Publications

2017

• Regulation, activation, and deactivation of soluble guanylate cyclase and NO-sensors
  
  • Petrova Olga
  , 2017. This thesis is devoted to the regulation of soluble guanylate cyclase (sGC), the endogenous nitric oxide (NO) receptor in mammals involved in signal transduction. The enzyme is activated by the binding of NO to its heme and catalyzes the formation of cGMP from GTP. While sGC is present in many mammalian cells, the homologous bacterial domain (H-NOX) is involved in NO detection and metabolism regulation. An important objective was to find sGC inhibitors to slow down tumor progression.The screening of natural compounds from a chemical library, tested on purified sGC activity, revealed six active inhibitors (Ki = 0.2 – 1 µM). Together with two agents for photodynamic therapy (hypericin and hypocrellin) we demonstrated that these inhibitors are allosteric modulators which bind neither to the heme nor to the catalytic and activator sites, revealing a new class of pharmacological compounds targetting the NO/cGMP signaling pathway.The structural transition induced in sGC by stimulator riociguat in synergy with CO was studied by transient absorption spectroscopy to demonstrate coordination changes of the heme. Two different activation states of sGC with CO 6c-heme and 5c-heme exist simultaneously in the presence of the stimulator which induces the breaking of the heme Fe-His bond, as does the sGC natural effector NO. In addition, the effect of isoliquiritigenin, which is sold as a sGC activator, was shown to be actually an inhibitor of sGC.The dynamics of the ligands CO, NO and O2 were measured over 12 orders of magnitude in time in wild type and mutant of a bacterial NO transporter (AXCP). The single mutation Leu16Ala increased 108-fold the CO affinity, ~106-fold the NO affinity and makes this protein reactive to O2. In the case of CO and NO, whose affinities for L16A-AXCP are the largest ever measured, the bimolecular rebinding was absolutely not detectable. Molecular dynamic simulations demonstrated that dissociated CO is constrained to stay within 4 Å from Fe2+ by Ala16, contrarily to wild-type Leu16.The dynamics of O2 in Tt-H-NOX proteins measured by transient absorption spectroscopy confirmed the hypothesis that Tt-H-NOX may not be a NO-sensor stricto sensu but a redox sensor. The properties of the Tt-H-NOX protein are not compatible with the role a mere NO-carrier.
• Discovery of a Novel and Selective Indoleamine 2,3-Dioxygenase (IDO-1) Inhibitor 3-(5-Fluoro-1 H -indol-3-yl)pyrrolidine-2,5-dione (EOS200271/PF-06840003) and Its Characterization as a Potential Clinical Candidate
  
  • Crosignani Stefano
  • Bingham Patrick
  • Bottemanne Pauline
  • Cannelle Hélène
  • Cauwenberghs Sandra
  • Cordonnier Marie
  • Dalvie Deepak
  • Deroose Frederik
  • Feng Jun Li
  • Gomes Bruno
  • Greasley Samantha
  • Kaiser Stephen
  • Kraus Manfred
  • Negrerie Michel
  • Maegley Karen
  • Miller Nichol
  • Murray Brion
  • Schneider Manfred
  • Soloweij James
  • Stewart Albert
  • Tumang Joseph
  • Torti Vince
  • van den Eynde Benoit
  • Wythes Martin
  Journal of Medicinal Chemistry, American Chemical Society, 2017, 60 (23), pp.9617 - 9629. (10.1021/acs.jmedchem.7b00974)
  DOI : 10.1021/acs.jmedchem.7b00974
• Conformational dynamics of G-quadruplex DNA probed by time-resolved circular dichroism
  
  • Schmid Marco
  , 2017. Guanine-quadruplexes (G4) are non-canonical DNA structures that result from the hydrophobic stacking of guanine quartets stabilized by metal cations (typically Na+ and K+). There is now an increasing body of experimental evidence of their occurrence in important cell functions correlated to their folding/unfolding mechanisms. However, only few studies have addressed the dynamical aspect of their formation. In this context, we have undertaken the study of several intramolecular G4 with a novel extension of temperature-jump experiments capable to measure the thermal denaturation and the consecutive renaturation of DNA over a time window spanning a few ten milliseconds to seconds. Conformational changes have been monitored by time-resolved circular dichroism (CD), which is known to be sensitive to the chiral arrangement of guanines in the G4 scaffolds.Prior to time-resolved measurements, within the frame of a collaboration with DISCO/SOLEIL, we have performed static synchrotron radiation CD measurements on several short G4-forming sequences, such as human telomere, thrombin-binding aptamer and c-MYC promoter sequences, displaying distinct topologies. Denaturation and renaturation kinetics are found to exhibit biphasic decays with time constants of a few hundred milliseconds and a few seconds, respectively. Those kinetics depend strongly on the amplitude of the temperature jump and the concentration of cations. Taken together these observations suggest the existence of multiple folding pathways on extremely rugged landscapes.
• How aging impacts skin biomechanics: a multiscale study in mice
  
  • Lynch Barbara
  • Bonod-Bidaud Christelle
  • Ducourthial Guillaume
  • Affagard Jean-Sébastien
  • Bancelin Stéphane
  • Psilodimitrakopoulos Sotiris
  • Ruggiero Florence
  • Allain Jean-Marc
  • Schanne-Klein Marie-Claire
  Scientific Reports, Nature Publishing Group, 2017, 7 (1). Skin aging is a complex process that strongly affects the mechanical behavior of skin. This study aims at deciphering the relationship between age-related changes in dermis mechanical behavior and the underlying changes in dermis microstructure. To that end, we use multiphoton microscopy to monitor the reorganization of dermal collagen during mechanical traction assays in ex vivo skin from young and old mice. The simultaneous variations of a full set of mechanical and microstructural parameters are analyzed in the framework of a multiscale mechanical interpretation. They show consistent results for wild-type mice as well as for genetically-modified mice with modified collagen V synthesis. We mainly observe an increase of the tangent modulus and a lengthening of the heel region in old murine skin from all strains, which is attributed to two different origins that may act together: (i) increased cross-linking of collagen fibers and (ii) loss of water due to proteoglycans deterioration, which impedes inner sliding within these fibers. In contrast, the microstructure reorganization upon stretching shows no age-related difference, which can be attributed to opposite effects of the decrease of collagen content and of the increase of collagen cross-linking in old mice. Aging is a complex process that affects the function of all organs and tissues and most often has an irreversible impact on their mechanical behavior. The most visible effects of aging are observed in skin and have been extensively studied for medical and cosmetic purposes. The three skin layers are affected both structurally and functionally. However, aging primary impacts the mechanical integrity of the dermis. At macroscopic scale, the mechanical behavior of aged dermis shows an increased stiffness and a decreased ability to recoil 1–3. At lower scales, a complex multi-parameters process eventually results in a decrease of collagen and elastin contents due to an imbalance between matrix proteins synthesis and degradation by matrix metalloproteinases, an increase of collagen cross-linking, a deterioration of proteoglycans and a subsequent loss of water 4–9. However, the link between these microstructural modifications and the mechanical changes has so far been inferred rather than experimentally demonstrated due to the technical issues encountered when trying to obtain multiscale data. Collagens are the main component of the dermis and other connective tissues 7,10. Fibril-forming collagens assemble into striated fibrils, the diameter and three-dimensional organization of which are tissue-specific. They form multiprotein networks with other matrix proteins such as the elastin fibers and non-fibrillar matrix (pro-teoglycans, glycoaminoglycans…) that determine the mechanical behavior of dermis and other collagen-rich tissues 11–18. Collagen fibers are usually heterotypic structures. In dermis, they are made of type I, III and V col-lagens. Type V collagen is a minor component that acts as a regulatory fibril-forming collagen 19,20. As such, it plays an important role in the pathogenesis of the classical Ehlers-Danlos (EDS) syndrome. This rare connective tissue disease illustrates the close link between collagen microstructure and tissue mechanics since it is caused by mutations in collagen V genes, while being primary characterized by skin hyperextensibility 19–21. Moreover, EDS patients show a prematurely aged skin, which illustrates the close link between collagen microstructure and skin aging. (10.1038/s41598-017-13150-4)
  DOI : 10.1038/s41598-017-13150-4
• Multicolor two-photon imaging of endogenous fluorophores in living tissues by wavelength mixing
  
  • Stringari Chiara
  • Abdeladim Lamiae
  • Malkinson Guy
  • Mahou Pierre
  • Solinas Xavier
  • Lamarre Isabelle
  • Brizion Sébastien
  • Galey Jean-Baptiste
  • Supatto Willy
  • Legouis Renaud
  • Pena Ana-Maria
  • Beaurepaire Emmanuel
  Scientific Reports, Nature Publishing Group, 2017, 7 (1), pp.3792 - 3792. Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750–1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development. Multiphoton microscopy is a powerful tool for label-free and non-invasive functional imaging in small organisms and tissues 1, 2. Pulsed near infrared excitation light allows in-depth imaging based on contrasts such as endog-enous fluorescence 2 , second harmonic generation (SHG) 3 and third harmonic generation (THG) 4. Endogenous fluorescence in living tissues arises from several intrinsic biomarkers that play important roles in physiological processes 2. The primary intracellular sources are NAD(P)H and FAD, the two major cofactors of redox reactions in the cell and central regulators of energy production and metabolism 5, 6. Their fluorescence reports on the metabolic activity of cells allowing tissue physiology and processes such as stem cell differentiation, cancer development and neurodegenerative diseases to being non-invasively monitored 7–12. The fluorescence lifetimes of NADH and FAD are different upon binding to the protein during the electron transport chain. FLIM provides sensitive measurements of the free and protein-bound NAD(P)H ratio and of the redox states (NADH/NAD +) of cells, and can be used to distinguish glycolytic and oxidative phosphorylation metabolic states 13–17. Monitoring lifetime of free and protein-bound FAD has also been exploited to quantify redox ratio FAD/FADH 2 , and used as a biomarker of precancerous epithelial cells 12. It is well established that retinoids play a crucial role in stem cell differentiation and embryo development 18, 19 and their concentration and gradients have been detected in vivo during zebrafish development 9, 20. Other intrinsic fluorophores such as porphyrin, collagen, elastin, keratin, (10.1038/s41598-017-03359-8)
  DOI : 10.1038/s41598-017-03359-8
• Easy xeno-free and feeder-free method for isolating and growing limbal stromal and epithelial stem cells of the human cornea
  
  • Ghoubay-Benallaoua Djida
  • de Sousa Céline
  • Martos Raphaël
  • Latour Gaël
  • Schanne-Klein Marie-Claire
  • Dupin Elisabeth
  • Borderie Vincent
  PLoS ONE, Public Library of Science, 2017, 12 (11), pp.e0188398. Epithelial and stromal stem cells are required to maintain corneal transparency. The aim of the study was to develop a new method to isolate and grow both corneal stromal (SSC) and epithelial limbal (LSC) stem cells from small human limbal biopsies under culture conditions in accordance with safety requirements mandatory for clinical use in humans. Superficial limbal explants were retrieved from human donor corneo-scleral rims. Human limbal cells were dissociated by digestion with collagenase A, either after epithelial scraping or with no scraping. Isolated cells were cultured with Essential 8 medium (E8), E8 supplemented with EGF (E8+) or Green’s medium with 3T3 feeder-layers. Cells were characterized by immunostaining, RT-qPCR, colony forming efficiency, sphere formation, population doubling, second harmonic generation microscopy and differentiation potentials. LSC were obtained from unscraped explants in E8, E8+ and Green’s media and were characterized by colony formation and expression of PAX6, ΔNP63α, Bmi1, ABCG2, SOX9, CK14, CK15 and vimentin, with a few cells positive for CK3. LSC underwent 28 population doublings still forming colonies. SSC were obtained from both scraped and unscraped explants in E8 and E8+ media and were characterized by sphere formation, expression of PAX6, SOX2, BMI1, NESTIN, ABCG2, KERATOCAN, VIMENTIN, SOX9, SOX10 and HNK1, production of collagen fibrils and differentiation into keratocytes, fibroblasts, myofibroblasts, neurons, adipocytes, chondrocytes and osteocytes. SSC underwent 48 population doublings still forming spheres, Thus, this new method allows both SSC and LSC to be isolated from small superficial limbal biopsies and to be primary cultured in feeder-free and xeno-free conditions, which will be useful for clinical purposes. (10.1371/journal.pone.0188398)
  DOI : 10.1371/journal.pone.0188398
• GANs for Biological Image Synthesis
  
  • Osokin Anton
  • Chessel Anatole
  • Salas Rafael E. Carazo
  • Vaggi Federico
  , 2017. In this paper, we propose a novel application of Generative Adversarial Networks (GAN) to the synthesis of cells imaged by fluorescence microscopy. Compared to natural images, cells tend to have a simpler and more geometric global structure that facilitates image generation. However, the correlation between the spatial pattern of different fluorescent proteins reflects important biological functions, and synthesized images have to capture these relationships to be relevant for biological applications. We adapt GANs to the task at hand and propose new models with casual dependencies between image channels that can generate multi-channel images, which would be impossible to obtain experimentally. We evaluate our approach using two independent techniques and compare it against sensible baselines. Finally, we demonstrate that by interpolating across the latent space we can mimic the known changes in protein localization that occur through time during the cell cycle, allowing us to predict temporal evolution from static images.
• Relative Contributions of Core Protein and Solvation Shell in the Terahertz Dielectric Properties of Protein Solutions
  
  • Grognot Marianne
  • Gallot Guilhem
  Journal of Physical Chemistry B, American Chemical Society, 2017, 121 (41), pp.9508 - 9512. The properties of the solvation shell surrounding biomolecules in a solution are fundamental to understand the modifications in the dynamics of the water molecules by peptides and proteins. The dynamics of the hydrogen bonding network typically occurs at the picosecond time scale, so terahertz spectroscopy is a unique tool to investigate the solvation shell. Here, we present the terahertz measurements of the refractive index and extinction coefficient of solutions of biomolecules of various molecular weights. We observe a clear correlation between the terahertz dielectric properties and the weight of the molecules. A three-component model is developed to analyze the relative contributions of the solute and the solvation shell to the total dielectric values. We find that the amino acids and short peptides (small molecules) domains are mainly governed by the solvation shell, whereas the solute properties are also implied in the protein domain (big molecules). (10.1021/acs.jpcb.7b06442)
  DOI : 10.1021/acs.jpcb.7b06442
• High-repetition-rate ultrafast source emitting concomitantly in the 1.3 and 1.7 μm SWIR water-transparency bands for dual-color 3-photon microscopy
  
  • Guesmi Khmaies
  • Abdeladim Lamiae
  • Jurkus Karolis
  • Rigaud Philippe
  • Hanna Marc
  • Mahou Pierre
  • Livet Jean
  • Supatto Willy
  • Georges Patrick
  • Beaurepaire Emmanuel
  • Druon Frédéric
  , 2017. We present a novel ultrafast laser design providing simultaneous excitation in the two water-transparency windows at 1.3 and 1.7 µm, and we demonstrate its potentiality for efficient 3-photon microscopy of nervous tissues. (10.1364/ASSL.2017.ATu6A.5)
  DOI : 10.1364/ASSL.2017.ATu6A.5
• Metrology of Multiphoton Microscopes Using Second Harmonic Generation Nanoprobes
  
  • Mahou Pierre
  • Malkinson Guy
  • Chaudan Élodie
  • Gacoin Thierry
  • Beaurepaire Emmanuel
  • Supatto Willy
  Small, Wiley-VCH Verlag, 2017, 13 (42), pp.1701442. In multiphoton microscopy, the ongoing trend toward the use of excitation wavelengths spanning the entire near‐infrared range calls for new standards in order to quantify and compare the performances of microscopes. This article describes a new method for characterizing the imaging properties of multiphoton microscopes over a broad range of excitation wavelengths in a straightforward and efficient manner. It demonstrates how second harmonic generation (SHG) nanoprobes can be used to map the spatial resolution, field curvature, and chromatic aberrations across the microscope field of view with a precision below the diffraction limit and with unique advantages over methods based on fluorescence. KTiOPO4 nanocrystals are used as SHG nanoprobes to measure and compare the performances over the 850–1100 nm wavelength range of several microscope objectives designed for multiphoton microscopy. Finally, this approach is extended to the post‐acquisition correction of chromatic aberrations in multicolor multiphoton imaging. Overall, the use of SHG nanoprobes appears as a uniquely suited method to standardize the metrology of multiphoton microscopes. (10.1002/smll.201701442)
  DOI : 10.1002/smll.201701442
• Endodermal germ-layer formation through active actin-driven migration triggered by N-cadherin
  
  • Giger Florence
  • David Nicolas
  Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences, 2017, 114 (38), pp.10143 - 10148. Germ-layer formation during gastrulation is both a fundamental step of development and a paradigm for tissue formation and remodeling. However, the cellular and molecular basis of germ-layer segregation is poorly understood, mostly because of the lack of direct in vivo observations. We used mosaic zebrafish embryos to investigate the formation of the endoderm. High-resolution live imaging and functional analyses revealed that endodermal cells reach their characteristic innermost position through an active, oriented, and actin-based migration dependent on Rac1, which contrasts with the previously proposed differential adhesion cell sorting. Rather than being attracted to their destination, the yolk syncytial layer, cells appear to migrate away from their neighbors. This migration depends on N-cadherin that, when imposed in ectodermal cells, is sufficient to trigger their internalization without affecting their fate. Overall, these results lead to a model of germ-layer formation in which, upon N-cadherin expression, endodermal cells actively migrate away from their epiblastic neighbors to reach their internal position, revealing cell-contact avoidance as an unexplored mechanism driving germ-layer formation. (10.1073/pnas.1708116114)
  DOI : 10.1073/pnas.1708116114
• The influence of manufacturing and alterations on skin-based artifacts as characterized by nonlinear optical microscopy
  
  • Robinet Laurianne
  • Thao Sylvie
  • Marie-Claire Schanne-Klein
  • Latour Gaël
  , 2017, pp.1609. Nonlinear optical (NLO) microscopy is an efficient technique to investigate artworks as it allows non-invasive, three-dimensional imaging with micrometer-scale resolution. This technique can combine two-photon excitation fluorescence (2PEF) from exogenous and endogenous fluorophores and second harmonic generation (SHG), which enables the visualization of unstained fibrillar collagen. The application of NLO microscopy to different skin-based materials encountered in cultural heritage showed the influence of manufacturing, in particular the type of tannin and fats introduced, on the morphological structure and collected signals. The data also showed that NLO microscopy was well-suited to non-invasive examination of the degradation state of collagen in all skinbased materials, particularly based on the SHG images, and could be used to identify artifacts most at risk in collections so suitable conservation measures could be taken.
• Identification of the TyrOH •+ Radical Cation in the Flavoenzyme TrmFO
  
  • Nag Lipsa
  • Sournia Pierre
  • Myllykallio Hannu
  • Liebl Ursula
  • Vos Marten H.
  Journal of the American Chemical Society, American Chemical Society, 2017, 139 (33), pp.11500 - 11505. Tyrosine (TyrOH) and tryptophan radicals play important roles as intermediates in biochemical charge-transfer reactions. Tryptophanyl radicals have been observed both in their protonated cation form and in their unprotonated neutral form, but to date, tyrosyl radicals have only been observed in their unprotonated form. With a genetically modified form of the flavoenzyme TrmFO as a suitable model system and using ultrafast fluorescence and absorption spectroscopy, we characterize its protonated precursor TyrOH•+, and we show this species to have a distinct visible absorption band and a transition moment that we suggest to lie close to the phenol symmetry axis. TyrOH•+ is formed in ∼1 ps by electron transfer to excited flavin and decays in ∼3 ps by charge recombination. These findings imply that TyrOH oxidation does not necessarily induce its concerted deprotonation. Our results will allow disentangling of photoproduct states in flavoproteins in often-encountered complex situations and more generally are important for understanding redox chains relying on tyrosyl intermediates. (10.1021/jacs.7b04586)
  DOI : 10.1021/jacs.7b04586
• Réponse impulsionnelle et inférence bayésienne pour la calibration de pinces optiques via la lumière retrodiffusée (Poster)
  
  • Gillant Flavie
  • Perronet Karen
  • Moreau Julien
  • Richly Maximilian U.
  • Alexandrou Antigoni
  • Westbrook Nathalie
  , 2017.
• Discovery of a new Mycobacterium tuberculosis thymidylate synthase X inhibitor with a unique inhibition profile
  
  • Abu El Asrar Rania
  • Margamuljana Lia
  • Klaassen Hugo
  • Nijs Marnik
  • Marchand Arnaud
  • Chaltin Patrick
  • Myllykallio Hannu
  • Becker Hubert F.
  • de Jonghe Steven
  • Herdewijn Piet
  • Lescrinier Eveline
  Biochemical Pharmacology, Elsevier, 2017, 135, pp.69 - 78. Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), is an infection that is responsible for roughly 1.5 million deaths per year. The situation is further complicated by the wide-spread resistance to the existing first- and second-line drugs. As a result of this, it is urgent to develop new drugs to combat the resistant bacteria as well as have lower side effects, which can promote adherence to the treatment regimens. Targeting the de novo synthesis of thymidylate (dTMP) is an important pathway to develop drugs for TB. Although Mtb carries genes for two families of thymidylate synthases (TS), ThyA and ThyX, only ThyX is essential for its normal growth. Both enzymes catalyze the conversion of uridylate (dUMP) to dTMP but employ a different catalytic approach and have different structures. Also, ThyA is the only TS found in humans. This is the rationale for identifying selective inhibitors against ThyX. We exploited the NADPH oxidation to NADP+ step, catalyzed by ThyX, to develop a spectrophotometric biochemical assay. Success of the assay was demonstrated by its effectiveness (average Z'=0.77) and identification of selective ThyX inhibitors. The most potent compound is a tight-binding inhibitor with an IC50 of 710nM. Its mechanism of inhibition is analyzed in relation to the latest findings of ThyX mechanism and substrate and cofactor binding order. (10.1016/j.bcp.2017.03.017)
  DOI : 10.1016/j.bcp.2017.03.017
• Image Data Resource: a bioimage data integration and publication platform
  
  • Williams Eleanor
  • Moore Josh
  • Li Simon
  • Rustici Gabriella
  • Tarkowska Aleksandra
  • Chessel Anatole
  • Leo Simone
  • Antal Bálint
  • Ferguson Richard
  • Sarkans Ugis
  • Brazma Alvis
  • Carazo Salas Rafael
  • Swedlow Jason
  Nature Methods, Nature Publishing Group, 2017, 14 (8), pp.775 - 781. Access to primary research data is vital for the advancement of science. To extend the data types supported by community repositories, we built a prototype Image Data Resource (IDR). IDR links data from several imaging modalities, including high-content screening, multi-dimensional microscopy and digital pathology, with public genetic or chemical databases and cell and tissue phenotypes expressed using controlled ontologies. Using this integration, IDR facilitates the analysis of gene networks and reveals functional interactions that are inaccessible to individual studies. To enable reanalysis, we also established a computational resource based on Jupyter notebooks that allows remote access to the entire IDR. IDR is also an open-source platform for publishing imaging data. Thus IDR provides an online resource and a software infrastructure that promotes and extends publication and reanalysis of scientific image data. (10.1038/nmeth.4326)
  DOI : 10.1038/nmeth.4326
• Label-free imaging of bone multiscale porosity and interfaces using third-harmonic generation microscopy
  
  • Genthial Rachel
  • Beaurepaire Emmanuel
  • Schanne-Klein Marie-Claire
  • Peyrin Françoise
  • Farlay Delphine
  • Olivier Cécile
  • Bala Yohann
  • Boivin Georges
  • Vial Jean-Claude
  • Débarre Delphine
  • Gourrier Aurélien
  Scientific Reports, Nature Publishing Group, 2017, 7 (1), pp.16 p.. Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization. (10.1038/s41598-017-03548-5)
  DOI : 10.1038/s41598-017-03548-5
• All-fiber femtosecond laser providing 9 nJ, 50 MHz pulses at 1650 nm for three-photon microscopy
  
  • Cadroas Patrick
  • Abdeladim L.
  • Kotov Leonid
  • Likhachev Mikhail
  • Lipatov D.
  • Gaponov Dmitry
  • Hideur A.
  • Tang Mincheng
  • Livet J.
  • Supatto W.
  • Beaurepaire E.
  • Fevrier Sébatien
  Journal of Optics, Institute of Physics (IOP), 2017, 19 (6), pp.065506. The spectral window lying between 1.6 and 1.7 mu m is interesting for in-depth multiphoton microscopy of intact tissues due to reduced scattering and absorption in this wavelength range. However, wide adoption of this excitation range will rely on the availability of robust and cost-effective high peak power pulsed lasers operating at these wavelengths. In this communication, we report on a monolithically integrated high repetition rate (50 MHz) all-fiber femtosecond laser based on a soliton self-frequency shift providing 9 nJ, 75 fs pulses at 1650 nm. We illustrate its potential for biological microscopy by recording three-photon-excited fluorescence and third-harmonic generation images of mouse nervous tissue and developing Drosophila embryos labeled with a red fluorescent protein. (10.1088/2040-8986/aa6f72)
  DOI : 10.1088/2040-8986/aa6f72
• Corrélation des propriétés mécaniques et micro-structurelles de la peau de souris à partir d'une sollicitation bi-axiale
  
  • Affagard Jean-Sébastien
  • Ducourthial Guillaume
  • Bonod-Bidaud Christelle
  • Ruggiero Florence
  • Schanne-Klein Marie-Claire
  • Allain Jean-Marc
  , 2017. Le derme, composant principal de la peau, est composée en majorité de collagène et présente une micro-structure très hiérarchisée qui influe sur son comportement mécanique aux différentes échelles. La prédiction de son comportement nécessite de caractériser l’influence de la micro-structure sur les propriétés mécaniques. Aussi, un test de traction bi-axiale couplé indépendamment à une mesure macroscopique et à une mesure microscopique a été développé. Ce travail a finalement permis d’identifier les paramètres d’une loi hyper-élastique anisotrope et de tester l’hypothèse de transformation affine sous-jacent au comportement adopté.
• Banding patterns: exploring a new feature in corneal stroma organization
  
  • Borderie Vincent M.
  • Ghoubay Djida
  • Georgeon Cristina
  • Bocheux Romain
  • Latour Gaël
  • Nguyen Thu-Mai
  • Andreiuolo Felipe
  • Schanne-Klein Marie-Claire
  • Sahel José-Alain
  • Grieve Kate
  , 2017, 58, pp.3906. Purpose : Vogt striae are one of the clinical indicators of keratoconus, and consist of dark, vertically oriented lines crossing the depth of the cornea. We observed similar banding patterns in corneas with pathologies other than keratoconus, and normal corneas. We postulate that these bands form spring-like links between zones of collagen lamellae, and their visibility is enhanced in pathologies where corneal shape is under stress. Methods : : Images of 24 normal and 47 pathological human corneas along with macaque and mouse corneas, were captured with a combination of in vivo confocal microscopy (IVCM), in vivo spectral domain optical coherence tomography (SD-OCT), ex vivo full-field optical coherence microscopy (FFOCM), and histology. Collagen types were immuno-labeled, and electron microscopy was performed. Optical coherence tomography shear wave elastography (OCT-SWE) provided stiffness maps. Second harmonic generation (SHG) microscopy allowed mapping of fibrillar collagen distribution. OCT-SWE and SHG measurements were made on corneas mounted in an artificial chamber while varying intraocular pressure (IOP) up to and beyond physiological levels to investigate behavior of the bands in relation to IOP changes. Results : Banding patterns were observed with all imaging modalities, in all species, with pathology-specific differences. In cross-sectional views of normal and non-keratoconic corneas, these dark bands depart from anchor points at Descemet’s membrane in the posterior stroma obliquely in a V-shape, whereas in keratoconus these bands depart vertically from posterior toward anterior stroma. In en face views the bands criss-crossed in the posterior stroma except in keratoconus where they tended to run parallel. OCT-SWE showed these bands are softer than surrounding stromal tissue, and their orientation was modified and visibility enhanced at non-physiological IOP. Immunohistology revealed these bands to predominantly contain collagen VI. Conclusions : Banding patterns are visible in most corneas, not only in keratoconus. Their visibility may be indicative of stresses to the regular arrangement of collagen lamellae. The role of these soft, elastic regions of collagen 6A1 linking sets of collagen lamellae may be to maintain corneal shape under normal IOP conditions and absorb shocks.
• Interaction of l-cysteine functionalized CdSe quantum dots with metallic cations and selective binding of cobalt in water probed by fluorescence
  
  • Ben Brahim Nassim
  • Bel Haj Mohamed Mohamed
  • Poggi Mélanie
  • Ben Chaâbane Rafik
  • Haouari Mohamed
  • Ben Ouada Hafedh
  • Négrerie Michel
  Sensors and Actuators B: Chemical, Elsevier, 2017, 243, pp.489 - 499. Water-soluble CdSe quantum dots (QDs) capped with l-cysteine (Cys-CdSe) were synthesized in aqueous medium and analyzed by X-ray diffraction, electronic microscopy, absorption spectroscopy and time-resolved fluorescence spectroscopy. We have measured the average diameter of Cys-CdSe QDs, 4.15 nm, the true molecular mass, 1.43 × 105 g mol−1 and the molar extinction coefficient, ε480 = 3 × 105 cm−1 M−1 at maximum of band edge (480 nm). The number of grafted l-cysteine chains per individual QD was measured to be ∼100. The interaction of these functionalized Cys-CdSe QDs at a concentration of 0.2 μM with seventeen different metal ions were evaluated by fluorescence. Only the interaction with Co2+ ions resulted in fluorescence quenching in the range 0.5–20 μM when the true concentration of QDs is 0.2 μM, with a saturation behavior at Co2+ concentration of ∼20 μM, in agreement with 100 grafted l-cysteines per QD. The quenching mechanism involves both static and dynamic fluorescence quenching processes. A model of interaction is derived for the selective binding of Co2+ to Cys-CdSe QDs, involving the carboxyl functions of l-cysteine. The comparison with other QD-systems shows the need for a systematic analysis of the parameters influencing the QD-ions interaction and fluorescence emission, especially their true concentration, in order to understand the fundamental mechanisms at the origin of the specificity for metal binding to a particular QD. (10.1016/j.snb.2016.12.003)
  DOI : 10.1016/j.snb.2016.12.003
• Cell viability and shock wave amplitudes in the endothelium of porcine cornea exposed to ultrashort laser pulses
  
  • Hussain Syed Asad
  • Milián Carles
  • Crotti Caroline
  • Kowalczuk Laura
  • Alahyane Fatima
  • Essaïdi Zacaria
  • Couairon Arnaud
  • Schanne-Klein Marie-Claire
  • Plamann Karsten
  Graefe's Archive for Clinical and Experimental Ophthalmology, Springer Verlag, 2017, 255 (5), pp.945 - 953. Purpose Some forms of keratoplasty assisted by ultrashort-pulse lasers require performing laser cuts close to the endothelium, which requires the knowledge of "safe" values concerning incision depth and pulse energy preserving endothelial cell viability. Our study aims to determine the thresholds for cell death in porcine corneas exposed to ultrashort laser pulses, in terms of laser pulse energy and nearness of the impacts to the endothelium. Methods Using a laboratory laser set-up, lamellar cuts were induced while varying pulse energies and distances from the endothelium. A fluorescent staining protocol was used to determine the percentage of surviving endothelial cells. Numerical simulations of the Euler equations for compressible fluids provided pressure level and axial and radial pressure gradient estimates at the endothelium. Results Ninety percent of the endothelial cells survived when using 16.5 mu J pulses no closer than 200 mu m to the endothelium, or pulses not exceeding 2 mu J at a distance of 50 mu m. The comparison of the observed percentage of surviving cells with the estimates of the shock wave amplitudes and gradients generated by the laser pulses yielded cell death thresholds at amplitudes in the megapascal range, or gradients of the order of 10(8) Pa/m. Conclusions Our results provide limits in terms of pulse energy and distance of the incision from the endothelium within which endothelial cell viability is preserved. Current forms of corneal laser surgery are compatible with these limits. However, these limits will need to be considered for the development of future laser routines working in close proximity to the endothelium. (10.1007/s00417-017-3583-3)
  DOI : 10.1007/s00417-017-3583-3
• ANALYSE MULTI-ECHELLE DE LA POROSITE OSSEUSE PAR MICROSCOPIE OPTIQUE NON LINEAIRE
  
  • Genthial Rachel
  • Beaurepaire Emmanuel
  • Schanne-Klein Marie-Claire
  • Peyrin Françoise
  • Débarre Delphine
  • Gourrier Aurélien
  , 2017. La porosité corticale est un marqueur important de la fragilité osseuse associée au vieillissement et à l’ostéoporose [1]. Les difficultés dans l’analyse du risque de fracture résident dans la complexité morphologique,multiéchellede la porosité ainsi que le lien avec la structure tissulaire. En particulier, la visualisation du réseau lacuno-canaliculaire (LCN) associé à la présence d’ostéocytes représente un défi important puisque les échelles associées sont de ̴ 10 – 30 μm pour les lacunes hébergeant les corps cellulaires, à ̴ 100 – 500 nm pour les canalicules autour des process dendritiques connectant ces cellules. En outre, l’analyse histologique du LCN requiertdes champs de vue de ̴ 1 mm2. À ce jour, l’observation 3D du LCN est essentiellement effectuée par microscopie confocale ou bi-photon de fluorescence [2]et par nanotomographie de rayons X synchrotron (SR-nanoCT) [3]. Cette présentation vise à introduire une nouvelle modalité d’imagerie optique non-linéaire par génération de troisième harmonique (THG) pour l’étude du LCN, combinée avec l’imagerie par génération de seconde harmonique (SHG) pour l’imagerie du collagène fibrillaire.
• Exposure to selenomethionine causes selenocysteine misincorporation and protein aggregation in Saccharomyces cerevisiae.
  
  • Plateau Pierre
  • Saveanu Cosmin
  • Lestini Roxane
  • Dauplais Marc
  • Decourty Laurence
  • Jacquier Alain
  • Blanquet Sylvain
  • Lazard Myriam
  Scientific Reports, Nature Publishing Group, 2017, 7, pp.44761. Selenomethionine, a dietary supplement with beneficial health effects, becomes toxic if taken in excess. To gain insight into the mechanisms of action of selenomethionine, we screened a collection of ≈5900 Saccharomyces cerevisiae mutants for sensitivity or resistance to growth-limiting amounts of the compound. Genes involved in protein degradation and synthesis were enriched in the obtained datasets, suggesting that selenomethionine causes a proteotoxic stress. We demonstrate that selenomethionine induces an accumulation of protein aggregates by a mechanism that requires de novo protein synthesis. Reduction of translation rates was accompanied by a decrease of protein aggregation and of selenomethionine toxicity. Protein aggregation was supressed in a ∆cys3 mutant unable to synthetize selenocysteine, suggesting that aggregation results from the metabolization of selenomethionine to selenocysteine followed by translational incorporation in the place of cysteine. In support of this mechanism, we were able to detect random substitutions of cysteinyl residues by selenocysteine in a reporter protein. Our results reveal a novel mechanism of toxicity that may have implications in higher eukaryotes. (10.1038/srep44761)
  DOI : 10.1038/srep44761
• High-Throughput Sequencing Reveals Circular Substrates for an Archaeal RNA ligase.
  
  • Becker Hubert F
  • Héliou Alice
  • Djaout Kamel
  • Lestini Roxane
  • Regnier Mireille
  • Myllykallio Hannu
  RNA Biology, Taylor & Francis, 2017, 14, pp.1075-1085. (10.1080/15476286.2017.1302640)
  DOI : 10.1080/15476286.2017.1302640