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

Unit publications

Year of publication 2018

Luis M.G. Abegão, Ruben D Fonseca, Tárcius N Ramos, Florence Mahuteau-Betzer, Sandrine PIGUEL, José J. Rodrigues Jr, Cleber R. Mendonca, Sylvio Canuto, Daniel Luis Silva, and Leonardo De Boni (2018 Apr 12)

Oxazole dyes with potential for photoluminescence bioprobes: A two-Photon absorption study

The Journal of Physical Chemistry C : 122 : 10526-10534 : DOI : 10.1021/acs.jpcc.8b01904 Learn more
Summary

In this work, six π-conjugated oxazole compounds dissolved in dichloromethane (DCM) were characterized with linear and nonlinear optical measurements. Z-Scan with femtosecond laser pulses was employed to determine the two-photon absorption (TPA) spectra. Other photophysical parameters, such as: absorbance, solvatochromism, lifetime fluorescence and fluorescence anisotropy were evaluated with linear optical techniques. The experimental TPA cross-section spectra were adjusted by Sum-Over-States (SOS) model, in which important parameters such as transition dipole moments and broadening parameters were determined. In order to better understand the TPA spectra of the oxazole compounds, quantum-chemical calculations using the response function formalism and the DFT level of theory were performed. Using the results provided by the quantum-chemical calculations and the broadening parameters estimated through the application of the SOS model, the TPA spectra were simulated by the superposition (summation) of individual homogeneous Lorentzian absorption profiles.

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Nian Zhang, Hui Chen, Yujiao Fan, Lu Zhou, Sylvain Trépout, Jia Guo, Min-Hui Li (2018 Apr 5)

Fluorescent Polymersomes with Aggregation-Induced Emission.

ACS nano : 12 : 4025-4035 : DOI : 10.1021/acsnano.8b01755 Learn more
Summary

Fluorescent polymersomes are interesting systems for cell/tissue imaging and in vivo study of drug distribution and delivery. We report on bright fluorescent polymersomes with aggregation-induced emission self-assembled by a series of tetraphenylethylene (TPE)-containing amphiphilic biodegradable block copolymers, where the hydrophilic block is a polyethylene glycol and hydrophobic block is a TPE-substituted trimethylenecarbonate polymer P(TPE-TMC). Their self-assemblies in water were prepared by nanoprecipitation using dioxane or tetrahydrofuran as co-solvent, and the self-assembling processes were studied in detail by cryo-electron microscopy, dynamic light scattering, and spectrofluorometer. The polymersomes are formed via the closure of bilayer lamellae self-assembled first by amphiphilic block copolymers. The polymersome membrane affords a nanosize bright fluorescent system with self-assembly induced emission in the thickness scale of 10-15 nm. The control of the whole size of polymersome is achieved by the choice of co-solvent for self-assembling and by the design of a suitable hydrophilic/hydrophobic ratio of block copolymers. These polymersomes can be potentially used as a stable fluorescent tool to monitor the transportation and distribution of drugs and bioconjugates in living cells.

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Trépout S., Tassin A.M., Marco S., Bastin P. (2018 Apr 1)

STEM tomography analysis of the trypanosome transition zone

Journal of Structural Biology : 202 : 51-60 : DOI : 10.1016/j.jsb.2017.12.005 Learn more
Summary

The protist Trypanosoma brucei is an emerging model for the study of cilia and flagella. Here, we used scanning transmission electron microscopy (STEM) tomography to describe the structure of the trypanosome transition zone (TZ). At the base of the TZ, nine transition fibres irradiate from the B microtubule of each doublet towards the membrane. The TZ adopts a 9+0 structure throughout its length of ∼300 nm and its lumen contains an electron-dense structure. The proximal portion of the TZ has an invariant length of 150 nm and is characterised by a collarette surrounding the membrane and the presence of electron-dense material between the membrane and the doublets. The distal portion exhibits more length variation (from 55 to 235 nm) and contains typical Y-links. STEM analysis revealed a more complex organisation of the Y-links compared to what was reported by conventional transmission electron microscopy. Observation of the very early phase of flagellum assembly demonstrated that the proximal portion and the collarette are assembled early during construction. The presence of the flagella connector that maintains the tip of the new flagellum to the side of the old was confirmed and additional filamentous structures making contact with the membrane of the flagellar pocket were also detected. The structure and potential functions of the TZ in trypanosomes are discussed, as well as its mode of assembly.

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Soumia Sid Ahmed, Zoubeida Messali, Florent Poyer, Livia Lumbroso-Le Rouic, Laurence Desjardins, Nathalie Cassoux, Carole D. Thomas, Sergio Marco, Stéphanie Lemaitre (2018 Mar 28)

Iterative Variance Stabilizing Transformation Denoising of Spectral Domain Optical Coherence Tomography Images Applied to Retinoblastoma.

Ophthalmic research : 59 : 164-169 : DOI : 10.1159/000486283 Learn more
Summary

BACKGROUND: Due to the presence of speckle Poisson noise, the interpretation of spectral domain-optical coherence tomography (SD-OCT) images frequently requires the use of data averaging to improve the signal-to-noise ratio. This implies long acquisition times and requires patient sedation in some cases. Iterative variance stabilizing transformation (VST) is a possible approach by which to remove speckle Poisson noise on single images.

METHODS: We used SD-OCT images of human and murine (LH Beta-Tag mouse model) retinas with and without retinoblastoma acquired with 2 different imaging devices (Bioptigen and Micron IV). These images were processed using a denoising workflow implemented in Matlab.

RESULTS: We demonstrated the presence of speckle Poisson noise, which can be removed by a VST-based approach. This approach is robust as it works in all used imaging devices and in both human and mouse retinas, independently of the tumor status. The implemented algorithm is freely available from the authors on demand.

CONCLUSIONS: On a single denoised image, the proposed method provides results similar to those expected from the SD-OCT averaging. Because of the friendly user interface, it can be easily used by clinicians and researchers in ophthalmology.

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Alexandra Garancher, Charles Y. Lin, Morgane Morabito, Wilfrid Richer, Nathalie Rocques, Magalie Larcher, Laure Bihannic, Kyle Smith, Catherine Miquel, Sophie Leboucher, Nirmitha I. Herath, Fanny Dupuy, Pascale Varlet, Christine Haberler, Christine Walczak, Nadine El Tayara, Andreas Volk, Stéphanie Puget, François Doz, Olivier Delattre, Sabine Druillennec, Olivier Ayrault, Robert J. Wechsler-Reya, Alain Eychène, Franck Bourdeaut, Paul A. Northcott, Celio Pouponnot (2018 Mar 12)

NRL and CRX Define Photoreceptor Identity and Reveal Subgroup-Specific Dependencies in Medulloblastoma

Cancer Cell : 33 : 435-449 : DOI : 10.1016/j.ccell.2018.02.006 Learn more
Summary

Cancer cells often express differentiation programs unrelated to their tissue of origin, although the contribution of these aberrant phenotypes to malignancy is poorly understood. An aggressive subgroup of medulloblastoma, a malignant pediatric brain tumor of the cerebellum, expresses a photoreceptor differentiation program normally expressed in the retina. We establish that two photoreceptor-specific transcription factors, NRL and CRX, are master regulators of this program and are required for tumor maintenance in this subgroup. Beyond photoreceptor lineage genes, we identify BCL-XL as a key transcriptional target of NRL and provide evidence substantiating anti-BCL therapy as a rational treatment opportunity for select MB patients. Our results highlight the utility of studying aberrant differentiation programs in cancer and their potential as selective therapeutic vulnerabilities.

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David Partouche, Antoine Malabirade, Thomas Bizien, Marisela Velez, Sylvain Trépout, Sergio Marco, Valeria Militello, Christophe Sandt, Frank Wien & Véronique Arluison (2018 Feb 27)

Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

Bacterial Regulatory RNA. Methods in Molecular Biology : 1737 : 321-340 : DOI : 10.1007/978-1-4939-7634-8_18 Learn more
Summary

Post-transcriptional control of gene expression by small regulatory noncoding RNA (sRNA) needs protein accomplices to occur. Past research mainly focused on the RNA chaperone Hfq as cofactor. Nevertheless, recent studies indicated that other proteins might be involved in sRNA-based regulations. As some of these proteins have been shown to self-assemble, we describe in this chapter protocols to analyze the nano-assemblies formed. Precisely, we focus our analysis on Escherichia coli Hfq as a model, but the protocols presented here can be applied to analyze any polymer of proteins. This chapter thus provides a guideline to develop commonly used approaches to detect prokaryotic protein self-assembly, with a special focus on the detection of amyloidogenic polymers.

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Ludivine Guyon, Marc Pirrotta, Katerina Duskova, Anton Granzhan, Marie-Paule Teulade-Fichou, David Monchaud (2018 Feb 16)

TWJ-Screen: an isothermal screening assay to assess ligand/DNA junction interactions in vitro

Nucleic Acids Research : 46 : e16 : DOI : 10.1093/nar/gkx1118 Learn more
Summary

The quest for chemicals able to operate at selected genomic loci in a spatiotemporally controlled manner is desirable to create manageable DNA damages. Mounting evidence now shows that alternative DNA structures, including G-quadruplexes and branched DNA (or DNA junctions), might hamper proper progression of replication fork, thus triggering DNA damages and genomic instability. Therefore, small molecules that stabilize these DNA structures are currently scrutinized as a promising way to create genomic defects that cannot be dealt with properly by cancer cells. While much emphasis has been recently given to G-quadruplexes and related ligands, we report herein on three-way DNA junctions (TWJ) and related ligands. We first highlight the biological implications of TWJ and their strategic relevance as triggers for replicative stress. Then, we describe a new in vitro high-throughput screening assay, TWJ-Screen, which allows for identifying TWJ ligands with both high affinity and selectivity for TWJ over other DNA structures (duplexes and quadruplexes), in a convenient and unbiased manner as demonstrated by the screening of a library of 25 compounds from different chemical families. TWJ-Screen thus represents a reliable mean to uncover molecular tools able to foster replicative stress through an innovative approach, thus providing new strategic opportunities to combat cancers.

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Chen, Su; Poyer, Florent; Garcia, Guillaume; Fiorini-Debuisschert, Céline; Rosilio, Véronique and Maillard, Philippe (2018 Feb 14)

Amphiphilic Glycoconjugated Porphyrin Heterodimers as Two-Photon Excitable Photosensitizers: Design, Synthesis, Photophysical and Photobiological Studies

ChemistrySelect : 3 : 1887-1897 : DOI : 10.1002/slct.201703013 Learn more
Summary

A new family of amphiphilic glycoconjugated porphyrin dimers usable in two-photon photodynamic therapy was designed, prepared and characterized. The physicochemical and biological data were presented and discussed. Their two-photon absorption cross section was determined, revealing quite good performances of the compounds (δ ≈ 300 GM at 880 nm for 2 and 3 and δ ≈ 1000 GM at 840 nm for 5 and 6). Despite their promising photophysical properties, porphyrin dimers are penalized by their low solubility and an insufficient penetration into tumour cells. Incorporation into nanocarriers such as lipid vesicles could be profitable, provided that the positioning and degree of freedom of the saccharide moieties are optimal to target the receptors of the cells of interest.

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Marius Mamone, Jessy Aziz, Julie Le Bescont, Sandrine Piguel (2018 Jan 18)

Aminocarbonylation of N-Containing Heterocycles with Aromatic Amines Using Mo(CO)6

Synthesis : 50 : 1521-1526 : DOI : 10.1055/s-0037-1609152 Learn more
Summary

We describe herein the palladium-catalyzed aminocarbonylation of nitrogen-containing heterocycles with aniline derivatives using molybdenum hexacarbonyl as a CO solid source, expanding the scope of the limited examples. This method is compatible with a variety of substitutions on the aniline moiety. The simple reaction conditions include easily available Pd(dppf)Cl2 catalyst, DBU as base in DMF at 120 °C for 3 hours in sealed tube thereby leading to the isolation of 21 compounds with yields ranging from 18 to 82%. We also show that double aminocarbonylation reactions are possible in satisfactory yields regarding both coupling partners.

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Ehlen A., Martin C., Miron S., Julien M., Theillet F.X., Boucherit V., Ropars V., Duchambon P., El Marjou A., Zinn Justin S., Carreira A. (2018 Jan 1)

Proper chromosome alignment depends on BRCA2 phosphorylation by PLK1

bioRxiv : Preprint : DOI : 10.1101/265934 Learn more
Summary

The BRCA2 tumor suppressor protein is involved in the maintenance of genome integrity through its role in homologous recombination in S/G2 phases of the cell cycle. A much less established function of BRCA2 takes place in mitosis where it interacts with the SAC component BUBR1 and is involved in cytokinesis by interaction with midbody components. Also in mitosis, BRCA2 is phosphorylated by the cell cycle regulator Polo-like kinase 1 (PLK1), so we asked whether this phosphorylation would have a role in the control of mitosis. Here we combined biophysical, biochemical and cell biology approaches to characterize the phenotype of BRCA2 variants that alter PLK1 phosphorylation. We identified T207 in BRCA2 as a bona fide docking site for PLK1. The 3D structure of the BRCA2 peptide bound to PLK1 Polo-box domain exhibits all the characteristics of an optimal and specific phosphopeptide-PLK1 interface. We show that this interaction is required for the phosphorylation of BUBR1 and pBUBR1 binding to the phosphatase PP2A-B56α, both critical for the establishment of proper kinetochore-microtubules attachments. Precluding T207 binding to PLK1 as observed in BRCA2 missense variants identified in breast cancer results in reduced phosphorylation of BUBR1 at PLK1-dependent sites and decreases the interaction of BUBR1 with the phosphatase PP2A-B56α. This leads to unaligned chromosomes, faulty chromosome segregation and aneuploidy. We thus reveal a direct mitotic role of BRCA2 in the alignment of chromosomes, distinct from its DNA repair function, with important consequences on chromosome stability. These findings may explain in part the aneuploidy observed in BRCA2-deficient tumors.

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Hammerer F., Poyer F., Fourmois L., Chen S., Garcia G., Teulade-Fichou M.P., Maillard P., Mahuteau-Betzer F. (2018 Jan 1)

Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy

Bioorganic & Medicinal Chemistry : 26 : 107-118 : DOI : 10.1016/j.bmc.2017.11.024 Learn more
Summary

The proof of concept for two-photon activated photodynamic therapy has already been achieved for cancer treatment but the efficiency of this approach still heavily relies on the availability of photosensitizers combining high two-photon absorption and biocompatibility. In this line we recently reported on a series of porphyrin-triphenylamine hybrids which exhibit high singlet oxygen production quantum yield as well as high two-photon absorption cross-sections but with a very poor cellular internalization. We present herein new photosensitizers of the same porphyrin-triphenylamine hybrid series but bearing cationic charges which led to strongly enhanced water solubility and thus cellular penetration. In addition the new compounds have been found localized in mitochondria that are preferential target organelles for photodynamic therapy. Altogether the strongly improved properties of the new series combined with their specific mitochondrial localization lead to a significantly enhanced two-photon activated photodynamic therapy efficiency.

Mitochondria-targeted cationic porphyrin-triphenylamine hybrids for enhanced two-photon photodynamic therapy

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Verguet A., Messaoudi C., Sorzano C.O.S., Marco S. (2018 Jan 1)

Alignment of Tilt Series

Cellular Imaging : 183-207 : DOI : 10.1007/978-3-319-68997-5 Learn more
Summary

Computing of three-dimensional reconstructions from images obtained by transmission electron tomography needs three main steps: data acquisition, projection alignment, and 3D reconstruction. In this chapter we will focus on the process of alignment moving from the justification of its need to the study of the different classical approaches (cross-correlation, use of added fiducial markers) that have been commonly used in this alignment process. We will also discuss the most recent algorithms (multiscale registration, invariant feature recognition) as they have been adapted to Electron Tomography and improved to increase the accuracy and resolution of the final tomograms.

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Year of publication 2017

Slodzian G., Wu T.D., Duprat J., Engrand C., Guerquin-Kern J.L. (2017 Dec 1)

Dynamic transfer applied to secondary ion imaging over large scanned fields with the nanoSIMS 50 at high mass resolution

Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms : 412 : 123-173 : DOI : 10.1016/j.nimb.2017.06.019 Learn more
Summary

Dynamic transfer is an adaptive optical approach used for coupling a scanning ion probe with the mass spectrometer designed for analyzing sputtered ions emanating from the probe impact. Its tuning is of crucial importance for getting uniform signal collection over large scanning fields and therefore scanning images free of vignetting in a context of high mass resolution. Revisiting the optical design of the NanoSIMS 50 instrument, where the same set of lenses focuses the primary ion probe on the sample and collects secondary ions from the sample, led us to develop novel experimental procedures to achieve dynamic transfer tuning and overcome instrumental imperfections. It is the case for scanning distortion that may be induced by the octopole used for correcting probe astigmatism and may cause irreducible vignetting on scanning images. We show that it is possible to develop complete tuning procedures by compromising temporarily on the sharpness of the probe focus. Most importantly, we show that, in a context of high mass resolution, the transfer does not significantly disturb isotopic ratios over large scanned fields provided external coils are properly adjusted to compensate ambient magnetic fields.

Deepening the procedures led us to demonstrate that the scanning center of the probe may not coincide with the imaging center of COOL, Coaxial Objective Lenses forming the probe and extracting secondary ions. We have checked that bringing those two centers into coincidence resulted in a better image quality over large fields.

In the present work, we show how to handle the secondary beam in order to position it before it enters the spectrometer. That capability is essential for optimizing transmission at high mass resolution by aligning the secondary beam axis on a given entrance axis of the spectrometer.

These results led us to propose several instrumental improvements including the crucial interest of an additional octopole upstream in the primary ion probe column to prevent scanning distortion when performing astigmatism correction and the possibility of offsetting primary beam deviating plates to bring scanning and imaging centers in coincidence.

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Chiara Mauriello-Jimenez, Maxime Henry, Dina Aggad, Laurence Raehm, Xavier Cattoën, Michel Wong Chi Man, Clarence Charnay, Serkan Alpugan, Vefa Ahsen, Deniz Kutlu Tarakci, Philippe Maillard, Marie Maynadier, Marcel Garcia, Fabienne Dumoulin, Magali Gary-Bobo, Jean-Luc Coll, Véronique Josserand, Jean-Olivier Durand (2017 Oct 31)

Porphyrin- or phthalocyanine-bridged silsesquioxane nanoparticles for two-photon photodynamic therapy or photoacoustic imaging.

Nanoscale : 9 : 16622-16626 : DOI : 10.1039/c7nr04677d Learn more
Summary

Porphyrin- or phthalocyanine-bridged silsesquioxane nanoparticles (BSPOR and BSPHT) were prepared. Their endocytosis in MCF-7 cancer cells was shown with two-photon excited fluorescence (TPEF) imaging. With two-photon excited photodynamic therapy (TPE-PDT), BSPOR was more phototoxic than BSPHT, which in contrast displayed a very high signal for photoacoustic imaging in mice.

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Michelle Newman, Rym Sfaxi, Abhijit Saha, David Monchaud, Marie-Paule Teulade-Fichou, Stéphan Vagner (2017 Oct 27)

The G-Quadruplex-Specific RNA Helicase DHX36 Regulates p53 Pre-mRNA 3′-End Processing Following UV-Induced DNA Damage.

Journal of Molecular Biology : 429 : 3121-3131 : DOI : 10.1016/j.jmb.2016.11.033 Learn more
Summary

Pre-mRNA 3′-end processing, the process through which almost all eukaryotic mRNAs acquire a poly(A) tail is generally inhibited during the cellular DNA damage response leading to a profound impact on the level of protein expression since unprocessed transcripts at the 3′-end will be degraded or unable to be transported to the cytoplasm. However, a compensatory mechanism involving the binding of the hnRNP H/F family of RNA binding proteins to an RNA G-quadruplex (G4) structure located in the vicinity of a polyadenylation site has previously been described to allow the transcript encoding the p53 tumour suppressor protein to be properly processed during DNA damage and to provide the cells with a way to react to DNA damage. Here we report that the DEAH (Asp-Glu-Ala-His) box RNA helicase DHX36/RHAU/G4R1, which specifically binds to and resolves parallel-stranded G4, is necessary to maintain p53 pre-mRNA 3′-end processing following UV-induced DNA damage. DHX36 binds to the p53 RNA G4, while mutation of the G4 impairs the ability of DHX36 to maintain pre-mRNA 3′-end processing. Stabilization of the p53 RNA G4 with two different G4 ligands ((PNA)DOTASQ and PhenDC3), which is expected from previous studies to prevent DHX36 from binding and unwinding G4s, also impairs p53 pre-mRNA 3′-end processing following UV. Our work identifies DHX36 as a new actor in the compensatory mechanisms that are in place to ensure that the mRNAs encoding p53 are still processed following UV.

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