Publications

2020

• Advances in fast multiphoton microscopy using light-sheet illumination
  
  • Malkinson Guy
  • Maioli Vincent
  • Boniface Antoine
  • Mahou Pierre
  • Ortas Júlia Ferrer
  • Abdeladim Lamiae
  • Chaudan Élodie
  • Sonay Ali
  • Gacoin Thierry
  • Pantazis Periklis
  • Beaurepaire Emmanuel
  • Supatto Willy
  , 2020, pp.MW1A.2. (10.1364/MICROSCOPY.2020.MW1A.2)
  DOI : 10.1364/MICROSCOPY.2020.MW1A.2
• Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy
  
  • Malkinson Guy
  • Mahou Pierre
  • Chaudan Élodie
  • Gacoin Thierry
  • Sonay Ali
  • Pantazis Periklis
  • Beaurepaire Emmanuel
  • Supatto Willy
  ACS photonics, American Chemical Society, 2020, 7 (4), pp.1036-1049. Two-photon light-sheet microscopy (2P-SPIM) provides a unique combination of advantages for fast and deep fluorescence imaging in live tissues. Detecting coherent signals such as second-harmonic generation (SHG) in 2P-SPIM in addition to fluorescence would open further imaging opportunities. However, light-sheet microscopy involves an orthogonal configuration of illumination and detection that questions the ability to detect coherent signals. Indeed, coherent scattering from micron-sized structures occurs predominantly along the illumination beam. By contrast, point-like sources such as SHG nanocrystals can efficiently scatter light in multiple directions and be detected using the orthogonal geometry of a light-sheet microscope. This study investigates the suitability of SHG light-sheet microscopy (SHG-SPIM) for fast imaging of SHG nanoprobes. Parameters that govern the detection efficiency of KTiOPO 4 and BaTiO 3 nanocrystals using SHG-SPIM are investigated theoretically and experimentally. The effects of incident polarization, detection numerical aperture, nanocrystal rotational motion, and second-order susceptibility tensor symmetries on the detectability of SHG nanoprobes in this specific geometry are clarified. Guidelines for optimizing SHG-SPIM imaging are established, enabling fast in vivo light-sheet imaging combining SHG and two-photon excited fluorescence. Finally, microangiography was achieved in live zebrafish embryos by SHG imaging at up to 180 frames per second and single-particle tracking of SHG nanoprobes in the blood flow. (10.1021/acsphotonics.9b01749)
  DOI : 10.1021/acsphotonics.9b01749
• Fast In Vivo Imaging of SHG Nanoprobes with Multiphoton Light-Sheet Microscopy
  
  • Malkinson Guy
  • Mahou Pierre
  • Chaudan Élodie
  • Gacoin Thierry
  • Sonay Ali
  • Pantazis Periklis
  • Beaurepaire Emmanuel
  • Supatto Willy
  ACS photonics, American Chemical Society, 2020, 7 (4), pp.1036-1049. (10.1021/acsphotonics.9b01749)
  DOI : 10.1021/acsphotonics.9b01749
• Motile cilia and left-right symmetry breaking: from images to biological insights
  
  • Supatto Willy
  , 2020. (10.1109/ISBI45749.2020)
  DOI : 10.1109/ISBI45749.2020
• Native Collagen: Electrospinning of Pure, Cross-Linker-Free, Self-Supported Membrane
  
  • Dems Dounia
  • Rodrigues da Silva Julien
  • Hélary Christophe
  • Wien Frank
  • Marchand Marion
  • Debons Nicolas
  • Muller Laurent
  • Chen Yong
  • Schanne-Klein Marie-Claire
  • Laberty-Robert Christel
  • Krins Natacha
  • Aimé Carole
  ACS Applied Bio Materials, ACS Publications, 2020. Rebuilding biological environments is crucial when facing the challenges of fundamental and biomedical research. Thus, preserving the native state of biomolecules is essential. We use electrospinning (ES), which is an extremely promising method for the preparation of fibrillar membranes to mimic the ECM of native tissues. Here, we report for the first time (1) the ES of pure and native collagen into a self-supported membrane in absence of crosslinker and polymer support, (2) the preservation of the membrane integrity in hydrated media in absence of crosslinker and (3) the preservation of the native molecular structure and recovery of the hierarchical assembly of collagen. We use a multiscale approach to characterize collagen native structure at the molecular level using circular dichroism, and to investigate collagen hierarchical organization within the self-supported membrane using a combination of multiphoton and electron microscopies. Finally, we show that the membranes are perfectly suited for cell adhesion and spreading, making them very promising candidates for the development of biomaterials and finding applications in biomedical research. (10.1021/acsabm.0c00006)
  DOI : 10.1021/acsabm.0c00006
• In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts
  
  • Crépin Alexandre
  • Jégou Jean-Francois
  • André Sonia
  • Ecale Florine
  • Croitoru Anastasia
  • Cantereau Anne
  • Berjeaud Jean-Marc
  • Ladram Ali
  • Verdon Julien
  Scientific Reports, Nature Publishing Group, 2020, 10 (1), pp.3978. Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K3]SHa, has been designed to improve those activities. SHa and [K3]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K3]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K3]SHa and exhibited low half maximal lethal concentrations (LC50). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments. (10.1038/s41598-020-60829-2)
  DOI : 10.1038/s41598-020-60829-2
• Terahertz sensing in biology and medicine
  
  • Gallot Guilhem
  Photoniques, EDP Sciences, 2020 (101), pp.53-58. Terahertz radiation offers new contrasts with biological systems, without markers or staining, at the molecular, cellular or tissue level. Thanks to technological advances, it is increasingly emerging as a solution of choice for directly probing the interaction with molecules and biological solutions. Applications range from dynamics of biological molecules to imaging of cancerous tissues, including ion, protein and membrane sensors. (10.1051/photon/202010153)
  DOI : 10.1051/photon/202010153
• Microscopie par génération de troisième harmonique résolue en polarisation pour l'imagerie de la myéline et des biocristaux
  
  • Morizet Joséphine
  , 2020. Cette thèse s’intéresse à l'imagerie multiphotonique par génération de troisième harmonique (THG) combinée à des mesures résolues en polarisation (PTHG) sur des systèmes biologiques pour accéder à des informations complémentaires sur la morphologie et la microstructure de structures anisotropes. Une première application vise à extraire des informations de la gaine de myéline qui est la gaine lipidique entourant les axones du système nerveux central (CNS) des vertébrés, et dont la destruction dans des pathologies comme la sclérose en plaques est a l’origine de perturbations dans la propagation des influx nerveux. Une seconde application concerne les biocristaux, dont nous traiterons ici l’exemple de l’otolithe situe dans l’oreille interne du poisson-zèbre. Grâce à sa forte sensibilité aux discontinuités des propriétés optiques et à l’anisotropie des matériaux, la microscopie THG apparaît comme un outil de choix pour visualiser ces deux structures biologiques non-marquées avec une résolution submicrométrique.Dans ce manuscrit, nous présentons d’abord un travail fondamental de caractérisation analytique, numérique (modèle FDTD) et expérimentale de la réponse PTHG sur des structures modèles. Nous avons notamment mis en évidence la présence d'aberrations de la réponse PTHG au voisinage d’interfaces parallèles à l’axe de propagation du faisceau d’excitation induites par des désaccords d’indices. L'évaluation de la modulation artefactuelle induite par ces aberrations a permis d’interpréter la composante de modulation PTHG sur ces interfaces associée à l’ordre moléculaire dans des structures lipidiques modèles. Ensuite, nous décrivons la mise en place d’un dispositif d’acquisition PTHG rapide dédié à l’imagerie in vivo de structures biologiques. Nous présentons de nouvelles applications biologiques de la microscopie PTHG en utilisant une méthode d’analyse des signaux THG polarimétriques par transformée de Fourier. Enfin, nous présentons une étude systématique de caractérisation des contrastes THG/PTHG sur les fibres myélinisées du CNS de poissons-zèbres et de souris.
• Ultrafast Dynamics and Vibrational Relaxation in Six-Coordinate Heme Proteins Revealed by Femtosecond Stimulated Raman Spectroscopy
  
  • Ferrante Carino
  • Batignani Giovanni
  • Pontecorvo Emanuele
  • Montemiglio Linda
  • Vos Marten H.
  • Scopigno Tullio
  Journal of the American Chemical Society, American Chemical Society, 2020, 142 (5), pp.2285-2292. Identifying the structural rearrangement and the active sites in photo-induced reactions is a fundamental challenge to understand from a microscopic perspective the underlying dynamics ruling the functional mechanisms of heme proteins. Here, femtosecond stimulated Raman spectroscopy is used to follow the ultrafast evolution of two 1 six-coordinate heme proteins. By exploiting the sensitivity of Raman spectra to the structural conguration, we investigate the eects of photolysis and binding of amino acid residues in cytochrome c and neuroglobin. Comparing the system response for different time delays and Raman pump resonances, we show how details of atomic motions and energy redistribution can be unveiled. (10.1021/jacs.9b10560)
  DOI : 10.1021/jacs.9b10560
• Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy
  
  • Karadi Kristof
  • Kapetanaki Sofia
  • Raics Katalin
  • Pecsi Ildiko
  • Kapronczai Robert
  • Fekete Zsuzsanna
  • Iuliano James
  • Collado Jinnette Tolentino
  • Gil Agnieszka
  • Orban Jozsef
  • Greetham Greg
  • Vos Marten H.
  • Tonge Peter
  • Meech Stephen
  • Lukacs Andras
  Scientific Reports, Nature Publishing Group, 2020, 10, pp.2061. Blue Light Using flavin (BLUf) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light-and darkadapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule. (10.1038/s41598-020-59073-5)
  DOI : 10.1038/s41598-020-59073-5
• Soluble Guanylate Cyclase Inhibitors Discovered among Natural Compounds
  
  • Petrova Olga
  • Lamarre Isabelle
  • Fasani Fabienne
  • Grillon Catherine
  • Negrerie Michel
  Journal of Natural Products, American Chemical Society, 2020, 83 (12), pp.3642-3651. Soluble guanylate cyclase (sGC) is the human receptor of nitric oxide (NO) in numerous kinds of cells and produces the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) upon NO binding to its heme. sGC is involved in many cell signaling pathways both under healthy conditions and under pathological conditions, such as angiogenesis associated with tumor growth. Addressing the selective inhibition of the NO/cGMP pathway is a strategy worthwhile to be investigated for slowing down tumoral angiogenesis or for curing vasoplegia. However, sGC inhibitors are lacking investigation. We have explored a chemical library of various natural compounds and have discovered inhibitors of sGC. The selected compounds were evaluated for their inhibition of purified sGC in vitro and sGC in endothelial cells. Six natural compounds, from various organisms, have IC50 in the range 0.2-1.5 μM for inhibiting the NO-activated synthesis of cGMP by sGC, and selected compounds exhibit a quantified antiangiogenic activity using an endothelial cell line. These sGC inhibitors can be used directly as tools to investigate angiogenesis and cell signaling or as templates for drug design. (10.1021/acs.jnatprod.0c00854)
  DOI : 10.1021/acs.jnatprod.0c00854
• Full-field optical coherence tomography—An educational setup for an undergraduate lab
  
  • Pieper Kai
  • Latour Gaël
  • Küchenmeister Jens
  • Bergmann Antje
  • Dengler Roman
  • Rockstuhl Carsten
  American Journal of Physics, American Association of Physics Teachers, 2020, 88 (12), pp.1132-1139. Optical coherence tomography, or in short OCT, is a measurement technique established in the early 1990s for the non-invasive imaging of interfaces in the bulk of biological tissues or other samples. A full-field OCT setup is built from a microscope combined with a Michelson interferometer, where the mirror in one arm is replaced by the sample. Using white light, which is temporally partially coherent, interference fringes disclose the presence of an interface whenever the lengths of both interferometer arms are nearly equal. Scanning one arm allows for a volumetric reconstruction of all interfaces inside the sample. While the importance of OCT in medicine is indisputable, it is hard to teach students the basic aspects of such technology as most available setups tend to be rather complex. It is our purpose to present a fully functional full-field OCT setup that is stripped-down to its essential components and to promote its use in an undergraduate lab course. The contribution is complemented by a description of the basic theory necessary to understand the working principle of OCT. (10.1119/10.0001755)
  DOI : 10.1119/10.0001755
• Differentiation of neural-type cells on multi-scale ordered collagen-silica bionanocomposites
  
  • Debons Nicolas
  • Dems Dounia
  • Hélary Christophe
  • Le Grill Sylvain
  • Picaut Lise
  • Renaud Flore
  • Delsuc Nicolas
  • Schanne-Klein Marie-Claire
  • Coradin Thibaud
  • Aimé Carole
  Biomaterials Science, Royal Society of Chemistry (RSC), 2020. Cells respond to biophysical and biochemical signals. We developed a composite filament from collagen and silica particles modified to interact with collagen and/or present a laminin epitope (IKVAV) crucial for cell–matrix adhesion and signal transduction. This combines scaffolding and signaling and shows that local tuning of collagen organization enhances cell differentiation. (10.1039/C9BM01029G)
  DOI : 10.1039/C9BM01029G
• A Push-Pull Mechanism Helps Design Highly Competent G-Quadruplex-DNA Catalysts
  
  • Chen Jielin
  • Wang Jiawei
  • van Der Lubbe Stephanie
  • Cheng Mingpan
  • Qiu Dehui
  • Monchaud David
  • Mergny Jean‐louis
  • Guerra Célia Fonseca
  • Ju Huangxian
  • Zhou Jun
  CCS Chemistry, Chinese Chemical Society, 2020, 2, pp.2183 - 2193. Massive efforts are currently being invested to improve the performance, versatility, and scope of applications of nucleic acid catalysts. G-quadruplex (G4)/hemin DNAzymes are of particular interest owing to their structural programmability and chemical robustness. However, optimized catalytic efficiency is still bottleneck and the activation mechanism is unclear. Herein, we have designed a series of parallel G4s with different proximal cytosine (dC) derivatives to fine-tune the hemin-binding pocket for G4-DNAzymes. Combining theoretical and experimental methods, we have assessed the dependence of catalytic enhancement on the electronic properties of proximal dCs and demonstrated how proximal dCs activate catalytic proficiency. These results provide interesting clues in recapitulating the push–pull mechanism as the basis of peroxidase activity and help to devise a new strategy to design highly competent DNA catalysts whose performances are of the same order as protease. (10.31635/ccschem.020.202000473)
  DOI : 10.31635/ccschem.020.202000473
• Introduction to Femtochemistry: Excited-State Proton Transfer from Pyranine to Water Studied by Femtosecond Transient Absorption
  
  • Changenet Pascale
  • Gustavsson Thomas
  • Lampre Isabelle
  Journal of Chemical Education, American Chemical Society, Division of Chemical Education, 2020, 97 (12), pp.4482–4489. In order to introduce students to the fascinating field of femtochemistry, we propose here a practical laboratory training course conceived for second-year master's students in chemistry. We describe the use of a broadband femtosecond transient absorption (pump−probe) experiment for monitoring a fast light-triggered chemical reaction in solution. The experiments are performed on the pyranine photoacid, which upon photo-excitation at 390 nm undergoes a proton transfer to the solvent in about 90 ps. While this practical course involves advanced equipment and techniques, the measured transient absorption data allow easy analysis and interpretation. The transient absorption spectra at a few selected delay times can be analyzed qualitatively in terms of bleach, induced absorption, and stimulated emission. Likewise, the transient absorption signals at a few chosen wavelengths can be quantitatively analyzed and explained with simple kinetic models to determine the time constant of the proton-transfer reaction. This training aims at giving the students the opportunity to face some of the current challenges in contemporary chemistry by learning the basics of ultrafast spectroscopy. (10.1021/acs.jchemed.0c01056)
  DOI : 10.1021/acs.jchemed.0c01056