin-Kern, M Duttine, T T Thuy, A Berdal, S Babajko (2019 Jul 23)

Disrupted Iron Storage in Dental Fluorosis.

Journal of dental research : 98 : 994-1001 : DOI : 10.1177/0022034519855650 Learn more
Summary

Enamel formation and quality are dependent on environmental conditions, including exposure to fluoride, which is a widespread natural element. Fluoride is routinely used to prevent caries. However, when absorbed in excess, fluoride may also lead to altered enamel structural properties associated with enamel gene expression modulations. As iron plays a determinant role in enamel quality, the aim of our study was to evaluate the iron metabolism in dental epithelial cells and forming enamel of mice exposed to fluoride, as well as its putative relation with enamel mechanical properties. Iron storage was investigated in dental epithelial cells with Perl’s blue staining and secondary ion mass spectrometry imaging. Iron was mainly stored by maturation-stage ameloblasts involved in terminal enamel mineralization. Iron storage was drastically reduced by fluoride. Among the proteins involved in iron metabolism, ferritin heavy chain (Fth), in charge of iron storage, appeared as the preferential target of fluoride according to quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry analyses. Fluorotic enamel presented a decreased quantity of iron oxides attested by electron spin resonance technique, altered mechanical properties measured by nanoindentation, and ultrastructural defects analyzed by scanning electron microscopy and energy dispersive x-ray spectroscopy. The in vivo functional role of Fth was illustrated with mice, which incorporated less iron into their dental epithelium and exhibited poor enamel quality. These data demonstrate that exposure to excessive fluoride decreases ameloblast iron storage, which contributes to the defective structural and mechanical properties in rodent fluorotic enamel. They raise the question of fluoride’s effects on iron storage in other cells and organs that may contribute to its effects on population health.

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Dapeng Zhang, Yujiao Fan, Hui Chen, Sylvain Trépout, Min-Hui Li (2019 Jul 22)

CO2-activated reversible transition between polymersomes and micelles with AIE fluorescence.

Angewandte Chemie (International ed. in English) : 58 : 10260-10265 : DOI : 10.1002/anie.201905089 Learn more
Summary

Fluorescent polymersomes with both aggregation-induced emission (AIE) and CO2-responsive properties were developed from amphiphilic block copolymer PEG-b-P(DEAEMA-co-TPEMA) in which the hydrophobic block was a copolymer made of tetraphenylethene functionalized methacrylate (TPEMA) and 2-(diethylamino)ethyl methacrylate (DEAEMA) with unspecified sequence arrangement. Four block copolymers with different DEAEMA/TPEMA and hydrophilic/hydrophobic ratios were synthesized and bright AIE polymersomes were prepared by nanoprecipitation in THF/water and dioxane/water systems. Polymersomes of PEG45-b-P(DEAEMA36-co-TPEMA6) were chosen to study the CO2-responsive property. Upon CO2 bubbling vesicles transformed to small spherical micelles, and upon Ar bubbling micelles returned to vesicles with the presence of a few intermediate morphologies. These polymersomes might have promising applications as sensors, nanoreactors or controlled release systems.

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Trépout S. (2019 Jul 16)

Tomographic Collection of Block-Based Sparse STEM Images: Practical Implementation and Impact on the Quality of the 3D Reconstructed Volume

Materials : 12 : 2281 : DOI : 10.3390/ma12142281 Learn more
Summary

The reduction of the electron dose in electron tomography of biological samples is of high significance to diminish radiation damages. Simulations have shown that sparse data collection can perform efficient electron dose reduction. Frameworks based on compressive-sensing or inpainting algorithms have been proposed to accurately reconstruct missing information in sparse data. The present work proposes a practical implementation to perform tomographic collection of block-based sparse images in scanning transmission electron microscopy. The method has been applied on sections of chemically-fixed and resin-embedded Trypanosoma brucei cells. There are 3D reconstructions obtained from various amounts of downsampling, which are compared and eventually the limits of electron dose reduction using this method are explored.

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