Publications

2016

• Ultrafast Light-Induced Processes in DNA Photolyase and Its Substrate-Bound Complex
  
  • Brettel Klaus
  • Byrdin Martin
  • Vos Marten H.
  , 2016.
• La méthylation flavine-dépendante d’acides nucléiques : aspects évolutifs, métaboliques, biochimiques et spectroscopiques
  
  • Sournia Pierre
  , 2016. La méthylation de l’uridine sur son carbone 5 est apparue au cours de l’évolution sous plusieurs formes. Tout d’abord, les thymidylate synthases permettent la synthèse de novo du dTMP, un précurseur essentiel de l’ADN des trois règnes du vivant. Deux familles de thymidylate synthases sont connues à ce jour : ThyA et la flavo-enzyme ThyX, codées par des gènes hétérologues et ayant des structures et mécanismes réactionnels radicalement différents. En outre, cette méthylation de l’uridine est apparue (probablement plus tard) sous forme de modifications post-transcriptionnelles des ARNt et ARNr. Cette thèse vise à questionner les contraintes évolutives ayant menés indépendament à ces quatres types de méthylation de l’uridine.Une première partie décrit l’identification d’une voie métabolique permetant la complémentation du phénotype d’auxotrophie pour la thymidine par des analogues nucléotidiques chez Escherichia coli. Une approche de biologie synthétique en vue d’établir une voie alternative de biosynthèse du thymidylate a aussi été mise en œuvre. Une technique de sélection de gènes de complémentation du phénotype d’auxotrophie pour la thymidine, issus de mutagénèse aléatoire, a pu être développée. Dans une seconde partie, des études biochimiques et sppectroscopiques ont été réalisées sur la méthyle-transférase flavine-dépendante TrmFO, responsable de la méthylation post-transciptionnelle de l’uridine 54 des ARNt de certains microorganismes.L’implication de certains résidus dans la fixation du substrat a pu être déterminée d’une part, et certains intermédiaires réactionnels potentiels ont été caractérisés spectralement d’autre part. Ces dernières observations s’appuient, en outre, sur des études en cours de spectroscopie résolue en temps et des simulations de dynamique moléculaire afin de mieux comprendre les flavoprotéines en général et les méthyle transférases flavine-dépendantes en particulier.
• Xanthines Studied via Femtosecond Fluorescence Spectroscopy
  
  • Changenet-Barret P.
  • Kovács L.
  • Markovitsi D.
  • Gustavsson T.
  Molecules, MDPI, 2016, 21 (12), pp.1668. Xanthines represent a wide class of compounds closely related to the DNA bases adenine and guanine. Ubiquitous in the human body, they are capable of replacing natural bases in double helices and give rise to four-stranded structures. Although the use of their fluorescence for analytical purposes was proposed, their fluorescence properties have not been properly characterized so far. The present paper reports the first fluorescence study of xanthine solutions relying on femtosecond spectroscopy. Initially, we focus on 3-methylxanthine, showing that this compound exhibits non-exponential fluorescence decays with no significant dependence on the emission wavelength. The fluorescence quantum yield (3 × 10−4) and average decay time (0.9 ps) are slightly larger than those found for the DNA bases. Subsequently, we compare the dynamical fluorescence properties of seven mono-, di- and tri-methylated derivatives. Both the fluorescence decays and fluorescence anisotropies vary only weakly with the site and the degree of methylation. These findings are in line with theoretical predictions suggesting the involvement of several conical intersections in the relaxation of the lowest singlet excited state. (10.3390/molecules21121668)
  DOI : 10.3390/molecules21121668
• The Dynamics Behind the Affinity: Controlling Heme-Gas Affinity via Geminate Recombination and Heme Propionate Conformation in the NO Carrier Cytochrome c ′
  
  • Andrew Colin R.
  • Petrova Olga N.
  • Lamarre Isabelle
  • Lambry Jean-Christophe
  • Rappaport Fabrice
  • Négrerie Michel
  ACS Chemical Biology, American Chemical Society, 2016, 11 (11), pp.3191 - 3201. Nitric oxide (NO) sensors are heme proteins which may also bind CO and O2. Control of heme-gas affinity and their discrimination are achieved by the structural properties and reactivity of the heme and its distal and proximal environments, leading to several energy barriers. In the bacterial NO sensor cytochrome c′ from Alcaligenes xylosoxidans (AXCP), the single Leu16Ala distal mutation boosts the affinity for gas ligands by a remarkable 106–108-fold, transforming AXCP from one of the lowest affinity gas binding proteins to one of the highest. Here, we report the dynamics of diatomics after photodissociation from wild type and L16A-AXCP over 12 orders of magnitude in time. For the L16A variant, the picosecond geminate rebinding of both CO and NO appears with an unprecedented 100% yield, and no exit of these ligands from protein to solvent could be observed. Molecular dynamic simulations saliently demonstrate that dissociated CO stays within 4 Å from Fe2+, in contrast to wild-type AXCP. The L16A mutation confers a heme propionate conformation and docking site which traps the diatomics, maximizing the probability of recombination and directly explaining the ultrahigh affinities for CO, NO, and O2. Overall, our results point to a novel mechanism for modulating heme-gas affinities in proteins. (10.1021/acschembio.6b00599)
  DOI : 10.1021/acschembio.6b00599
• Dynamique cellulaire des protéines de la réplication chez l'archée halophile Haloferax volcanii
  
  • Delpech Floriane
  , 2016. Ce travail de thèse porte sur l’étude de la réplication chez les archées, qui constituent le troisième domaine du vivant avec les bactéries et les eucaryotes. L’organisme modèle que nous avons utilisé est l'archée halophile Haloferax volcanii car les outils génétiques disponibles permettent d’exprimer des protéines fusionnées à la Protéine Fluorescente Verte (GFP) dans cet organisme mésophile et aérobe et ainsi de localiser les protéines d’intérêt dans des cellules vivantes. Nous nous sommes ainsi intéressés à la localisation cellulaire de quatre protéines de la réplication qui ont été fusionnées à la GFP et exprimées sous contrôle de leur propre promoteur : (i) la protéine ‘Flap Endonuclease 1’ (FEN1), qui intervient dans la maturation des fragments d’Okazaki, (ii) la protéine ‘Origin Recognition Complex’ (ORC1) impliquée dans la reconnaissance des origines de réplication, (iii) la protéine ‘Proliferating Cellular Nuclear Antigen’ (PCNA), anneau de processivité des ADN polymérases réplicatives, et (iv) la protéine de fixation à l’ADN simple-brin ‘Replication Protein A’ (RPA2) essentielle à la réplication chez H. volcanii. Seule la protéine PCNA n’a pu être exprimée en fusion avec la GFP, suggérant que la protéine fusion n’est pas fonctionnelle. GFP::Orc1 et GFP::Fen1 ont été exprimées dans la cellule mais ne présentent pas de localisation spécifique reflétant un rôle de ces protéines dans la réplication de l’ADN. En revanche des foyers de fluorescence de la protéine fusion GFP::Rpa2 ont été observés, dont le nombre augmente significativement dans des cellules exposées à l’aphidicoline, drogue inhibant la synthèse de l’ADN et induisant ainsi un stress réplicatif. Cependant une localisation différente de la protéine GFP::Rpa2 a été observée lorsque les cellules sont exposés à la phléomycine, qui induit notamment des cassures double-brin de l‘ADN. Dans ces cellules, GFP::Rpa2 forme un foyer de fluorescence massif qui colocalise avec l’ADN compacté dans la grande majorité des cellules observées. Nos résultats suggèrent donc que la localisation spécifique observée pour GFP::Rpa2 reflète son rôle dans la réparation de l’ADN et/ou le redémarrage des fourche de réplication arrêtées.
• Unusual Dynamics of Ligand Binding to the Heme Domain of the Bacterial CO Sensor Protein RcoM-2
  
  • Bouzhir-Sima Latifa
  • Motterlini Roberto
  • Gross Julia
  • Vos Marten H.
  • Liebl Ursula
  Journal of Physical Chemistry B, American Chemical Society, 2016, 120 (41), pp.10686 - 10694. The aerobic Gram-negative bacterium Burkholderia xenovorans expresses two highly homologous carbon monoxide (CO)-responsive transcriptional regulators, RcoM-1 and RcoM-2, which display extraordinarily high CO affinities, even under oxygenic conditions. To gain insight into the origin and perspectives of this feature, we characterized the ligand-binding properties of the N-terminal, heme-binding Per/Arnt/Sim sensor domain of RcoM-2 by time-resolved spectroscopy. We show that upon photodissociation of the heme–ligand bond, CO geminately rebinds to the heme with picosecond time constants and more than 99% rebinding yield, an unprecedented property of native heme proteins. Remarkably, the rebinding kinetics speeds up when the protein motions are slowed by cooling or solvent viscosity. This indicates that the origin of the observed efficient rebinding is a protein-imposed CO configuration in the heme pocket that is highly favorable for binding, a feature strongly in contrast to that of hemoglobins. The binding of CO to the ferrous heme from the solvent requires dissociation of the methionine axial heme ligand. From the kinetics of ligand binding and the extreme stability of the CO complex, we deduce that the dissociation constant for CO is lower than 100 pM. Finally, we show that when the ferric complex is exposed to CO gas or a CO-releasing molecule under oxygenic conditions formation of the ferrous carbonyl complex can occur on a time scale of minutes in the presence of a redox mediator. These findings pave the way for possible applications of the RcoM-2 heme domain as a CO sensor and/or scavenger. (10.1021/acs.jpcb.6b08160)
  DOI : 10.1021/acs.jpcb.6b08160
• Imagerie quantitative de biopolymères par génération de second harmonique résolue en polarisation.
  
  • Teulon Claire
  , 2016. Le collagène est un élément majeur de l'architecture des organes chez les mammifères. Cette protéine s'organise en structures tridimensionnelles (3D) spécifiques à chaque tissu et responsables de leurs propriétés biophysiques et biomécaniques. La microscopie multiphoton permet de visualiser le collagène fibrillaire dans les tissus biologiques, sans aucun marquage, grâce aux signaux de génération de second harmonique (SHG). Cette thèse présente des mesures SHG résolues en polarisation (P-SHG), dans le but de caractériser la structure 3D du collagène dans divers tissus, de l'échelle moléculaire à l'échelle macroscopique.Nous avons d'abord étudié la sensibilité et la fiabilité des mesures P-SHG, afin de valider cette technique comme un outil quantitatif d'observation de la structure 3D du collagène dans des tissus intacts.En collaboration avec le Laboratoire de Chimie de la Matière Condensée de Paris, cette technique a ensuite été appliquée à l'étude de systèmes modèles de collagène présentant une organisation de type cristal liquide, afin de caractériser les conditions physico-chimiques menant à des phases proches de celles observées à l’état stabilisé dans la cornée.Enfin, nous présentons une imagerie SHG en différence circulaire (CD-SHG), permettant de déterminer la polarité des fibrilles de collagène par rapport au plan de l'image. Ces mesures sont complémentaires de l'information obtenue en P-SHG. Une première mise en place expérimentale de cette technique est présentée dans des coupes histologiques de cornée humaine. Nous présentons de plus les résultats préliminaires d'une imagerie corrélative CD-SHG/I-SHG, en collaboration avec l'INRS, donnant une information complète sur la polarité des fibrilles de collagène.
• Imagerie térahertz par réflexion interne totale pour la biologie. : Application à l'étude de la perméabilisation cellulaire.
  
  • Grognot Marianne
  , 2016. Les ondes térahertz s’étendent de 0.1 à 10x1012 Hz, à la frontière entre les domaines de l’optique et des radiofréquences. Cette position intermédiaire originale en a longtemps rendu l’accès difficile : les technologies térahertz n’ont pris leur essor qu’au cours des années 90. Le domaine n’a pas encore atteint la maturité des domaines des microondes ou de l’infrarouge qui le jouxtent. Cependant, les motivations exploratoires sont fortes, de par la sensibilité spectroscopique du térahertz aux états moléculaires (rotationnels, vibrationnels..) et aux liaisons faibles établies dans et entre les molécules. Dans le cas des objets biologiques, le térahertz est particulièrement sensible à l’eau : sa quantité, son état physico-chimique et ses solutés.Nous avons mis en œuvre un montage d’imagerie en réflexion interne totale atténuée (ATR) pour pouvoir distinguer des cellules vivantes de leur milieu physiologique. Au cours de ce travail, le montage d’imagerie ATR a été caractérisé théoriquement, puis expérimentalement. La première démonstration de l’origine du contraste sur ces images térahertz a été réalisée. Il provient du contenu intracellulaire, plus spécifiquement des protéines et peptides dissouts dans le cytoplasme.Une analyse fine des mécanismes sous-jacents à la nature protéique du contraste térahertz a également été développée. Elle donne accès à des informations spectroscopiques inédites sur l’eau, les protéines dissoutes et la couche de solvatation les entourant.Mettant à profit cette compréhension de notre montage térahertz, nous l’avons proposé comme outil non invasif de suivi quantitatif de la perméabilisation de cellules en conditions physiologiques. Lors de la perméabilisation, augmentation des transferts moléculaires à travers la membrane, notre outil permet de quantifier le passage des peptides et protéines. La perméabilisation de cellules vivantes a une gamme d’application vaste, de l’entrée de fluorochromes pour l’imagerie ou de médicaments à la thérapie génique. Afin d’assurer ces passages à travers la membrane des cellules, il est nécessaire d’altérer ses propriétés, sans pour autant compromettre la viabilité cellulaire. L’étude de deux types de perméabilisation avec notre outil térahertz est proposée : la perméabilisation chimique et l’électroporation. Dans les deux cas, des mécanismes d’effet dose ont été caractérisés quantitativement. Notre outil térahertz a démontré de grands avantages devant les méthodes actuellement utilisées pour quantifier ces dynamiques de perméabilisation et en caractériser la réversibilité.
• Thermal stability and binding energetics of thymidylate synthase ThyX
  
  • Krumova Sashka
  • Todinova Svetla
  • Tileva Milena
  • Bouzhir-Sima Latifa
  • Vos Marten H.
  • Liebl Ursula
  • Taneva Stefka G.
  International Journal of Biological Macromolecules, Elsevier, 2016, 91, pp.560 - 567. The bacterial thymidylate synthase ThyX is a multisubstrate flavoenzyme that takes part in the de novo synthesis of thymidylate in a variety of microorganisms. Herein we study the effect of FAD and dUMP binding on the thermal stability of wild type (WT) ThyX from the mesophilic Paramecium bursaria chlorella virus-1 (PBCV-1) and from the thermophilic bacterium Thermotoga maritima (TmThyX), and from two variants of TmThyX, Y91F and S88W, using differential scanning calorimetry. The energetics underlying these processes was characterized by isothermal titration calorimetry. The PBCV-1 protein is significantly less stable against the thermal challenge than the TmThyX WT. FAD exerted stabilizing effect greater for PBCV-1 than for TmThyX and for both mutants, whereas binding of dUMP to FAD-loaded proteins stabilized further only TmThyX. Different thermodynamic signatures describe the FAD binding to the WT ThyX proteins. While TmThyX binds FAD with a low μM binding affinity in a process characterized by a favorable entropy change, the assembly of PBCV-1 with FAD is governed by a large enthalpy change opposed by an unfavorable entropy change resulting in a relatively strong nM binding. An enthalpy-driven formation of a high affinity ternary ThyX/FAD/dUMP complex was observed only for TmThyX. (10.1016/j.ijbiomac.2016.05.083)
  DOI : 10.1016/j.ijbiomac.2016.05.083
• Probing living cells composition by THz attenuated total reflection — Application to quantitative permeabilization measurement
  
  • Grognot M.
  • Gallot Guilhem
  , 2016, pp.1-2. (10.1109/IRMMW-THZ.2016.7758371)
  DOI : 10.1109/IRMMW-THZ.2016.7758371
• Synthesis, characterization and spectral temperature-dependence of thioglycerol-CdSe nanocrystals
  
  • Ben Brahim Nassim
  • Poggi Mélanie
  • Haj Mohamed Mohamed
  • Ben Chaâbane Rafik
  • Haouari Mohamed
  • Negrerie Michel
  • Ben Ouada Hafedh
  Journal of Luminescence, Elsevier, 2016, 177, pp.402 - 408. Water-soluble CdSe quantum dots (QDs) have been synthesized with thioglycerol as a stabilizer through a novel hydrothermal route. The obtained thioglycerol capped CdSe (TG-CdSe) nanocrystals were characterized regarding their morphology and structural, thermal and optical properties. The resulting nanocrystals were synthesized in the cubic structure with a near spherical shape, as confirmed by X-ray diffraction and transmission electron microscopy. Combining transmission electron microscopy imaging and calculations using UV–visible absorption spectrum and X-ray diffraction pattern, the diameter of the synthesized nanocrystals was estimated to 2.26 nm. As confirmed by its Fourier transform IR spectrum, thioglycerol was successfully liganded on the surface of the resulting nanocrystals. Band structure parameters of the TG-CdSe nanoparticles were determined and quantum confinement effect was evidenced by optical absorption, fluorescence and Raman measurements. The thermal properties of the TG-CdSe were explored by thermal gravimetric analysis and differential scanning calorimetry. The temperature dependence of both the absorption and fluorescence spectra in the physiological range makes the TG-CdSe nanocrystals sensitive temperature markers, a property that must be taken into account when developing any probing applications, especially for cellular imaging. (10.1016/j.jlumin.2016.05.026)
  DOI : 10.1016/j.jlumin.2016.05.026
• Transient Two-Dimensional Infrared Spectroscopy in a Vibrational Ladder
  
  • Kemlin Vincent
  • Bonvalet Adeline
  • Daniault Louis
  • Joffre Manuel
  Journal of Physical Chemistry Letters, American Chemical Society, 2016, 7 (17), pp.3377 - 3382. We report on transient 2D Fourier transform infrared spectroscopy (2DIR) after vibrational ladder climbing induced in the CO-moiety longitudinal stretch of carboxyhemoglobin. The population distribution, spreading up to seven vibrational levels, results in a nonequilibrium 2DIR spectrum evidencing a large number of peaks that can be easily attributed to individual transitions thanks to the anharmonicity of the vibrational potential. We discuss the physical origin of the observed peaks as well as the qualitative behavior of the subsequent dynamics governed by population relaxation in the vibrational ladder. (10.1021/acs.jpclett.6b01535)
  DOI : 10.1021/acs.jpclett.6b01535
• Efficient second-harmonic imaging of collagen in histological slides using Bessel beam excitation
  
  • Vuillemin Nelly
  • Mahou Pierre
  • Débarre Delphine
  • Gacoin Thierry
  • Tharaux Pierre-Louis
  • Schanne-Klein Marie-Claire
  • Supatto Willy
  • Beaurepaire Emmanuel
  Scientific Reports, Nature Publishing Group, 2016, 6 (29863 (2016)). econd-harmonic generation (SHG) is the most specific label-free indicator of collagen accumulation in widespread pathologies such as fibrosis, and SHG-based measurements hold important potential for biomedical analyses. However, efficient collagen SHG scoring in histological slides is hampered by the limited depth-of-field of usual nonlinear microscopes relying on focused Gaussian beam excitation. In this work we analyze theoretically and experimentally the use of Bessel beam excitation to address this issue. Focused Bessel beams can provide an axially extended excitation volume for nonlinear microscopy while preserving lateral resolution. We show that shaping the focal volume has consequences on signal level and scattering directionality in the case of coherent signals (such as SHG) which significantly differ from the case of incoherent signals (two-photon excited fluorescence, 2PEF). We demonstrate extended-depth SHG-2PEF imaging of fibrotic mouse kidney histological slides. Finally, we show that Bessel beam excitation combined with spatial filtering of the harmonic light in wave vector space can be used to probe collagen accumulation more efficiently than the usual Gaussian excitation scheme. These results open the way to SHG-based histological diagnoses. (10.1038/srep29863)
  DOI : 10.1038/srep29863
• Three-photon microscopy with a monolithic all-fiber laser format laser emitting at 1650 nm
  
  • Cadroas Patrick
  • Kotov Leonid
  • Abdeladim Lamiae
  • Likhachev Mikhail
  • Lipatov D.
  • Hideur Ammar
  • Supatto Willy
  • Livet Jean
  • Beaurepaire Emmanuel
  • Fevrier Sébastien
  , 2016.
• Comparison between experimental and affine reorientation of collagen fibers in plane fibrous tissues
  
  • Jayyosi Charles
  • Bruyère-Garnier Karine
  • Coret Michel
  • Affagard Jean-Michel
  • Bancelin Stéphane
  • Ducourthial Guillaume
  • Schanne-Klein Marie-Claire
  , 2016.
• Réponse impulsionnelle et inférence bayésienne pour la calibration de pinces optiques via la lumière rétrodiffusée (Poster)
  
  • Gillant Flavie
  • Perronet Karen
  • Moreau Julien
  • Richly Maximilian U.
  • Alexandrou Antigoni
  • Westbrook Nathalie
  , 2016.
• Simultaneous microstructural and mechanical characterization of human corneas at increasing pressure
  
  • Benoit Aurélie
  • Latour Gaël
  • Marie-Claire Schanne-Klein
  • Allain Jean-Marc
  Journal of the mechanical behavior of biomedical materials, Elsevier, 2016, 60, pp.93-105. The cornea, through its shape, is the main contributor to the eye׳s focusing power. Pathological alterations of the cornea strongly affect the eye power. To improve treatments, complex biomechanical models have been developed based on the architecture and mechanical properties of the collagen network in the stroma, the main layer of the cornea. However, direct investigations of the structure of the stroma, as well as its link to the mechanical response, remained limited. We propose here an original set up, associating nonlinear optical imaging and mechanical testing. By using polarization resolved Second Harmonic signals, we simultaneously quantified micrometer (orientation of the collagen lamellae) and nanometer (local disorder within lamellae) scale corneal organization. We showed that the organization of the lamellae changes along the stroma thickness. Then, we measured simultaneously the deformation on the epithelial side of the cornea and the reorientation of the collagen lamellae for increasing intraocular pressure levels, from physiological ones to pathological ones. We showed that the observed deformation is not correlated to initial orientation, but to the reorganization of the lamellae in the stroma. Our results, by providing a direct multi-scale observation, will be useful for the development of more accurate biomechanical model (10.1016/j.jmbbm.2015.12.031)
  DOI : 10.1016/j.jmbbm.2015.12.031
• Optique non-linéaire
  
  • Hache François
  , 2016.
• Evolution of the Skin Microstructural Organization During a Mechanical Assay
  
  • Lynch Barbara
  • Bancelin Stéphane
  • Bonod Bidaud Christelle
  • Marie-Claire Schanne-Klein
  • Allain Jean-Marc
  , 2016, 6, pp.45-52. Skin is a complex multi-layered tissue, consisting of three main parts: the epidermis, the dermis and the hypodermis. The dermis is responsible for most of the complex mechanical properties of skin, such as viscoelasticity, non-linearity and anisotropy. At the microscopic level the dermis consists for the greater part of extracellular matrix, compounded mainly of collagen fibers forming an orderless network. The mechanical properties of skin have been studied in the past, but their exact link with the microscopic organization is still an open question. The goal of our study is to measure the evolution of the microstructure during a mechanical assay and to improve existing mechanical models of skin with relevant parameters identified at the microscopic level. We perform uniaxial tensile test on ex vivo mouse skin. The mechanical tests are performed in situ under a second harmonic generation microscope. This allows us to determine quantitatively and simultaneously the mechanical response and the microstructural reorganization of the tissue. This technique can be used to better understand the link between pathological alterations of collagen synthesis, fibers organization, and alteration of the biomechanical properties of skin, as in the Ehlers-Danlos syndrome (EDS). (10.1007/978-3-319-41351-8_7)
  DOI : 10.1007/978-3-319-41351-8_7
• High repetition rate ultrafast MIR fiber laser system for deep multiphoton microscopy (poster)
  
  • Guesmi Khmaies
  • Jurkus Karolis
  • Rigaud Philippe
  • Supatto Willy
  • Beaurepaire Emmanuel
  • Hanna Marc
  • Druon Frédéric
  • Georges Patrick
  , 2016.
• Ultrafast Electron Transfer in Complexes of Doxorubicin with Human Telomeric G-Quadruplexes and GC Duplexes Probed by Femtosecond Fluorescence Spectroscopy
  
  • Changenet-Barret P.
  • Gustavsson T.
  • Markovitsi D.
  • Manet I.
  ChemPhysChem, Wiley-VCH Verlag, 2016, 17 (9), pp.1264–1272. Doxorubicin (DOX) is a natural anthracycline widely used in chemotherapy; its combined application as a chemotherapeutic and photodynamic agent has been recently proposed. In this context, understanding the photoinduced properties of DOX complexes with nucleic acids is crucial. Herein, the study of photoinduced electron transfer in DOX–DNA complexes by femtosecond fluorescence spectroscopy is reported. The behaviour of complexes with two model DNA structures, a G-quadruplex (G4) formed by the human telomeric sequence (Tel21) and a d(GC) duplex, is compared. The DOX affinity for these two sequences is similar. Although both 1:1 and 2:1 stoichiometries have been reported for DOX–G4 complexes, only 1:1 complexes form with the duplex. The steady-state absorption indicates a strong binding interaction with the duplex due to drug intercalation between the GC base pairs. In contrast, the interaction of DOX with Tel21 is much weaker and arises from drug binding on the G4 external faces at two independent binding sites. As observed for DOX–d(GC) complexes, fluorescence of the drug in the first binding site of Tel21 exhibits decays within a few picoseconds following a biphasic pattern; this is attributed to the existence of two drug conformations. The fluorescence of the drug in the second binding site of Tel21 shows slower decays within 150 ps. These timescales are consistent with electron transfer from the guanines to the excited drug, as favoured by the lower oxidation potential of the stacked guanines of G4 with respect to those in the duplex. (10.1002/cphc.201501091)
  DOI : 10.1002/cphc.201501091
• 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
  , 2016, 57, pp.1207. Purpose : Clinical surgical laser systems based on ultrashort pulse lasers have become increasingly widespread in corneal surgery. Some forms of keratoplasty require performing laser cuts close to the endothelium, using “safe” values concerning incision depth and pulse energy in order to avoid damaging the endothelium. We present a study on the viability of endothelial cells of porcine corneas exposed to ultrashort laser pulses. The thresholds for cell death in terms of laser pulse energy and closeness of the impacts to the endothelium were compared to numerical simulations of the maximum shock wave amplitude at the corneal endothelium. Methods : Experiments were performed with a laboratory set-up based on a laser emitting 730 fs pulses at a wavelength of 1030 nm in corneas from eyes of pigs aged of about 6 months. Lamellar cuts were induced, first, using pulse energies of 16.5 µJ at distances from the endothelium from 50 µm to 400 µm, and then, at a fixed distance of 50 µm from the endothelium with pulse energies from 2 µJ to 14 µJ. The triple Hoechst 33342-Ethidium-Calcein staining protocol was used to determine the viability of endothelial cells after the laser cut. Numerical simulations of the Euler equations for compressible fluids provided pressure level estimates at the corneal endothelium. The broadening of the laser spot in the volume of the cornea was taken into account for the calculations. Results : Ninety percent of the endothelial cells survived when using 16.5 µJ pulses not closer than 200 µm to the endothelium, or pulses not exceeding 2 µJ at a distance of 50 µm. The comparison of the observed cell viability percentage with the calculated estimates of the shock wave amplitudes generated by the laser pulses yielded cell death thresholds at amplitudes in the Megapascal range. Conclusions : Our results permit to define limits in terms of pulse energy and distance of the incision to the endothelium within which the viability of the endothelial cells is preserved. The parameters used for current forms of corneal surgery assisted by ultrashort pulse lasers are compatible with these limits. However, these limits will need to be taken into account for the development of future laser routines working closely to the endothelium as cell death threshold may be reached under those conditions.
• Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments
  
  • Latour Gaël
  • Robinet Laurianne
  • Dazzi Alexandre
  • Portier François
  • Deniset-Besseau Ariane
  • Schanne-Klein Marie-Claire
  Scientific Reports, Nature Publishing Group, 2016, 6, pp.26344. This paper presents the correlative imaging of collagen denaturation by nonlinear optical microscopy (NLO) and nanoscale infrared (IR) spectroscopy to obtain morphological and chemical information at different length scales. Such multiscale correlated measurements are applied to the investigation of ancient parchments, which are mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, the ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, which may also occur in biological tissues. Key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, correlated to the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals, which are a structural probe of the fibrillar organization of the collagen at the micrometer scale. NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non-invasive way. It should provide a relevant method to assess or monitor the condition of collagen-based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material. (10.1038/srep26344)
  DOI : 10.1038/srep26344
• A Primer on the Bayesian Approach to High-Density Single-Molecule Trajectories Analysis
  
  • El beheiry Mohamed
  • Türkcan Silvan
  • Richly Maximilian u.
  • Triller Antoine
  • Alexandrou Antigone
  • Dahan Maxime
  • Masson Jean-Baptiste
  Biophysical Journal, Biophysical Society, 2016, 110 (6), pp.1209 - 1215. (10.1016/j.bpj.2016.01.018)
  DOI : 10.1016/j.bpj.2016.01.018
• Microscopie optique non-linéaire quantitative
  
  • Beaurepaire Emmanuel
  • Schanne-Klein Marie-Claire
  • Vuillemin Nelly
  • Supatto Willy
  • Mahou Pierre
  , 2016.