Publications

2021

• Core–Shell Pure Collagen Threads Extruded from Highly Concentrated Solutions Promote Colonization and Differentiation of C3H10T1/2 Cells
  
  • Picaut Lise
  • Trichet Lea
  • Hélary Christophe
  • Ducourthial Guillaume
  • Bonnin Marie-Ange
  • Haye Bernard
  • Ronsin Olivier
  • Schanne-Klein Marie-Claire
  • Duprez Delphine
  • Baumberger Tristan
  • Mosser Gervaise
  ACS Biomaterials Science and Engineering, ACS, 2021. The elaboration of scaffolds able to efficiently promote cell differentiation toward a given cell type remains challenging. Here, we engineered dense type I collagen threads with the aim of providing scaffolds with specific morphological and mechanical properties for C3H10T1/2 mesenchymal stem cells. Extrusion of pure collagen solutions at different concentrations (15, 30, and 60 mg/mL) in a PBS 5× buffer generated dense fibrillated collagen threads. For the two highest concentrations, threads displayed a core-shell structure with a marked fibril orientation of the outer layer along the longitudinal axis of the threads. Young's modulus and ultimate tensile stress as high as 1 and 0.3 MPa, respectively, were obtained for the most concentrated collagen threads without addition of any cross-linkers. C3H10T1/2 cells oriented themselves with a mean angle of 15-24° with respect to the longitudinal axis of the threads. Cells penetrated the 30 mg/mL scaffolds but remained on the surface of the 60 mg/mL ones. After three weeks of culture, cells displayed strong expression of the tendon differentiation marker Tnmd, especially for the 30 mg/mL threads. These results suggest that both the morphological and mechanical characteristics of collagen threads are key factors in promoting C3H10T1/2 differentiation into tenocytes, offering promising levers to optimize tissue engineering scaffolds for tendon regeneration. (10.1021/acsbiomaterials.0c01273)
  DOI : 10.1021/acsbiomaterials.0c01273
• Probing living cells permeabilization dynamics by terahertz attenuated total reflection
  
  • Gallot Guilhem
  , 2021, pp.118270S. Using attenuated total reflection (ATR) in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of live epithelial cells by saponin detergent and after electropermeabilization. The origin of the contrast observed between cells and culture medium is addressed by both experimental and theoretical approaches, and gives access to permeabilization dynamics of live cells in real time. We show that terahertz modalities are more sensitive than fluorescence microscopy which is the reference optical technique for electropermeabilization. We propose analytical models for the influx and efflux of non-permeant molecules through permeabilized cell membranes. (10.1117/12.2596166)
  DOI : 10.1117/12.2596166
• SARS-CoV-2 Nsp3 unique domain SUD interacts with guanine quadruplexes and G4-ligands inhibit this interaction
  
  • Lavigne Marc
  • Helynck Olivier
  • Rigolet Pascal
  • Boudria-Souilah Rofia
  • Nowakowski Mireille
  • Baron Bruno
  • Brülé Sébastien
  • Hoos Sylviane
  • Raynal Bertrand
  • Guittat Lionel
  • Beauvineau Claire
  • Petres Stéphane
  • Granzhan Anton
  • Guillon Jean
  • Pratviel Geneviève
  • Teulade-Fichou Marie-Paule
  • England Patrick
  • Mergny Jean‐louis
  • Munier-Lehmann Hélène
  Nucleic Acids Research, Oxford University Press, 2021, 49 (13), pp.7695–7712. The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds. (10.1093/nar/gkab571)
  DOI : 10.1093/nar/gkab571
• Digital micromirror device for holographic and Fourier optics applications
  
  • Douet Brice
  • Tedoldi Téo
  • Kabacinski Adeline
  • Morana Ambra
  • Gallot Guilhem
  • Bouganne Raphaël
  Emergent Scientist, EDP Sciences, 2021, 5 (4), pp.1-7. The electromagnetic wavefront diffracted by an object carries information about the shape of the object from which the wave was emitted. Being able to record the phase and intensity of such a wave thus allows to reconstruct the object from the information carried by the wave, even if the object is no longer present. Among the reconstruction techniques, holography plays a big part. However the waves may experience a great variety of distortions on their way from the object to the measurement apparatus. Thus being able to shape the wavefront at will is key in holography. Micromirror light modulators are powerful tools for that matter and are well known for holographic applications. This paper explores the fundamental principles for digitally reconstructing a precise image of an object, but also for digitally correcting an imperfectly shaped wavefront, by exploiting the diffraction properties of light on a reflective surface. The methods presented here have been implemented as part of practical work for 2nd year students at the Ecole Polytechnique (last year of undergraduate program). (10.1051/emsci/2021003)
  DOI : 10.1051/emsci/2021003
• QM/MM with Auxiliary DFT
  
  • Samaniego-Rojas Juan D
  • Hernández-Segura Luis-Ignacio
  • López-Sosa Luis
  • Delgado-Venegas Rogelio I
  • Gomez Badhin
  • Lambry Jean-Christophe
  • de la Lande Aurelien
  • Mineva Tzonka
  • Alejandre José
  • Zúñiga-Gutiérrez Bernardo A
  • Flores-Moreno Roberto
  • Calaminici Patrizia
  • Geudtner Gerald
  • Köster Andreas M
  , 2021, 22, pp.1 - 54. This chapter describes the theoretical background of the quantum mechanical/molecular mechanical (QM/MM) implementation in deMon2k within the framework of auxiliary density functional theory (ADFT). It aims to give the reader an overview of the current state of the art of this QM/MM implementation and perspectives for its future development. To this end, we first derive the ADFT working equations for the QM and QM/MM energy and gradient expressions. Based on the joint QM/MM gradient expression, we present algorithms for QM/MM structure optimizations, transition-state searches and molecular dynamics simulations. The use of auxiliary density perturbation theory (ADPT) in the framework of QM/MM is discussed using illustrative implementations including analytic second-order ADFT energy derivatives, nuclear magnetic resonance chemical shift calculations and excited state calculations using time-dependent ADFT. The chapter closes with the description of a transformation program used to generate deMon2k QM/MM inputs. (10.1039/9781839164668-00001)
  DOI : 10.1039/9781839164668-00001
• L’imagerie optique de nanoparticules luminescentes : de la détection de biomolécules au diagnostic in vitro
  
  • Mousseau Fanny
  • Yu Chao
  • Alexandrou Antigoni
  • Bouzigues Cédric
  Photoniques, EDP Sciences, 2021 (106), pp.30-33. Pour détecter des biomolécules et pathogènes (protéines, virus, bactéries, …) avec des sensibilités satisfaisantes, il est actuellement nécessaire d’utiliser des appareils de laboratoires coûteux. En combinant les remarquables propriétés optiques des ions lanthanides à un lecteur simple couplé à un smartphone, nous démontrons comment développer un système de détection portable, rapide et ultrasensible. (10.1051/photon/202110630)
  DOI : 10.1051/photon/202110630
• Photochemical processes in flavo-enzymes as a probe for active site dynamics: TrmFO of Thermus thermophilus
  
  • Zhuang Bo
  • Nag Lipsa
  • Sournia Pierre
  • Croitoru Anastasia
  • Ramodiharilafy Rivo
  • Lambry Jean-Christophe
  • Myllykallio Hannu
  • Aleksandrov Alexey
  • Liebl Ursula
  • Vos Marten
  Photochemical & Photobiological Sciences, Springer, 2021, 20 (5), pp.663–670. Quenching of flavin fluorescence by electron transfer from neighboring aromatic residues is ubiquitous in flavoproteins. Apart from constituting a functional process in specific light-active systems, time-resolved spectral characterization of the process can more generally be employed as a probe for the active site configuration and dynamics. In the C51A variant of the bacterial RNA-transforming flavoenzyme TrmFO from the bacterium Thermus thermophilus, fluorescence is very short-lived (~ 1 ps), and close-by Tyr343 is known to act as the main quencher, as confirmed here by the very similar dynamics observed in protein variants with modified other potential quenchers, Trp283 and Trp214. When Tyr343 is modified to redox-inactive phenylalanine, slower and highly multiphasic kinetics are observed on the picosecond-nanosecond timescale, reflecting heterogeneous electron donor–acceptor configurations. We demonstrate that Trp214, which is located on a potentially functional flexible loop, contributes to electron donor quenching in this variant. Contrasting with observations in other nucleic acid-transforming enzymes, these kinetics are strikingly temperature-independent. This indicates (a) near-barrierless electron transfer reactions and (b) no exchange between different configurations on the timescale up to at least 2 ns, despite the presumed flexibility of Trp214. Results of extensive molecular dynamics simulations are presented to explain this unexpected finding in terms of slowly exchanging protein configurations (10.1007/s43630-021-00052-8)
  DOI : 10.1007/s43630-021-00052-8
• The catalytic properties of DNA G-quadruplexes rely on their structural integrity
  
  • Chen Jielin
  • Cheng Mingpan
  • Wang Jiawei
  • Qiu Dehui
  • Monchaud David
  • Mergny Jean‐louis
  • Ju Huangxian
  • Zhou Jun
  Chinese Journal of Catalysis, Elsevier, 2021, 42 (7), pp.1102-1107. The influence of the G-quartet structural integrity on the catalytic activity of the G-quadruplex (G4) was investigated by comparing the G4-DNAzyme performances of a series of G4s with a G-vacancy site and a G-triplex (G-tri). The results presented herein not only confirm that the structural integrity of the 3’-end G-quartet is necessary for G4s to be catalytically competent but also show how to remediate G-vacancy-mediated catalytic activity losses via the addition of guanine surrogates in an approach referred to as G-vacancy complementation strategy that is applicable to parallel G4s only. Furthermore, this study demonstrates that the terminal G-quartet could act as a proximal coordinating group and cooperate with the flanking nucleotide to activate the hemin cofactor. (10.1016/S1872-2067(20)63744-5)
  DOI : 10.1016/S1872-2067(20)63744-5
• Methylselenol Produced In Vivo from Methylseleninic Acid or Dimethyl Diselenide Induces Toxic Protein Aggregation in Saccharomyces cerevisiae
  
  • Dauplais Marc
  • Bierla Katarzyna
  • Maizeray Coralie
  • Lestini Roxane
  • Lobinski Ryszard
  • Plateau Pierre
  • Szpunar Joanna
  • Lazard Myriam
  International Journal of Molecular Sciences, MDPI, 2021, 22 (5), pp.2241. Methylselenol (MeSeH) has been suggested to be a critical metabolite for anticancer activity of selenium, although the mechanisms underlying its activity remain to be fully established. The aim of this study was to identify metabolic pathways of MeSeH in Saccharomyces cerevisiae to decipher the mechanism of its toxicity. We first investigated in vitro the formation of MeSeH from methylseleninic acid (MSeA) or dimethyldiselenide. Determination of the equilibrium and rate constants of the reactions between glutathione (GSH) and these MeSeH precursors indicates that in the conditions that prevail in vivo, GSH can reduce the major part of MSeA or dimethyldiselenide into MeSeH. MeSeH can also be enzymatically produced by glutathione reductase or thioredoxin/thioredoxin reductase. Studies on the toxicity of MeSeH precursors (MSeA, dimethyldiselenide or a mixture of MSeA and GSH) in S.cerevisiae revealed that cytotoxicity and selenomethionine content were severely reduced in a met17 mutant devoid of O-acetylhomoserine sulfhydrylase. This suggests conversion of MeSeH into selenomethionine by this enzyme. Protein aggregation was observed in wild-type but not in met17 cells. Altogether, our findings support the view that MeSeH is toxic in S. cerevisiae because it is metabolized into selenomethionine which, in turn, induces toxic protein aggregation. (10.3390/ijms22052241)
  DOI : 10.3390/ijms22052241
• Synthesis of acyclic nucleoside phosphonates targeting Flavin-Dependent Thymidylate Synthase in Mycobacterium tuberculosis
  
  • Biteau Nicolas G
  • Roy Vincent
  • Lambry J.-C.
  • Becker Hubert F
  • Myllykallio Hannu
  • Agrofoglio Luigi A
  Bioorganic and Medicinal Chemistry Letters, Elsevier, 2021, 46, pp.116351. Flavin-Dependent Thymidylate Synthase (FDTS) encoded by ThyX gene was discovered as a new class of thymidylate synthase involved in the de novo synthesis of dTMP named only in 30 % of human pathogenic bacteria. This target was pursed for the development of new antibacterial agents against multiresistant pathogens. We have developed a new class of ANPs based on the mimic of two natural’s cofactors (dUMP and FAD) as inhibitors against Mycobacterium tuberculosis ThyX. Several synthetic efforts were performed to optimize regioselective N1-alkylation, cross-coupling metathesis and Sonogashira cross-coupling. Compound 19c showed a poor 31.8% inhibitory effect on ThyX at 200 μM. (10.1016/j.bmc.2021.116351)
  DOI : 10.1016/j.bmc.2021.116351
• Simultaneous NAD(P)H and FAD fluorescence lifetime microscopy of long UVA–induced metabolic stress in reconstructed human skin
  
  • Ung Thi Phuong Lien
  • Lim Seongbin
  • Solinas Xavier
  • Mahou Pierre
  • Chessel Anatole
  • Marionnet Claire
  • Bornschlögl Thomas
  • Beaurepaire Emmanuel
  • Bernerd Françoise
  • Pena Ana-Maria
  • Stringari Chiara
  Scientific Reports, Nature Publishing Group, 2021, 11, pp.22171. Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P) + /NAD(P)H and FAD/FADH 2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring. (10.1038/s41598-021-00126-8)
  DOI : 10.1038/s41598-021-00126-8
• Objective assessment of corneal transparency in the clinical setting with standard SD-OCT devices
  
  • Vilbert Maëlle
  • Bocheux Romain
  • Georgeon Cristina
  • Borderie Vincent
  • Pernot Pascal
  • Irsch Kristina
  • Plamann Karsten
  , 2021. (10.48550/arXiv.2112.00528)
  DOI : 10.48550/arXiv.2112.00528
• FRET-MC: a fluorescence melting competition assay for studying G4 structures in vitro
  
  • Luo Yu
  • Granzhan Anton
  • Verga Daniela
  • Mergny Jean-Louis
  Biopolymers, Wiley, 2021, 112 (4), pp.e23415. G-quadruplexes (G4) play crucial roles in biology, analytical chemistry and nanotechnology. The stability of G4 structures is impacted by the number of G-quartets, the length and positions of loops, flanking motifs, as well as additional structural elements such as bulges, capping base pairs, or triads. Algorithms such as G4Hunter or Quadparser may predict if a given sequence is G4-prone by calculating a quadruplex propensity score; however, experimental validation is still required. We previously demonstrated that this validation is not always straightforward, and that a combination of techniques is often required to unambiguously establish whether a sequence forms a G-quadruplex or not. In this article, we adapted the well-known FRETmelting assay to characterize G4 in batch, where the sequence to be tested is added, as an unlabeled competitor, to a system composed of a dual-labeled probe (F21T) and a specific quadruplex ligand. PhenDC3 was preferred over TMPyP4 because of its better selectivity for G-quadruplexes. In this so-called FRET-MC (melting competition) assay, G4-forming competitors lead to a marked decrease of the ligand-induced stabilization effect (∆Tm), while non-specific competitors (e.g., single-or double-stranded sequences) have little effect. Sixtyfive known sequences with different typical secondary structures were used to validate the assay, which was subsequently employed to assess eight novel sequences that were not previously characterized. (10.1002/bip.23415)
  DOI : 10.1002/bip.23415